AU2004201501A1 - Recombinational cloning using engineered recombination sites - Google Patents

Description

P/00/0 II Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: INVITROGEN CORPORATION James L. HARTLEY; Michael A. BRASCH CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia RECOMBINATIONAL CLONING USING ENGINEERED RECOMBINATION SITES The following statement is a full description of this invention, including the best method of performing it known to us:- 07104/04,Document4,1 Recombinational Cloning Using Engineered Recombination Sites Background of the Invention Field of the Invention The present invention relates to recombinant DNA technology. DNA and vectors having engineered recombination sites are provided for use in a recombinational cloning method that enables efficient and specific recombination of DNA segments using recombination proteins. The DNAs, vectors and methods are useful for a variety of DNA exchanges, such as subcloning of DNA, in vitro or in vivo.

RelatedArt Site specific recombinases. Site specific recombinases are enzymes that are present in some viruses and bacteria and have been characterized to have both endonuclease and ligase properties. These recombinases (along with associated proteins in some cases) recognize specific sequences of bases in DNA and exchange the DNA segments flanking those segments. The recombinases and associated proteins are collectively referred to as "recombination proteins" (see, Landy, Current Opinion in Biotechnology 3:699-707 (1993)).

Numerous recombination systems from various organisms have been described. See, Hoess et al., Nucleic Acids Research 14(6):2287 (1986); Abremski et al., J. Biol. Chem.261(1):391 (1986); Campbell, J -2- Bacteriol. 174(23):7495 (1992); Qian et J Biol. Chem. 267(11):7794 (1992); Araki et al.,J Mol. Biol. 225(1):25 (1992); Maeser and Kahnmann (1991) Mol Gen. Genet. 230:170-176).

Many of these belong to the integrase family of recombinases (Argos et al. EMBO J. 5:433-440 (1986)). Perhaps the best studied of these are the Integrase/att system from bacteriophage (Landy, A. Current Opinions in Genetics andDevel. 3:699-707 (1993)), the CrelloxP system from bacteriophage P I (Hoess and Abremski (1990) In Nucleic Acids and Molecular Biology, vol. 4.

Eds.: Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT system from the Saccharomyces cerevisiae 2 p circle plasmid (Broach et al. Cell 29:227-234 (1982)).

While these recombination systems have been characterized for particular organisms, the related art has only taught using recombinant DNA flanked by recombination sites, for in vivo recombination.

Backman Patent No. 4,673,640) discloses the in vivo use of I recombinase to recombine a protein producing DNA segment by enzymatic sitespecific recombination using wild-type recombination sites attB and attP.

Hasan and Szybalski (Gene 56:145-151 (1987)) discloses the use of A Int recombinase in vivo for intramolecular recombination between wild type attP and attB sites which flank a promoter. Because the orientations of these sites are inverted relative to each other, this causes an irreversible flipping of the promoter region relative to the gene of interest.

Palazzolo et al. Gene 88:25-36 (1990), discloses phage lambda vectors having bacteriophage A arms that contain restriction sites positioned outside a cloned DNA sequence and between wild-type loxP sites. Infection ofE coli cells that express the Cre recombinase with these phage vectors results in recombination between the loxP sites and the in vivo excision of the plasmid replicon, including the cloned cDNA.

P6sfai et al. (Nucl. Acids Res. 22:2392-2398 (1994)) discloses a method for inserting into genomic DNA partial expression vectors having a selectable marker, flanked by two wild-type FRT recognition sequences. FLP site-specific recombinase as present in the cells is used to integrate the vectors into the genome at predetermined sites. Under conditions where the replicon is functional, this cloned genomic DNA can be amplified.

Bebee etal. Patent No. 5,434,066) discloses the use of site-specific recombinases such as Cre for DNA containing two loxP sites is used for in vivo recombination between the sites.

Boyd (Nucl. Acids Res. 2/:817-821 (1993)) discloses a method to facilitate the cloning of blunt-ended DNA using conditions that encourage intermolecular ligation to a dephosphorylated vector that contains a wild-type loxP site acted upon by a Cre site-specific recombinase present in E coli host cells.

Waterhouse et al. (PCTNo. 93/19172 and Nucleic Acids Res. 21 (9):2265 (1993)) disclose an in vivo method where light and heavy chains of a particular antibody were cloned in different phage vectors between loxP and loxP 511 sites and used to transfect new E. coli cells. Cre, acting in the host cells on the two parental molecules (one plasmid, one phage), produced four products in equilibrium: two different cointegrates (produced by recombination at either loxP or loxP 511 sites), and two daughter molecules, one of which was the desired product In contrast to the other related art, Schlake Bode (Biochemistry 33:12746-12751 (1994)) discloses an in vivo method to exchange expression cassettes at defined chromosomal locations, each flanked by a wild type and a spacer-mutated FRT recombination site. A double-reciprocal crossover was mediated in cultured mammalian cells by using this FLP/FRT system for sitespecific recombination.

Transposases. The family of enzymes, the transposases, has also been used to transfer genetic information between replicons. Transposons are structurally variable, being described as simple or compound, but typically encode the recombinase gene flanked by DNA sequences organized in inverted orientations. Integration of transposons can be random or highly specific.

Representatives such as Tn7, which are highly site-specific, have been applied to the in vivo movement of DNA segments between replicons (Lucklow et al., J. Virol. 67:4566-4579 (1993)).

-4- Devine and Boeke NucL Acids Res. 22:3765-3772 (1994), discloses the construction of artificial transposons for the insertion of DNA segments, in vitro, into recipient DNA molecules. The system makes use of the integrase of yeast TYI vinrs-like particles. The DNA segment of interest is cloned, using standard methods, between the ends of the transposon-like element TYI. In the presence of the TYI integrase, the resulting element integrates randomly into a second target DNA molecule.

DNA cloning. The cloning of DNA segments currently occurs as a daily routine in many research labs and as a prerequisite step in many genetic analyses.

The purpose of these clonings is various, however, two general purposes can be considered: the initial cloning of DNA from large DNA or RNA segments (chromosomes, YACs, PCR fragments, nRNA, etc.), done in a relative handful of known vectors such as pUC, pGem, pBlueScript, and the subcloning of these DNA segments into specialized vectors for functional analysis. A great deal of time and effort is expended both in the initial cloning of DNA segments and in the transfer of DNA segments from the initial cloning vectors to the more specialized vectors. This transfer is called subcloning.

The basic methods for cloning have been known for many years and have changed little during that time. A typical cloning protocol is as follows: digest the DNA of interest with one or two restriction enzymes; gel purify the DNA segment of interest when known; prepare the vector by cutting with appropriate restriction enzymes, treating with alkaline phosphatase, gel purify etc., as appropriate; ligate the DNA segment to vector, with appropriate controls to estimate background of uncut and self-ligated vector, introduce the resulting vector into an E. colt host cell; pick selected colonies and grow small cultures overnight; make DNA minipreps; and analyze the isolated plasmid on agarose gels (often after diagnostic restriction enzyme digestions) or by PC.

The specialized vectors used for subeloning DNA segments are functionally diverse. These include but are not limited to: vectors for expressing genes in various organisms; for regulating gene expression; for providing tags to aid in protein purification or to allow tracking of proteins in cells; for modifying the cloned DNA segment generating deletions); for the synthesis of probes riboprobes); for the preparation of templates for DNA sequencing; for the identification of protein coding regions; for the fusion of various protein-coding regions; to provide large amounts of the DNA of interest, etc. It is common that a particular investigation will involve subcloning the DNA segment of interest into several different specialized vectors.

As known in the art, simple subclonings can be done in one day the DNA segment is not large and the restriction sites are compatible with those of the subcloning vector). However, many other subclonings can take several weeks, especially those involving unknown sequences, long fragments, toxic genes, unsuitable placement of restriction sites, high backgrounds, impure enzymes, etc. Subcloning DNA fragments is thus often viewed as a chore to be done as few times as possible.

Several methods for facilitating the cloning of DNA segments have been described, as in the following references.

Ferguson, et a. Gene 16:191 (1981), discloses a family of vectors for subcloning fragments of yeast DNA. The vectors encode kanamycin resistance.

Clones of longer yeast DNA segments can be partially digested and ligated into the subeloning vectors. If the original cloning vector conveys resistance to ampidllin, no purification is necessary prior to transformation, since the selection will be for kanamycin.

Hashimoto-Gotoh, et al. Gene 41:125 (1986), discloses a suboloning vector with unique cloning sites within a streptomycin sensitivity gene; in a streptomycin-resistant host, only plasmids with inserts or deletions in the dominant sensitivity gene will survive streptomycin selection.

Accordingly, traditional subeloning methods, using restriction enzymes and ligase, are time consuming and relatively unreliable. Considerable labor is expended, and if two or more days later the desired subclone can not be found among the candidate plasmids, the entire process must then be repeated with alternative conditions attempted. Although site specific recombinases have been used to recombine DNA in vivo, the successful use of such enzymes in vitro was expected to suffer from several problems. For example, the site specificities and efficiencies were expected to differ in vitro; topologically-linked products were expected; and the topology of the DNA substrates and recombination proteins was expected to differ significantly in vitro (see, Adams et al, J.

Mol. Biol. 226:661-73 (1992)). Reactions that could go on for many hours in vivo were expected to occur in significantly less time in vitro before the enzymes became inactive.

Multiple DNA recombination products were expected in the biological host used, resulting in unsatisfactory reliability, specificity or efficiency of subcloning. In vitro recombination reactions were not expected to be sufficiently efficient to yield the desired levels of product.

Accordingly, there is a long felt need to provide an alternative subcloning system that provides advantages over the known use of restriction enzymes and ligases.

The present application is a divisional application of Australian Patent Application No.

10062/01 (the "parent" application), the specification of which is herein incorporated by reference. The parent application is itself a divisional application of Australian Patent No.

724922. The present application claims priority from both Application No. 10062/01 and Patent No. 724922.

Summary of the Invention The present invention provides nucleic acid, vectors and methods for obtaining chimeric nucleic acid using recombination proteins and engineered recombination sites, in vitro or in vivo. These methods are highly specific, rapid, and less labor intensive than what is disclosed or suggested in the related background art. The improved specificity, speed and yields of the present invention facilitates DNA or RNA subcloning, regulation or exchange useful for any related purpose. Such purposes include in vitro recombination of DNA segments and in vitro or in vivo insertion or modification of transcribed, replicated, isolated or genomic DNA or RNA.

The present invention relates to nucleic acids, vectors and methods for moving or exchanging segments of DNA using at least one engineered recombination site and at least one recombination protein to provide chimeric DNA molecules which have the desired characteristic(s) and/or DNA segment(s).

07/04/04.at 11797.specipg -7- Generally, one or more parent DNA molecules are recombined to give one or more daughter molecules, at least one of which is the desired Product DNA segment or vector. The invention thus relates to DNA, RNAi, vectors and methods to effect the exchange and/or to select for one or more desired products.

One embodiment of the present invention relates to a method of making chimeric DNA, which comprises combining in vitro or in viva an Insert Donor DNA molecule, comprising a desired DNA segment flanked by a first recombination site and a second recombination site, wherein the first and second recombination sites do not recombine with each other, (ii) a Vector Donor DNA molecule containing a third recombination site and a fourth recombination site, wherein the third and fourth recombination sites do not recombine with each other; and (iii) one or more site specific recombination proteins capable of recombining the first and third recombinational sites and/or the second and fourth recombinationial sites; thereby allowing recornbination to occur, so as to produce at least one Cointegrate DNA molecule, at least one desired Product DNA molecule which comprises said desired DNA segment, and optionally a Byproduct DNA molecule; and then, optionally, selecting for the Product or Byproduct DNA molecule.

Another embodiment of the present invention relates to a kit comprising a catrer or receptacle being compartmentalized to receive and hold therein at least one container, wherein a first container containts a DNA molecule comprising a vector having at least two recombination sites flanking a cloning site or a Selectable marker, as described herein. The kit optionally further comprises: a second container containing a Vector Donor plasinid comprising a subcloning vector and/or a Selectable marker of which one or both are flanked by one or more engineered recombination sites; and/or (ii) a third container containing at least one recombination protein which recognizes and is capable of recombining at least one of said recombination sites.

Other embodiments include DNA and vectors usefuil in the methods of the present invention. In particular, Vector Donor molecules are provided in one embodiment, wherein DNA segments within the Vector Donor are separated either by, in a circular Vector Donor, at least two recombination sites, or (ii) in a linear Vector Donor, at least one recomubination site, where the recombination sites are preferably engineered to enhance specificity or efficiency of recombination.

One Vector Donor embodiment comprises a first DNA segment and a second DNA. segment, the first or second segment comprising a Selectable marker. A second Vector Donor embodiment comprises a first DNA segment and a second DNA segment, the first or second DNA segment comprising a toxic gene. A third Vector Donor embodiment comprises a first DNA segment and a second DNA segment, the first or second DNA segment comprising an inactive fr-agment of at least one Selectable marker, wherein the inactive fragment of the Selectable marker is capable of reconstituting a functional Selectable marker when recombined across the first or second recomnbination site with another inactive fr-agment of at least one Selectable marker.

The present recombinational cloning method possesses several advantages over previous In vlvo methods. Since single molecules of recombination products can be introduced into a biological host propagation of the desired Product DNA in the absence of other DNA molecules starting molecules, intermediates, and by-products) is more readily realized. Reaction coziditions can be freely adjusted in vitro to optimize enzyme activities. DNA molecules can be incompatible with the desired biological host YACs, genomic DNA, etc.), can be used. Recombination proteins from diverse sources can be employed, together or sequentially.

Other embodiments will be evident to those of ordinary skill in the art from the teachings contained herein in combination with what is known to the art -9- Brief Description of the Figures Figure 1 depicts one general method of the present invention, wherein the starting (parent) DNA molecules can be circular or linear. The goal is to exchange the new subcloning vector D for the original cloning vector B. It is desirable in one embodiment to select for AD and against all the other molecules, including the Cointegrate. The square and circle are sites of recombination: e.g., loxP sites, att sites, etc. For example, segment D can contain expression signals, new drug markers, new origins of replication, or specialized functions for mapping or sequencing DNA.

Figure 2A depicts an in vitro method of recombining an Insert Donor plasmid (here, pEZC705) with a Vector Donor plasmid (here, pEZC726), and obtaining Product DNA and Byproduct daughter molecules. The two recombination sites are attP and loxP on the Vector Donor. On one segment defined by these sites is a kanamycin resistance gene whose promoter has been replaced by the tetOP operator/promoter from transposon Tnl0. See Sizemore et al., Nucl. Acids Res. 18(10):2875 (1990). In the absence of tet repressor protein, E. coli RNA polymerase transcribes the kanamycin resistance gene from the tetOP. If tet repressor is present, it binds to tetOP and blocks transcription of the kanamycin resistance gene. The other segment of pEZC726 has the tet repressor gene expressed by a constitutive promoter. Thus cells transformed by pEZC726 are resistant to chloramphenicol, because of the chloramphenicol acetyl transferase gene on the same segment as tetR, but are sensitive to kanamycin.

The recombinase-mediated reactions result in separation of the tetR gene from the regulated kanamycin resistance gene. This separation results in kanamycin resistance in cells receiving only the desired recombination products. The first recombination reaction is driven by the addition of the recombinase called Integrase. The second recombination reaction is driven by adding the recombinase Cre to the Cointegrate (here, pEZC7 Cointegrate).

Figure 2B depicts a restriction map of pEZC705.

Figure 2C depicts a restriction map of pEZC726.

Figure 2D depicts a restriction map of pEZC7 Cointegrate.

Figure 2E depicts a restriction map of Intprod.

Figure 2F depicts a restriction map ofIntbypro.

Figure 3A depicts an in vitro method of recombining an Insert Donor plasmid (here, pEZC602) with a Vector Donor plasmid (here, pEZC629), and obtaining Product (here, EZC6prod) and Byproduct (here, EZC6Bypr) daughter molecules. The two recombination sites are laxP and loxP 511. One segment of pEZC629 defined by these sites is a kanamycin resistance gene whose promoter has been replaced by the tetOP operator/promoter from transposon Tnl0. In the absence of tet repressor protein, E. coli RNA polymerase transcribes the kanamycin resistance gene from the tetOP. If tet repressor is present, it binds to tetOP and blocks transcription of the kanamycin resistance gene. The other segment of pEZC629 has the tet repressor gene expressed by a constitutive promoter. Thus cells transformed by pEZC629 are resistant to chloramphenicol, because of the chloramphenicol acetyl transferase gene on the same segment as tetR, but are sensitive to kanamycin. The reactions result in separation of the tetR gene from the regulated kanamycin resistance gene. This separation results in kanamycin resistance in cells receiving the desired recombination product. The first and the second recombination events are driven by the addition of the same recombinase, Cre.

Figure 3B depicts a restriction map of EZC6Bypr.

Figure 3C depicts a restriction map of EZC6prod.

Figure 3D depicts a restriction map of pEZC602.

Figure 3E depicts a restriction map of pEZC629.

Figure 3F depicts a restriction map ofEZC6coint.

Figure 4A depicts an application of the in vitro method of recombinational cloning to subclone the chloramphenicol acetyl transferase gene into a vector for expression in eukaryotic cells. The Insert Donor plasmid, pEZC843, is comprised of the chloramphenicol acetyl transferase gene of E coll, cloned between loxP and attB sites such that the loxP site is positioned at the 5'-end of the gene. The Vector Donor plasmid, pEZC1003, contains the cytomegalovirus eukaryotic promoter apposed to a loxP site. The supercoiled plasmids were combined with lambda Integrase and Cre recombinase in vitro.

-11 After incubation, competent E. coli cells were transformed with the recombinational reaction solution. Aliquots of transformations were spread on agar plates containing kanamycin to select for the Product molecule (here CMVProd).

Figure 4B depicts a restriction map of pEZC843.

Figure 4C depicts a restriction map ofpEZC 1003.

Figure 4D depicts a restriction map of CMVBypro.

Figure 4E depicts a restriction map of CMVProd.

Figure 4F depicts a restriction map of CMVcoint.

Figure 5A depicts a vector diagram ofpEZC1301.

Figure 5B depicts a vector diagram ofpEZCI305.

Figure 5C depicts a vector diagram ofpEZC1309.

Figure 5D depicts a vector diagram ofpEZC1313.

Figure SE depicts a vector diagram of pEZC1317.

Figure 5F depicts a vector diagram ofpEZC1321.

Figure 5G depicts a vector diagram ofpEZC1405.

Figure 5H depicts a vector diagram ofpEZC1502.

Figure 6A depicts a vector diagram ofpEZC1603.

Figure 6B depicts a vector diagram ofpEZC1706.

Figure 7A depicts a vector diagram of pEZC2901.

Figure 7B3 depicts a vector diagram ofpEZC2913 Figure 7C depicts a vector diagram ofpEZC3101.

Figure 7D depicts a vector diagram of pEZC1 802.

Figure 8A depicts a vector diagram ofpGEX-2TK.

Figure 8B depicts a vector diagram ofpEZC3501: Figure 8C depicts a vector diagram ofpEZC3601.

Figure 8D depicts a vector diagram ofpEZC3609.

Figure 8E depicts a vector diagram ofpEZC3617.

Figure 8F depicts a vector diagram of pEZC3606.

Figure 8G depicts a vector diagram ofpEZC3613.

Figure 8H depicts a vector diagram ofpEZC362l.

Figure 81 depicts a vector diagram of GST-CAT.

-12- Figure 8J depicts a vector diagram of GST-phoA.

Figure SK depicts a vector diagram of pEZC3201.

Detailed Description of the Preferred Embodiments It is unexpectedly discovered in the present invention that subcloning reactions can be provided using recombinational cloning. Recombination cloning according to the present invention uses DNAs, vectors and methods, in vitro and in vtvo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins. These methods provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

The present invention thus provides nucleic acid, vectors and methods for obtaining chimeric nucleic acid using recombination proteins and engineered recombination sites, in vitro or in vivo. These methods are highly specific, rapid, and less labor intensive than what is disclosed or suggested in the related background art. The improved specificity, speed and yields of the present invention facilitates DNA or RNA subcloning, regulation or exchange useful for any related purpose. Such purposes include in vitro recombination of DNA segments and in vitro or in vivo insertion or modification of transcribed, replicated, isolated or genomic DNA or RNA.

Definitions In the description that follows, a number of terms used in recombinant DNA technology are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Byproduct: is a daughter molecule (a new clone produced after the second recombination event during the recombinational cloning process) lacking the DNA which is desired to be subcloned.

-13- Cointegrate: is at least one recombination intermediate DNA molecule of the present invention that contains both parental (starting) DNA molecules.

It will usually be circular. In some embodiments it can be linear.

Host: is any prokaryotic or eukaryotic organism that can be a recipient of the recombinational cloning Product. A "host," as the term is used herein, includes prokaryotic or eukaryotic organisms that can be genetically engineered.

For examples of such hosts, see Maniatis el al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).

Insert: is the desired DNA segment (segmentA of Figure 1) which one wishes to manipulate by the method of the present invention. The insert can have one or more genes.

Insert Donor: is one of the two parental DNA molecules of the present invention which carries the Insert. The Insert Donor DNA molecule comprises the Insert flanked on both sides with recombination signals. The Insert Donor can be linear or circular. In one embodiment of the invention, the Insert Donor is a circular DNA molecule and further comprises a cloning vector sequence outside of the recombination signals (see Figure 1).

Product: is one or both the desired daughter molecules comprising the A and D or B and C sequences which are produced after the second recombination event during the recombinational cloning process (see Figure The Product contains the DNA which was to be cloned or subcloned.

Promoter: is a DNA sequence generally described as the 5'-region of a gene, located proximal to the start codon. The transcription of an adjacent DNA segment is initiated at the promoter region. A repressible promoter's rate of transcription decreases in response to a repressing agent An inducible promoter's rate of transcription increases in response to an inducing agent. A.'constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.

-14- Recognition sequence: Recognition sequences are particular DNA sequences which a protein, DNA, or RNA molecule restriction endonuclease, a modification methylase, or a recombinase) recognizes and binds.

For example, the recognition sequence for Cre recombinase is loxP which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence. See Figure I of Sauer, Current Opinion in Biotechnology 5:521-527 (1994). Other examples of recognition sequences are the attB, attP, attL, and attR sequences which are recognized by the recombinase enzyme Integrase. attB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region. attP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins IHF, FIS, and Xis. See Landy, Current 'Opinion in Biotechnology 3:699-707 (1993). Such sites are also engineered according to the present invention to enhance methods and products.

Recombinase: is an enzyme which catalyzes the exchange of DNA segments at specific recombination sites.

Recombinational Cloning: is a method described herein, whereby segments of DNA molecules are exchanged, inserted, replaced, substituted or modified, in vitro or in vivo.

Recombination proteins: include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites. See, Landy (1994), infra.

Repression cassette: is a DNA segment that contains a repressor of a Selectable marker present in the subeloning vector., Selectable marker: is a DNA segment that allows one to select for or against a molecule or a cell that contains it, often under particular conditions.

These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like.

Examples of Selectable markers include but are not limited to: DNA segments that encode products which provide resistance against otherwise toxic compounds antibiotics); DNA segments that encode products which are otherwise lacking in the recipient cell tRNA genes, auxotrophic markers); DNA segments that encode products which suppress the activity of a gene product; DNA segments that encode products which can be readily identified phenotypic markers such as P-galactosidase, green fluorescent protein (GFP), and cell surface proteins); DNA segments that bind products which arc otherwise detrimental to cell survival and/or function; DNA segments that otherwise inhibit the activity of any of the DNA segments described in Nos. above antisense oligonucleotides); DNA segments that bind products that modify a substrate restriction endonucleases); DNA segments that can be used to isolate a desired molecule specific protein binding sites); (9) DNA segments that encode a specific nucleotide sequence which can be otherwise non-functional for PCR amplification of subpopulations of molecules); and/or (10) DNA segments, which when absent, directly or indirectly confer sensitivity to particular compounds.

Selection scheme: is any method which allows selection, enrichment, or identification of a desired Product or Product(s) from a mixture containing the Insert Donor, Vector Donor, and/or any intermediates, a Cointegrate) Byproducts. The selection schemes of one preferred embodiment have at least two components that are either linked or unlinked during recombinational cloning. One component is a Selectable marker. The other component controls the expression in vitro or in v.ivo of the Selectable marker, or survival of the cell harboring the plasmid carrying the Selectable marker. Generally, this controlling element will be a repressor or inducer of the Selectable marker, but other means for controlling expression of the Selectable marker can 'be used. Whether a repressor or activator is used will depend on whether the marker is for a positive or negative selection, and the exact arrangement of the various DNA segments, as will be readily apparent to those skilled in the art. A preferred requirement is that the selection scheme results in selection of or enrichment for only one or more desired Products. As defined herein, to select for a DNA molecule includes selecting or enriching for the presence of the desired DNA molecule, and (b) 16 selecting or enriching against the presence of DNA molecules that are not the desired DNA molecule.

In one embodiment, the selection schemes (which can be carried out reversed) will take one of three forms, which will be discussed in terms of Figure 1. The firt exemplified herein with a Selectable marker and a repressor therefor, selects for molecules having segment D and lacking segment C The second selects against molecules having segment C and for molecules having segment D. Possible embodiments of the second form would have a DNA segment carrying a gene toxic to cells into which the in vitro reaction products are tobe introduced. A toxic gene can be aDNA that iscexpressed as atoxic gene product (a toxic protein or RNA), or can be toxic in and of itself. (In the latter cae, the toxic gene is understood to carry its classical definition of "heritable trait") Examples of such toxic gene products are well known in the art, and include, but are not limited to, restriction endonucleases PpnI) and genes that kill hosts in the absence of a suppressing function, kicE. A toxic gene can alternatively be selectable in vitro, e.g, a restriction site.

In the second form, segment D carries a Selectable marker. The toxic gene would eliminate transformants harboring the Vector Donor, Cointegrate, and Byproduct molecules, while the Selectable marker can be used to select for cells containing the Product and against cells harboring only the Insert Donor.

Thethirdformnselects for cellsthat have both segments A and Din cison the same molecule, but not for cells that have both segments in trans on different molecules. This could be embodied by a Selectable marker that is split into two inactive firagments, one each on segments A and DA The fragments are so arranged relative to the recombination sites that when the segments are brought together by the recombination event, they reconstitute a functional Selectable marker. For example, the recombinational event can link a promoter with a structural gene, can link two fragments of a structural gene, or can link genes that encode a heterodimeric gene product needed for survival, or can link portions of a replicon.

-17- Site-specific recombinase: is a type of recombinase which typically has at least the following four activities: recognition of one or two specific DNA sequences; cleavage of said DNA sequence or sequences; DNA topoisomerase activity involved in strand exchange; and DNA ligase activity to reseal the cleaved strands of DNA. See Saucr, Current Opinions in Biotechnology 5:521-527 (1994). Conservative site-specific recombination is distinguished from homologous recombination and transposition by a high degree of specificity for both partners. The strand exchange mechanism involves the cleavage and rejoining of specific DNA sequences in the absence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem 58:913-949).

Subcloning vector: is a cloning vector comprising a circular or linear DNA molecule which includes an appropriate replicon. In the present invention, the subcloning vector (segment D in Figure 1) can also contain functional and/or regulatory elements that arc desired to be incorporated into the final product to act upon or with the cloned DNA Insert (segmentA in Figure The subcloning vector can also contain a Selectable marker (contained in segment C in Figure 1).

Vector: is a DNA that provides a useful biological or biochemical property to an Insert. Examples include plasmids, phages, and other DNA sequences which arc able to replicate or be replicated in vitro or in a host cell, or to convey a desired DNA segment to a desired location within a host cell. A Vector can have one or more restriction endonuclease recognition sites at which the DNA sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a DNA fragment can be spliced in order to bring about its replication and cloning. Vectors can further provide primer sites, for PCR, transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, Selectable markers, etc. Clearly, methods of inserting a desired DNA fragment which do not require the use of homologous recombination or restriction enzymes (such as, but not limited to, UDC cloning of PCR fragments Patent No. 5,334,575, entirely incorporated herein by reference), T:A cloning, and the like) can also be applied to clone a fragment of DNAinto a cloning vector to be used according to the -18.present invention. The cloning vector can further contain a Selectable marker suitable for use in the identification of cells transformed with the cloning vector.

Vector Donor: is one of the two parents] DNA molecules of the present invention which carries the DNA segments encoding the DNA vector which is to become part of the desired Product The Vector Donor comprises a subcloning vector D (or it can be called the clong vector if the Insert Donor does not already contain a cloning vector) and a segment C flanked by recombination sites (see Figure Segments C and/or D can contain elements that contribute to selection for the desired Product daughter molecule, as described above for selection schemes. The recombination signals can be the same or different, and can be acted upon by the same or different recombinases. In addition, the Vector Donor can be linear or circular.

Description One general scheme for an in vitro or in vivo method of the invention is shown in Figure 1, where the Insert Donor and the Vector Donor can be either circular or linear DNA, but is shown as circular. Vector D is exchanged for the original cloning vector A. It is desirable to select for the daughter vector containing elements A and D and against other molecules, including one or more Cointegrate(s). The square and circle are different sets of recombination sites lox sites or aft sites). Segment A or D can contain at least one Selection Marker, expression signals, origins of replication, or specialized functions for detecting, selecting, expressing, mapping or sequencing DNA, where D is used in this example.

Examples of desired DNA segments that can be part of Element A or D include, but are not limited to, PCR products, large DNA segments, genoinic clones or fragments, cDNA clones, functional elements, etc., and genes or partial genes, which encode useful nucleic acids or proteins. Moreover, the recombinational cloning of the present invention can be used to make ex vivo and in vivo gene transfer vehicles for protein expression and/or gene therapy.

-19- In Figure 1, the scheme provides the desired Product as containing vectors D and A, as follows. The Insert Donor (containing A and B) is first recombined at the square recombination sites by recombination proteins, with the Vector Donor (containing C and to form a Co-integrate having each of A-D- C-B. Next, recombination occurs at the circle recombination sites to form Product DNA (A and D) and Byproduct DNA (C and However, if desired, two or more different Co-integrates can be formed to generate two or more Products.

In one embodiment of the present in vitro or in vivo recombinational cloning method, a method for selecting at least one desired Product DNA is provided. This can be understood by consideration of the map of plasmid pEZC726 depicted in Figure 2. The two exemplary recombination sites are attP and loxP. On one segment defined by these sites is a kanamycin resistance gene whose promoter has been replaced by the tetOP operator/promoter from transposon Tn 10. In the absence oftet repressor protein, E coli RNA polymerase transcribes the kanamycin resistance gene from the tetOP. If tet represser is present, it binds to tetOP and blocks transcription of the kanamycin resistance gene. The other segment of pEZC726 has the tet represser gene expressed by a constitutive promoter. Thus cells transformed by pEZC726 are resistant to chloramphenicol, because of the chloramphenicol acetyl transferase gene on the same segment as tetR, but are sensitive to kanamycin. The recombination reactions result in separation of the tetR gene from the regulated kanamycin resistance gene. This separation results in kanamycin resistance in cells receiving the desired recombination Product Two different sets of plasmids were constructed to demonstrate the in vitro method. One set, for use with Cre recombinase only (cloning vector 602 and subcloning vector 629 (Figure contained loxP and loxP 511 sites. A second set, for use with Cre and integrase (cloning vector 705 and subcloning vector 726 (Figure contained loxP and at sites. The efficiency of production of the desired daughter plasmid was about 60 fold higher using both enzymes than using Cre alone. Nineteen of twenty four colonies from the Cre-only reaction contained the desired product, while thirty eight of thirty eight colonies from the integrase plus Cre reaction contained the desired product plasmid.

Other Selection Schemes A variety of selection schemes can be used that are known in the art as they can suit a particular purpose for which the recombinational cloning is carried out. Depending upon individual preferences and needs, a number of different types of selection schemes can be used in the recombinational cloning method of the present invention. The skilled artisan can take advantage of the availability of the many DNA segments or methods for making them and the different methods of selection that are routinely used in the art. Such DNA segments include but are not limited to those which encodes an activity such as, but not limited to, production of RNA, peptide, or protein, or providing a binding site for such RNA, peptide, or protein. Examples of DNA molecules used in devising a selection scheme are given above, under the definition of "selection scheme" Additional examples include but are not limited to: Generation of new primer sites for PCR juxtaposition of two DNA sequences that were not previously juxtaposed); (ii) Inclusion of a DNA sequence acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, ribozyme, etc.; (iii) Inclusion of a DNA sequence recognized by a DNA binding protein, RNA, DNA, chemical, etc.) for use as an affinity tag for selecting for or excluding from a population) (Davis, Nuc7.

Acids Res. 24:702-706 (1996); J Virol 69: 8027-8034 (1995)); (iv) In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Bar vinis-expressed RNA by using randomized and cDNA-derived RNA libraries; (vi) The positioning of functional elements whose activity requires a specific orientation or juxtaposition a recombination site which reacts poorly in tram, but when placed in cis, in the presence of the appropriate proteins, results in recombination that destroys certain populations of molecules; reconstitution of -21a promoter sequence that allows in vitro RNA synthesis). The RNA can be used directly, or can be reverse transcribed to obtain the desired DNA construct; (vii) Selection of the desired product by size fractionation) or other physical property of the molecule(s); and (viii) Inclusion of a DNA sequence required for a specific modification inethylation) that allows its identification.

After formation of the Product and Byproduct in the method of the present invention, the selection step can be carried out either in vitro or in viva depending upon the particular selection scheme which has been optionally devised in the particular recombinational. cloning procedure.

For example, an in vitro method of selection can be devised for the Insert Donor and Vector Donor DNA molecules. Such scheme can involve engineering a rare restriction site in the starting circular vectors in such a way that after the is recombination event the rare cutting sites end up in the Byproduct. Hence, when the restriction enzyme which binds andeuats atthe rare restriction site is added to the reaction mixture in vitro, all of the DNA molecules carrying the rare cutting site, iLe., the starting DNA molecules, the Cointegrate, and the Byproduct, will be cut and rendered nonreplicable in the intended host cell. For example, cutting sites in segments R and C (see Figure 1) can be used to select against all molecules except the Product Alternatively, only a cutting site in C is needed if one is able to select for segment D, by a drug resistance gene not found on B.

Similarly, an in vitro selection method can be devised when dealing with linear DNA molecules. DNA sequences complementary to a PCR primer sequence can be so engineered that they are transferred, through the recombinational cloning method, only to the Product molecule;. After the reactions are completed, the appropriate primers are added to the reaction solution and the sample is subjected to PCR Hence, all or part of the Product molecule is amplified.

Other in viva selection schemes can be used with a variety of E. coi cell lines. One is to put a repressor gene on one segment of the subcloning plasmnid, and a drug marker controlled by that repressor on the other segment of the same -22plasmid. Another is to put a killer gene on segment C of the subcloning plasmid (Figure Of course a way must exist for growing such a plasmid, Le., there must exist circumstances under which the killer gene will not kill. There are a number of these genes known which require particular strains of E. coli. One such scheme is to use the restriction enzyme DpnI, which will not cleave unless its recognition sequence GATC is methylated. Many popular common E. coli strains methylate GATC sequences, but there are mutants in which cloned DpnI can be expressed without harm.

Of course analogous selection schemes can be devised for other host organisms. For example, the tet repressor/operator of TnlO has been adapted to control gene expression in eukaryotes (Gossen, and Bujard, Proc. Natl.

Acad Sci. USA 89:5547-5551 (1992)). Thus the same control of drug resistance by the tet repressor exemplified herein can be applied to select for Product in eukaryotic cells.

Recombination Proteins In the present invention, the exchange of DNA segments is achieved by the use of recombination proteins, including recombinases and associated co-factors and proteins. Various recombination proteins are described in the art.

Examples of such recombinases include: Cre: A protein from bacteriophage PI (Abremski and Hoess, J. Biol Chem. 259(3):1509-1514 (1984)) catalyzes the exchange causes recombination) between 34 bp DNA sequences called loxP (locus of crossover) sites (See Hoess et al., Nucl. Acids Res. 14(5):2287 (1986)). Cre is available commercially (Novagen, Catalog No. 69247-1). Recombination mediated by Cre is freely reversible. From thermodynamic considerations it is not surprising that Cre-mediated integration (recombination between two molecules to form one molecule) is much less efficient than Cre-mediated excision (recombination between two loxP sites in the same molecule to form two daughter molecules).

Cre works in simple buffers with either magnesium or spermidine as a cofactor, as is well known in the art. The DNA substrates can be either linear or -23supercoiled. A number of mutant loxP sites have been described (Hoess et al., supra). One of these, loxP 511, recombines with another loxP 511 site, but will not recombine with a loxP site.

Integrase: A protein firom bacteriophage lambda that mediates the integration of the lambda genome into the E. coli chromosome. The bacteriophage X. Int recombinational proteins promote irreversible recombination between its substrate art sites as part of the formation or induction of a lysogenic state. Reversibility of the recombination reactions results from two independent pathways for integrative and exdcisive recombination. Each pathway uses a unique, but overlapping, set of the 15 protein binding sites that comprise art site DNAs. Cooperative and competitive interactions involving four proteins (Int, Xis, IHF aLnd FIS) determine the direction of recombination.

Integrative recombination involves the Int and IHF proteins and sites attP (240 bp) and attB (25 bp). Recombination results in the formation of two new sites: atiL and attR. Excisive recombination requires Int, IRF, and Xis, and sites attL and attR to generate attP and attB. Under certain conditions, FIS stimulates excisive recombination. In addition to these normal reactions, it should be appreciated that attP and attB, when placed on the same molecule, can promote excisive recombination to generate two excision products, one with attL and one with attR. Similarly, intermolecular recombination between molecules containing attL and attR, in the presence of Int, IHF and Xis, can result in integrative recombination and the generation attP and attB. Hence, by flanking DNA segments with appropriate combinations of engineered ant sites, in the presence of the appropriate recombination proteins, one can direct excisive or integrative recombination, as reverse reactions of each other.

Each of the att sites contains a 15 bp core sequence; individual sequence elements of functional significance lie within, outside, and across the boundaries of this common core (Landy, Ann Rev. Biochem. 58:913 (1989)). Efficient recombination between the various aft sites requires that the sequence of the central common region be identical between the recombining partners, however, the exact sequence is now found to be modifiable. Consequently, derivatives of -24the art site with changes within the core are now discovered to recombine as least as efficiently as the native core sequences.

Integrase acts to recombine the attP site on bacteriophage lambda (about 240 bp) with the attB site on the E. coli genome (about 25 bp) (Weisberg, RA.

and Landy, A. in Lambda I, p. 2 11 (1983), Cold Spring Harbor Laboratory)), to produce the integrated lambda genome flanked by attL (about 100 bp) and attR (about 160 bp) sites. In the absence of Xis (see below), this reaction is essentially irreversible. The integration reaction mediated by integrase and IHF works in vitro, with simple buffer containing spermidine. Integrase can be obtained as described by Nash, HA., Methods of Enymology 100:210-216 (1983). IHF can be obtained as described by Fihutowicz, et al., Gene 147:149-150 (1994).

In the presence of the A protein Xis (excise) integrase catalyzes the reaction of atR and attL to form attP and attB, it promotes the reverse of the reaction described above. This reaction can also be applied in the present invention.

Other Recombination Systems. Numerous recombination systems from various organisms can also be used, based on the teaching and guidance provided herein. See, Hoess et al., Nucleic Acids Research 14(6):2287 (1986); Abremski et al., J Biol. Chem.261(l):391 (1986); Campbell, J Bacteriol.

174(23):7495 (1992); Qian et at, J Biol. Chem 267(11):7794 (1992); Araki et al., J. Mol. Biol. 225(1):25 (1992)). Many of these belong to the integrase family of recombinases (Argos et al. EMBOJ 5:433-440 (1986)). Perhaps the best studied of these are the Integraselatt system from bacteriophage A (Landy, A. (1993) Current Opinions in Genetics and Devel. 3:699-707), the CrelloxP system from bacteriophage Pl (Hoess and Abremski (1990)' In Nucleic Acids and Molecular Biology, vol. 4. Eds.: Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT system from the Saccharomyces cerevisiae 2 it circle plasmid (Broach et al. Cell 29:227-234 (1982)).

Members of a second family of site-specific recombinases, the resolvase family y6, Tn3 resolvase, Hin, Gin, and Cin) are also known. Members of this highly related family of recombinases are typically constrained to intramolecular reactions inversions and excisions) and can require hostencoded factors. Mutants have been isolated that relieve some of the requirements for host factors (Maeser and Kahnmann (1991) MoL Gen. Genet.

230:170-176), as well as some of the constraints of intramolecular recombination.

Other site-specific recombinases similar to A Int and similar to PI Cre can be substituted for Int and Cre. Such recombinases are known. In many cases the purification of such other recombinases has been described in the art. In cases when they are not known, cell extracts can be used or the enzymes can be partially purified using procedures described for Cre and Int.

While Cre and Int are described in detail for reasons of example, many related recombinase systems exist and their application to the described invention is also provided according to the present invention. The integrase family of sitespecific recombinases can be used to provide alternative recombination proteins and recombination sites for the present invention, as site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and Pl. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves.

A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. These residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining. See. Argos, P. et al., EMBOJ. 5:433-40 (1986).

Alternatively, IS231 and other Bacillus thuringiensis transposable elements could be used as recombination proteins and recombination sites.

Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of delta-endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBTI and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been -26 shown to be active and participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity.

Structural analysis ofthe iso-IS231 elements indicates that they are related to IS 1151 from Clostridium perfringens and distantly related to IS4 and IS186 from Escherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses.

Moreover, functional data gathered from IS231A in Escherichia coli indicate a non-replicative mode of transposition, with a preference for specific targets. Similar results were also obtained in Bacillus subtilis and B.

thuringiensis. See, Mahillon, et aL, Genetica 93:13-26 (1994); Campbell, J. Bacteriol. 7495-7499 (1992).

The amount of recombinase which is added to drive the recombination reaction can be determined by using known assays. Specifically, titration assay is used to determine the appropriate amount of a purified recombinase enzyme, or the appropriate amount of an extract.

Engineered Recombination Sites. The above recombinases and corresponding recombinase sites are suitable for use in recombination cloning according to the present invention. However, wild-type recombination sites contain sequences that reduce the efficiency or specificity of recombination reactions as applied in methods of the present invention. For example, multiple stop codons in attB, attR, attP, attL and loxP recombination sites occur in multiple reading frames on both strands, so recombination efficiencies are reducted, where the coding sequence must cross the recombination sites, (only one reading frame is available on each strand of loxP and attB sites) or impossible (in attP, attR or attL).

Accordifigly, the present invention also provides -engineered recombination sites that overcome these problems. For example, att sites can be engineered to have one or multiple mutations to enhance specificity or efficiency of the recombination reaction and the properties of Product DNAs attl, att2, and att3 sites); to decrease reverse reaction removing P 1 and HI from attB).

The testing of these mutants determines which mutants yield sufficient -27 recombinational activity to be suitable for recombination subcloning according to the present invention.

Mutations can therefore be introduced into recombination sites for enhancing site specific recombination. Such mutations include, but are not limited to: recombination sites without translation stop codons that allow fusion proteins to be encoded; recombination sites recognized by the same proteins but differing in base sequence such that they react largely or exclusively with their homologous partners allow multiple reactions to be contemplated. Which particular reactions take place can be specified by which particular partners are present in the reaction mixture. For example, a tripartite protein fusion could be accomplished with parental plasmids containing recombination sites attRI and attR2; attLi and attL3; and/or attIR3 and attL2.

There are well known procedures for introducing specific mutations into nucleic acid sequences. A number of these are descnbed in Ausubel, FM. et al., Current Protocols in Molecular Biology, Wiley Interscience, New York (1989- 1996). Mutations can be designed into oligonucleotides, which can be used to modify existing cloned sequences, or in amplification reactions. Random mutagenesis can also be employed if appropriate selection methods are available to isolate the desired mutant DNA or RNA. The presence of the desired mutations can be confirmed by sequencing the nucleic acid by well. known methods.

The following non-limiting methods can be used to engineer a core region of a given recombination site to provide mutated sites suitable for use in the present invention: 1. By recombination of two parental DNA sequencei by site-specific (e.g.

attL and attR to give attB) or other homologous) recombination mechanisms. The DNA parental DNA segments containing one or more base alterations resulting in the final core sequence; 2. By mutation or mutagenesis (site-specific, PCR, random, spontaneous, etc) directly of the desired core sequence; 28 3. By niutagenesis (site-specific, PCR, random, spontanteous, etc) of parental DNA sequences, which are recombined to generate a desired core sequence; and 4. By reverse transcription of an RNA encoding the desired core sequence.

The functionality of the mutant recombination sites can be demonstrated in ways that depend on the particular characteristic that is desired. For example, the lack of translation stop codons in a recombination site can be demonstrated by expressing the appropriate fusion proteins. Specificity of recombination between homologous partners can be demonstrated by introducing the appropriate molecules into in vitro reactions, and assaying for recombination products as described herein or known in the art. Other desired mutations in reconmbination sites might include the presence or absence of restriction sites, translation or transcription start signals, protein binding sites, and other known functionalities of nucleic. acid base sequences. Genetic selection schemes for particular flmctional attributes in the recombination sites can be used according to known method steps. For example, the modification of sites to provide (from a pair of sites that do not interact) partners that do interact could be achieved by requiring deletion, via recomnbination between the sites, of a DNA sequence encoding a toxic substance. Similarly, selection for sites that remove translation stop sequences, the presence or absence of protein binding sites, etc., can be easily devised by those skilled in the art.

Accordingly, the present invention provides a nucleic acid molecule, comprising at least one DNA segment having at least two engineered recombination sites flanking a Selectable marker and/or a desired DNA segment, wherein at least one of said recombination sites comprises a core region having at least one engineered mutation that enhances recombination in vitro in the formation of a Cointegrate DNA or a Product DNA.

The nucleic acid molecule can have at least one mutation that confers at least one enhancement of said recombination, said enhancement selected from the group consisting of substantially favoring excisive integration; (ii) favoring excisive recombination; (ii) relieving the requirement for host factors; (iii) -29increasing the efficiency of said Cointegrate DNA or Product DNA formation; and (iv) increasing the specificity of said Cointegrate DNA or Product DNA formation.

The nucleic acid molecule preferably comprises at least one recombination site derived from attB, attP, attL or attR. More preferably the att site is selected from attl, att2, or att3, as described herein.

In a preferred embodiment, the core region comprises a DNA sequence selected from the group consisting of: RKYCWGCTITYKTRTACNAASTSGB (m-at) (SEQ ID NO:1); AGCCWGCTITYKTRTACNAAC TSGB (m-attB) (SEQ ID NO:2); GTTCAGCTTTCKTRTACNAACTSGB (m-attR) (SEQ ID NO:3); AGCCWGCITCKTRTACNAAGTSGB (m-attL) (SEQ ID NO:4); GTTCAGCTITYKTRTACNAAGTSGB(m-attP1) (SEQ ID or a corresponding or complementary DNA or RNA sequence, wherein R=A or G; K=G or T/U; Y=C or T/U; W=A or T/U; N=A or C or G or T/U; S=Cor G; and B=C or G or T/U, as presented in 37 C.F.R. §1.822, which is entirely incorporated herein by reference, wherein the core region does not contain a stop codon in one or more reading frames.

The core region also preferably comprises a DNA sequence selected from the group consisting of: AGCCTGCTTITIGTACAAACTTGT(ttBl) (SEQ IDNO:6); AGCCTGCTITCTGTACAAACTTGT(attB2)(SEQ ID NO:7); ACCCAGCTITCTGTACAAACTTGT (attB3) (SEQ ID NO:8); GTTCAGCTTITGTACAAACTTGT( 1)(SEQIDNO:9); GTTCAGCTICTTGTACAAACTTGT (a 2) (SEQID NO: 0); GTTCAGCTTTCTIGTACAAAGTTGG (attR3) (SEQ ID NO:11); AGCCTGCITTrGTACAAAGTTGG (attLl) (SEQ ID NO:12); AGCCTGCTTTCTTGTACAAAGTTGG (attL2) (SEQ ID NO:13); ACCCAGCTITCTTGTACAAAGTTGG (attL3) (SEQ ID NO:14); 0) GTTCAGCTTTCTTGTACAAAGTTGG (attP2,P3) (SEQ ID NO: 16); or a corresponding or complementary DNA or RNA sequence.

The present invention thus also provides a method for making a nucleic acid molecule, comprising providing a nucleic acid molecule having at least one engineered recombination site comprising at least one DNA sequence having at least 80-99% homology (or any range or value therein) to at least one of SEQ ID NOS:1-16, or any suitable recombination site, or which hybridizes under stringent conditions thereto, as known in the art.

Clearly, there are various types and permutations of such well-known in vitro and in vivo selection methods, each of which are not described herein for the sake of brevity. However, such variations and permutations are contemplated and considered to be the different embodiments of the present invention.

It is important to note that as a result of the preferred embodiment being in vitro recombination reactions, non-biological molecules such as PCR products can be manipulated via the present recombinational cloning method. In one example, it is possible to clone linear molecules into circular vectors.

There are a number of applications for the present invention. These uses include, but are not limited to, changing vectors, apposing promoters with genes, constructing genes for fusion proteins, changing copy number, changing replicons, cloning into phages, and cloning, PCR products (with an attB site at one end and a loxP site at the other end), genomic DNAs, and cDNAs.

-31 The following examples are intended to further illustrate certain preferred embodiments of the invention and are not intended to be limiting in nature.

Examples The present recombinational cloning method accomplishes the exchange of nucleic acid segments to render something useful to the user, such as a change of cloning vectors. These segments must be flanked on both sides by recombination signals that are in the proper orientation with respect to one another. In the examples below the two parental nucleic acid molecules plasnids) are called the Insert Donor and the Vector Donor. The Insert Donor contains a segment that will become joined to a new vector contributed by the Vector Donor. The recombination intermediate(s) that contain(s) both starting molecules is called the Cointegrate(s). The second recombination event produces two daughter molecules, called the Product (the desired new clone) and the Byproduct.

Buffers Various known buffers can be used in the reactions of the present invention. For restriction enzymes, it is advisable to use the buffers recommended by the manufacturer. Alternative buffers can be readily found in the literature or can be devised by those of ordinary skill in the art.

Examples 1-3. One exemplary buffer for lambda integrase is comprised of 50 mM Tris-HCI, at pH 7.5-7.8, 70 mM KC1, 5 mM spermidine, 0.5 mM EDTA, and 0.25 mg/ml bovine serum albumin, and optionally, 10% glycerol.

One preferred buffer for P1 Cre recombinase is comprised of 50 mM Tris-HCl at pH 7.5, 33 mM NaCl, 5 mM spermidine, and 0.5 mg/ml bovine serum albumin.

The buffer for other site-specific recombinases which are similar to lambda Int and P1 Cre are either known in the art or can be determined -32empirically by the skilled artisans, particularly in light of the above-described buffers.

Example 1: Recombinational Cloning Using Cre and Cre Int Two pairs ofplasmids were constructed to do the in vitro recombinational cloning method in two different ways. One pair, pEZC705 and pEZC726 (Figure 2A), was constructed with loxP and att sites, to be used with Cre and X integrase. The other pair, pEZC602 and pEZC629 (Figure 3A), contained the loxP (wild type) site for Cre, and a second mutant lox site, loxP 511, which differs from loxP in one base (out of 34 total). The minimum requirement for recombinational cloning of the present invention is two recombination sites in each plasmid, in general X and Y, and X and r. Recombinational cloning takes place if either or both types of site can recombine to form a Cointegrate X and and if either or both (but necessarily a site different from the type forming the Cointegrate) can recombine to excise the Product and Byproduct plasmids from the Cointegrate Y and It is important that the recombination sites on the same plasmid do not recombine. It was found that the present recombinational cloning could be done with Cre alone.

Cre-Only Two plasmids were constructed to demonstrate this conception (see Figure 3A). pEZC629 was the Vector Donor plasmid. It contained a constitutive drug marker (chloramphenicol resistance), an origin of ieplication, loxP and loxP 511 sites, a conditional drug marker (kanamycin resistance whose expression is controlled by the operator/promoter of the tetracycline resistance operon of transposon TnlO), and a constitutively expressed gene for the tet repressor protein, tetR. E. coli cells containing pEZC629 were resistant to chloramphenicol at 30 pg/ml, but sensitive to kanamycin at 100 pg/ml. pEZC602 was the Insert Donor plasmid, which contained a different drug marker -33- (ampicillin resistance), an origin, and loxP and loxP 511 sites flanking a multiple cloning site.

This experiment was comprised of two parts as follows: Part I: About 75 ng each of pEZC602 and pEZC629 were mixed in a total volume of 30 pl of Cre buffer (50 mM Tris-HCI pH 7.5, 33 mM NaCI, mM spermidine-HCl, 500 pg/ml bovine serum albumin). Two 10 pl aliquots were transferred to new tubes. One tube received 0.5 pl of Cre protein (approx.

4 units per pl; partially purified according to Abremski and Hoess, J Biol. Chem.

259:1509 (1984)). Both tubes were incubated at 37C for 30 minutes, then for 10 minutes. Aliquots of each reaction were diluted and transformed into Following expression, aliquots were plated on 30 lig/ml chloramphenicol; 100 pg/ml ampicillin plus 200 pg/ml methicillin; or 100 pg/m kanamycin. Results: See Table 1. The reaction without Cre gave 1.1 lxl0 ampicillin resistant colonies (from the Insert Donor plasmid pEZC602); 7.8x10 chloramphenicol resistant colonies (from the Vector Donor plasmid pEZC629); and 140 kanamycin resistant colonies (background). The reaction with added Cre gave 7.5x105 ampicillin resistant colonies (from the Insert Donor plasmid pEZC602); 6.1xl0 5 chloramphenicol resistant colonies (from the Vector Donor plasmid pEZC629); and 760 kanamycin resistant colonies (mixture of background colonies and colonies from the recombinational cloning Product plasmid). Analysis: Because the number of colonies on the kanamycin plates was much higher in the presence of Cre, many or most of them were predicted to contain the desired Product plasmid.

Table 1 Enzyme Ampicillin Chloramphenicol Kanamycin Efficiency None 1.1x10 7.8x10' 140 140/7.8x10= 0.02% Cre 7.5x10' 6.1x10' 760 760/6.1xlO'- 0.12% Part II: Twenty four colonies from the Cre" kanamycin plates were picked and inoculated into medium containing 100 pg/ml kanamycin. Minipreps were done, and the miniprep DNAs, uncut or cut with Smal or HindIII, were -34electrophoresed. Results: 19 of the 24 minipreps showed supercoiled plasmid of the size predicted for the Product plasmid. Al 19 showed the predicted Smal and HindIII restriction fragments. Analysis: The Cre only scheme was demonstrated. Specifically, it was determined to have yielded about 70% (19 of 24) Product clones. The efficiency was about 0.1% (760 kanamycin resistant clones resulted from 6.1xl0' chloramphenicol resistant colonies).

Cre Plus Integrase The plasmids used to demonstrate this method are exactly analogous to those used above, except that pEZC726, the Vector Donor plasmid, contained an atP site in place ofloxP 511, and pEZC705, the Insert Donor plasmid, contained an attB site in place of loxP 511 (Figure 2A).

This experiment was comprised of three parts as follows: Part I: About 500 ng of pEZC705 (the Insert Donor plasmid) was cut with Scal, which linearized the plasmid within the ampicillin resistance gene.

(This was done because the integrase reaction has been historically done with the attB plasmid in a linear state Nash, personal communication). However, it was found later that the integrase reaction proceeds well with both plasmids supercoiled.) Then, the linear plasmid was ethanol precipitated and dissolved in pl of I integrase buffer (50 mM Tris-HCl, about pH 7.8, 70 mM KCI, 5 mM spermidine-HCI, 0.5 mM EDTA, 250 pg/ml bovine serum albumin). Also, about 500 ng of the Vector Donor plasmid pEZC726 was ethanol precipitated and dissolved in 20 pl I integrase buffer. Just before use, I integrase (2 pl, 393 pg/ml) was thawed and diluted by adding 18 uI cold A integrase buffer.

One pl IHF (integration host factor, 2.4 mg/ml, an accessory protein) was diluted into 150 pl cold A integrase buffer. Aliquots (2 pI) of each DNA were mixed with l integrase buffer, with or without I pl each u integrase and IHF, in a total of 10 pl. The mixture was incubated at 25*C for 45 minutes, then at 70*C for minutes. Half of each reaction was applied to an agarose gel. Result: In the presence of integrase and IHF, about 5% of the total DNA was converted to a linear Cointegrate form. Analysis: Activity of integrase and IHF was confirmed.

Part II: Three microliters of each reaction with or without integrase and IHF) were diluted into 27 p1 of Cre buffer (above), then each reaction was split into two 10 pi aliquots (four altogether). To two of these reactions, 0.5 1p of Cre protein (above) were added, and all reactions were incubated at 37*C for 30 minutes, then at 70C for 10 minutes. TE buffer (90 pl; TE: 10 mM Tris-HCI, pH 7.5, 1 mM EDTA) was added to each reaction, and 1 pl each was transformed into E. coli DH5a. The transformation mixtures were plated on 100 pgg/nl ampicillin plus 200 pg/ml methicillin; 30 pg/ml chloramphenicol; or 100 pg/ml kanamycin. Results: See Table 2.

Table 2 Enzyme Ampicillin Chloramphenicol Kanamycin Efficiency None 990 20000 4 4/2xl0 0.02% Cre only 280 3640 0 0 Integrase* 1040 27000 9 9 2.7x10 4 0.03% only Integrase* 110 1110 76 76 1.1xl0' 6.9% +Cre Integrase reactions also contained IHF.

Analysis: The Cre protein impaired transformation. When adjusted for this effect, the number of kanamycin resistant colonies, compared to the control reactions, increased more than 100 fold when both Cre and Integrase were used.

This suggests a specificity of greater than 99%.

Part III: 38 colonies were picked from the Integrase plus Cre plates, miniprep DNAs were made and cut with HindI to give diagnostic mapping information. Result: All 38 had precisely the expected fragment sizes.

Analysis: The Cre plus integrase method was observed to have much higher specificity than Cre-alone. Conclusion: The Cre plus I integrase method was demonstrated. Efficiency and specificity were much higher than for Cre only.

-36- Example 2: Using in vitro Recombinational Cloning to Subclone the Chloramphenicol Acetyl Transferase Gene into a Vector for Expression in Eukaryotic Cells (Figure 4A) An Insert Donor plasmid, pEZC843, was constructed, comprising the chloramphenicol acetyl transferase gene of coli, cloned between loxP and attB sites such that the loxP site was positioned at the 5'-end of the gene (Figure 4B).

A Vector Donor plasmid, pEZC1003, was constructed, which contained the cytomegalovirus eukaryotic promoter apposed to a loxP site (Figure 4C). One microliter aliquots of each supercoiled plasmid (about 50 ng crude miniprep DNA) were combined in a ten microliter reaction containing equal parts of lambda integrase buffer (50 mM Tris-HCl, pH 7.8, 70 mM KC1, 5 mM spermidine, 0.5 mM EDTA, 0.25 mg/ml bovine serum albumin) and Cre recombinase buffer (50 mM Tris-HCl, pH 7.5, 33 mM NaC1, 5 mM spermidine, mg/ml bovine serum albumin), two units of Crc recombinase, 16 ng integration host factor, and 32 ng lambda integrase. After incubation at 30'C for minutes and 75C for 10 minutes, one microliter was transformed into competent E coli strain DH5a (Life Technologies, Inc.). Aliquots of transformations were spread on agar plates containing 200 pg/ml kanamycin and incubated at 37*C overnight An otherwise identical control reaction contained the Vector Donor plasmid only. The plate receiving 10% of the control reaction transformation gave one colony, the plate receiving 10% of the recombinational cloning reaction gave 144 colonies. These numbers suggested that greater than 99% of the recombinational cloning colonies contained the desired product plasmid. Miniprep DNA made from six recombinational cloning colonies gave the predicted size plasmid (5026 base pairs), CMVProd. Restriction digestion with NcoI gave the fragments predicted for the chloramphenicol acetyl transferase cloned downstream of the CMV promoter for all six plasmids.

-37- Example 3: Subcloned DNA Segments Flanked by attB Sites Without Stop Codons Part I: Background The above examples are suitable for transcriptional fusions, in which transcription crosses recombination sites. However, both attR and loxP sites contain multiple stop codons on both strands, so translational fusions can be difficult, where the coding sequence must cross the recombination sites, (only one reading fiame is available on each strand of loxP sites) or impossible (in attR or attL).

A principal reason for subcloning is to fuse protein domains. For example, fusion of the glutathione S-transferase (GST) domain to a protein of interest allows the fusion protein to be purified by affinity chromatography on glutathione agarose (Pharmacia, Inc., 1995 catalog). If the protein of interest is fused to runs of consecutive histidines (for example His6), the fusion protein can be purified by affinity chromatography on chelating resins containing metal ions (Qiagen, Inc.). It is often desirable to compare amino terminal and carboxy terminal fusions for activity, solubility, stability, and the like.

The attB sites of the bacteriophage I integration system were examined as an alternative to loxP sites, because they are small (25 bp) and have some sequence flexibility (Nash, HA. et al., Proc. Natl. Acad Sci. USA 84:4049-4053 (1987). It was not previously suggested that multiple mutations to remove all stop codes would result in useful recombination sites for recombinational subcloning.

Using standard nomenclature for site specific recombination in lambda bacteriophage (Weisber, in Lambda III, Hendrix, et al., eds., Cold Spring Harbor -38- Laboratory, Cold Spring Harbor, NY (1989)), the nucleotide regions that participate in the recombination reaction in an E. coli host cell are represented as follows: attP attB Int, IHF II Xis, Int, IHF attR attL B-O-C--H -P'1--P2--P where: O represents the 15 bp core DNA sequence found in both the phage and E coli genomes; B and B' represent approximately 5 bases adjacent to the core in the E coli genome; and PI, H1, P2, X, H2, C, IH, P'l, P2, and P'3 represent known DNA sequences encoding protein binding domains in the bacteriophage A genome.

The reaction is reversible in the presence of the protein Xis (excisionase); recombination between attL and attR precisely excise the I genome from its integrated state, regenerating the circular I genome containing attP and the linear E coli genome containing attB.

Part H: Construction and Testing ofPlasmids Containing Mutant att Sites Mutant attL and attR sites were constructed. Importantly, Landy et aL (Ann. Rev. Biochem. 58:913 (1989)) observed that deletion of the P1 and H1 domains of attP facilitated the excision reaction and eliminated the integration reaction, thereby making the excision reaction irreversible. Therefore, as mutations were introduced in attR, the P1 and HI domains were also deleted.

attR sites in the present example lack the PI and HI regions and have the NdeI site removed (base 27630 changed from C to and contain sequences -39corresponding to bacteriophage coordinates 27619-27738 (GenBank release 92.0, bg:LAMCG, "Complete Sequence of Bacteriophage Lambda").

The sequence of attB produced by recombination of wild type attL and attR sites is: B 0 B' attBwt: 5' AGCCT GCTTTTTTATACfA CTTGA 3' (SEQ. ID NO:31) 3' TCGGA CGAAAAAAaTgT GAACT The stopcodons are italicized and underlined. Note that sequences of attL,.attR, and attP can be derived from the attB sequence and the boundaries of bacteriophage contained within attL and attR (coordinates 27619 to 27818).

When mutant attRl and attL1 sites were recombined the sequence attB 1 was produced (mutations in bold, large font): B 0 B' attBl: 5' AGCCT GCTTrI-fT GTACAAA CTTGT 3 (SEQ. ID NO:6) 3' TCGGA CGAAAAAACATGTTT GAACA Note that the four stop codons are gone.

When an additional mutation was introduced in the attRI and attLl sequences (bold), attR2 and attL2 sites resulted. Recombination of attR2 and attL2 produced the attB2 site: B O B' attB2: 5' AGCCT GTTTCTTGTACAAA CT T G T 3' (SEQ. ID NO:7) 3' TCGGA CGAAAGAACATGTTT GAACA The recombination activities of the above attL and attR sites were assayed as follows. The attB site of plasmid pEZC705 (Figure 2B) was replaced with attLwt, attL1, or attL2. The attP site of plasmid pEZC726 (Figure 2C) was replaced with attRwt (lacking regions P1 and HI), attR1, or attR2. Thus, the resulting plasmids could recombine via their loxP sites, mediated by Cre, and via their attR and attL sites, mediated by Int, Xis, and IHF. Pairs of plasmids were mixed and reacted with Cre, Int, Xis, and IHF, transformed into E coli competent cells, and plated on agar containing kanamycin. The results are presented in Table 3: Table 3 Vector donor att site Gene donor att site ofkanamycin resistant colonies* attRwt (pEZC1301) None 1 (background) attLwt (pEZC1313) 147 attLl(pEZC1317) 47 attL2 (pEZC1321) 0 attRl (pEZC1305) None 1 (background) attLwt (pEZC1313) 4 attL1 (pEZC1317) 128 attL2 (pEZC1321) 0 attR2 (pEZC1309) None 9 (background) attLwt (pEZC1313) 0 .0 attL2 (pEZC1317) 0 attL2 (pEZC1321) 209 of each transformation was spread on a kanamycin plate.) The above data show that whereas the wild type att and attl sites recombine to a small extent, the atti and att2 sites do not recombine detectably with each other.

Part I. Recombination was demonstrated when the core region of both attB sites flanking the DNA segment of interest did not contain stop cbdons. The physical state of the participating plasmids was discovered to influence recombination efficiency.

0 The appropriate att sites were moved into pEZC705 and pEZC726 to make the plasmids pEZC1405 (Figure 5G) (attRI and attR2) and pEZC1502 (Figure 5H) (attL1 and attL2). The desired DNA segment in this experiment was a copy of the chloramphenicol resistance gene cloned between the two attL sites -41of pEZC1502. Pairs of plasmids were recombined in vitro using Int, Xis, and IHF (no Cre because no loxP sites were present). The yield of desired kanamycin resistant colonies was determined when both parental plasmids were circular, or when one plasmid was circular and the other linear as preseated in Table 4: Table 4 Vector donor' Gene donor' Kanamycin resistant colonies 2 Circular pEZC1405 None Circular pEZC1405 Circular pEZC 1502 2680 Linear pEZC1405 None Linear pEZC1405 Circular pEZC1502 172000 Circular pEZC1405 Linear pEZC 1502 73000 DNAs were purified with Qiagen columns, concentrations determined by A260, and linearized with Xba I (pEZC1405) or AlwN I (pEZC1502). Each reaction contained 100 ng of the indicated DNA. All reactions (10 pl total) contained 3 p of enzyme mix (Xis, Int, and IHF). After incubation (45 minutes at 25", 10 minutes at one pl was used to transform E. coli DHSa cells.

2 Number of colonies expected if the entire transformation reaction (1 ml) had been plated. Either 100 pl or 1 pl of the transformations were actually plated.

Analysis: Recombinational cloning using mutant attR and attL sites was 0 confirmed. The desired DNA segment is subcloned between attB sites that do not contain any stop codons in either strand. The enhanced yield of Product DNA (when one parent was linear) was unexpected because of earlier observations that the excision reaction was more efficient when both participating molecules were supercoiled and proteins were limiting (Nunes-Duby et al., Cell 50:779-788 (1987).

Example 4: Demonstration of Recombinational Cloning Without Inverted Repeats Part I: Rationale The above Example 3 showed that plasmids containing inverted repeats i0 of the appropriate recombination sites (for example, attL1 and attL2 in plasmid pEZC1502) (Figure 5H) could recombine to give the desired DNA segment -42flanked by attB sites without stop codons, also in inverted orientation. A concern was the in vivo and in vitro influence of the inverted repeats. For example, transcription of a desired DNA segment flanked by attB sites in inverted orientation could yield a single stranded RNA molecule that might form a hairpin structure, thereby inhibiting translation.

Inverted orientation of similar recombination sites can be avoided by placing the sites in direct repeat arrangement att sites. If parental plasmids each have a wild type attL and wild type attR site, in direct repeat the Int, Xis, and IF proteins will simply remove the DNA segment flanked by those sites in an intramolecular reaction. However, the mutant sites described in the above Example 3 suggested that it might be possible to inhibit the intramolecular reaction while allowing the intermolecular recombination to proceed as desired.

Part H1 Structure of Plasmids Without Inverted Repeats for Recombinational aConing The attR2 sequence in plasmid pEZCI405 (Figure 5G) was replaced with attL2, in the opposite orientation, to make pEZC1603 (Figure 6A). The attL2 sequence of pEZC1502 (Figure 5H) was replaced with attR2, in the opposite orientation, to make pEZCI706 (Figure 6B). Each of these plasmids contained mutations in the core region that make intramolecular reactions between atti and att2 cores very inefficient (see Example 3, above).

Plasmids pEZC1405, pEZC1502, pEZC1603 and pEZC1706 were purified on Qiagen columns (Qiagen, Inc.). Aliquots ofplasmids pEZC1405 and pEZC1603 were linearized with Xba I. Aliquots of plasmids pEZC1502 and pEZC1706 were linearized with AlwN I. One hundred ng of plasmids were mixed in buffer (equal volumes of 50 mM Tris HCI pH 7.5, 25 mM Tris HCI pH 70 mM KC1, 5 mM spermidine, 0.5 mM EDTA, 250pg/ml BSA, glycerol) containing Int (43.5 ng), Xis (4.3 ng) and IHF (8.1 ng) in a final volume of 10 pl. Reactions were incubated for 45 minutes at 25°C, 10 minutes at 65 "C, and 1 pl was transformed into E. coli DH5a. After expression, aliquots were spread on agar plates containing 200 pg/ml kanamycin and incubated at 37°C.

-43- Results, expressed as the number of colonies per 1 pI1 of recombination reaction are presented in Table Table Vector Donor Gene Donor Colonies Predicted product Circular 1405 100 Circular 1405 Circular 1502 3740 3640/3740 =97% Linear 1405 Linear 1405 Circular 1502 172,000 171,910/172,000=99.9% Circular 1405 Linear 1502 73,000 72,900/73,000 99.9% Circular 1603 80 Circular 1603 Circular 1706 410 330/410 Linear 1603 270 Linear 1603 Circular 1706 7000 6730/7000 96% Circular 1603 Linear 1706 10,800 10,530/10,800=97% Analysis. In all configurations, circular or linear, the pEZC1405 x pEZC 1502 pair (with att sites in inverted repeat configuration) was more efficient than pEZC1603 x pEZC1706 pair (with att sites mutated to avoid hairpin formation). The pEZC1603 x pEZC1706 pair gave higher backgrounds and lower efficiencies than the pEZC1405 x pEZC1502 pair. While less efficient, or more of the colonies from the pEZC1603 x pEZCI706 reactions were expected to contain the desired plasmid product. Making one partner linear stimulated the reactions in all cases.

Part I: Confirmation of Product Plasmids' Structure Six colonies each from the linear pEZC1405 (Figure 5G) x circular pEZC1502 (Figure 5H), circular pEZC1405 x linear pEZC1502, linear pEZC1603 (Figure 6A) x circular pEZC1706 (Figure 6B), and circular pEZC1603 x linear pEZC1706 reactions were picked into rich medium and -44miniprep DNAs were prepared. Diagnostic cuts with Ssp I gave the predicted restriction fragments for all 24 colonies.

Analysis. Recombination reactions between plasmids with mutant attL and attR sites on the same molecules gave the desired plasmid products with a high degree of specificity.

Example 5: Recombinational Cloning with a Toxic Gene Part I: Background Restriction enzyme Dpn I recognizes the sequence GATC and cuts that sequence only if the A is methylated by the dam methylase. Most commonly used E. coil strains are dam*. Expression of Dpn I in dam* strains of E coli is lethal because the chromosome of the cell is chopped into many pieces.

However, in dam" cells expression of Dpn I is innocuous because the chromosome is immune to Dpn I cutting.

In the general recombinational cloning scheme, in which the vector donor contains two segments C and D separated by recombination sites, selection for the desired product depends upon selection for the presence of segment D, and the absence of segment C. In the original Example segment D contained a drug resistance gene (Kin) that was negatively controlled by a represser gene found on segment C. When C was present, cells containing D were not resistant to kanamycin because the resistance gene was turned off The Dpn I gene is an example of a toxic gene that can replace the represser gene of the above embodiment If segment C expresses the Dpn I gene product, transforming plasmid CD into a dam* host kills the cell. If-segment D is transferred to a new plasmid, for example by recombinational cloning, then selecting for the drug marker will be successful because the toxic gene is no longer present.

Part II: Construction of a Vector Donor Using Dpn I as a Toxic Gene The gene encoding Dpn I endonuclease was amplified by PCR using primers 5'CCA CCA CAA ACG CGT CCA TGG AAT TAC ACT TTA ATT TAG3' (SEQ. ID NO: 17) and 5'CCA CCA CAA GTC GAC GCA TGC CGA CAG CCT TCC AAA TGT3'(SEQ. IDNO:18) and a plasmid containing the Dpn I gene (derived from plasmids obtained from Sanford A. Lacks, Brookhaven National Laboratory, Upton, New York; also available from American Type Culture Collection as ATCC 67494) as the template.

Additional mutations were introduced into the B and B' regions of attL and attR, respectively, by amplifying existing attL and attR domains with primers containing the desired base changes. Recombination of the mutant attL3 (made with oligo Xisl 15) and attR3 (made with oligo Xis 12) yielded attB3 with the following sequence (differences from attBl in bold): B 0 B' ACCCA GCTTTCTTGTACAAA GTGGT (SEQ. ID NO:8) TGGGT CGAAAGAACATGTTT CACCA The attL3 sequence was cloned in place of attL2 of an existing Gene Donor plasmid to give the plasmid pEZC2901 (Figure 7A). The attR3 sequence was cloned in place of attR2 in an existing Vector Donor plasmid to give plasmid pEZC2913 (Figure 7B) Dpa I gene was cloned into plasmid pEZC2913 to replace the tet repressor gene. The resulting Vector Donor plasmid was named pEZC3101 (Figure 7C). When pEZC3101 was transformed into the dam- strain SCS 110 (Stratagene), hundreds of colonies resulted. When the same plasmid was transformed into the dam+ strain DHSa, only one colony was produced, even though the DH5a cells were about 20 fold more competent than the SCS cells. When a related plasmid that did not contain the Dpn I gene was transformed into the same two cell lines, 28 colonies were produced from the SCSI 10 cells, while 448 colonies resulted from the DH5a cells. This is evidence -46that the Dpn I gene is being expressed on plasmid pEZC3101 (Figure 7C), and that it is killing the dam* DH5a cells but not the dam- SCS 10 cells.

Part 1I: Demonstration ofRecombinational Cloning Using Dpn I Selection A pair ofplasmids was used to demonstrate recombinational cloning with selection for product dependent upon the toxic gene Dpn I. Plasmid pEZC3101 (Figure 7C) was linearized with Mlu I and reacted with circular plasmid pEZC2901 (Figure 7A). A second pair of plasmids using selection based on control of drug resistance by a repressor gene was used as a control: plasmid pEZC1802 (Figure 7D) was linearized with Xba I and reacted with circular plasmid pEZC1502 (Figure 5H). Eight microliterreactions containing the same buffer and proteins Xis, Int, and IHF as in previous examples were incubated for minutes at 25 C, then 10 minutes at 75"C, and 1 pl aliquots were transformed into DH5a dam+) competent cells, as presented in Table 6.

Table 6 Reaction Vector donor Basis of selection Gene donor Colonies I pEZC3101/MIu Dpn I toxicity 3 2 pEZC3101/Mlu Dpn I toxicity Circular pEZC2901 4000 3 pEZC1802/Xba Tet repressor 0 4 pEZC1802/Xba Tetrepressor Cirular pEZC1502 12100 Miniprep DNAs were prepared from four colonies from reaction and cut with restriction enzyme Ssp I. All gave the predicted fragments.

Analysis: Subcloning using selection with -a toxic gene was demonstrated. Plasmids of the predicted structure were produced.

-47- Example 6: Cloning of Genes with Uracil DNA Glycosylase and Subcloning of the Genes with Recombinational Cloning to Make Fusion Proteins Part I: Converting an Existing Expression Vector to a Vector Donor for Recombinational Cloning A cassette useful for converting existing vectors into functional Vector Donors was made as follows. Plasmid pEZC3 101 (Figure 7C) was digested with Apa I and Kpn I, treated with T4 DNA polymerase and dNTPs to render the ends blunt, further digested with Sma I, Hpa I, and AlwN I to reader the undesirable DNA fragments small, and the 2.6 kb cassette containing the attRI Cm R Dpn I attR-3 domains was gel purified. The concentration of the purified cassette was estimated to be about 75 ng DNA/I.

Plasmid pGEX-2TK (Figure 8A) (Pharmacia) allows fusions between the protein glutathione S transferase and any second coding sequence that can be inserted in its multiple cloning site. pGEX-2TK DNA was digested with Sma I and treated with alkaline phosphatase. About 75 ng of the above purified DNA cassette was ligated with about 100 ng of the pGEX-2TK vector for 2.5 hours in a 5 pl ligation, then 1 ul was transformed into competent BRL 3056 cells (a dam" derivative of DH10B; dam- strains commercially available include DM1 from Life Technologies, Inc., and SCS 110 from Stratagene). Aliquots of the transformation mixture were plated on LB agar containing 100 pg/ml ampicillin (resistance gene present on pGEX-2TK) and 30 gg/ml chloramphenicol (resistance gene present on the DNA cassette). Colonies were picked and miniprep DNAs were made. The orientation of the cassette in pGEX-2TK was determined by diagnostic cuts with EcoR I. A plasmid with the desired orientation was named pEZC3501 (Figure 8B).

-48- Part I: Cloning Reporter Genes Into an Recombinational Cloning Gene Donor Plasmid in Three Reading Frames Uracil DNA glycosylase (UDG) cloning is a method for cloning PCR amplification products into cloning vectors patent No. 5,334,515, entirely incorporated herein by reference). Briefly, PCR amplification of the desired DNA segment is performed with primers that contain uracil bases in place of thymidine bases in their 5' ends. When such PCR products are incubated with the enzyme UDG, the uracil bases are specifically removed. The loss of these bases weakens base pairing in the ends of the PCR product DNA, and when incubated at a suitable temperature 37C), the ends of such products are largely single stranded. If such incubations are done in the presence of linear cloning vectors containing protruding 3' tails that are complementary to the 3' ends of the PCR products, base pairing efficiently anneals the PCR products to the cloning vector.

When the annealed product is introduced into E. coli cells by transformation, in vivo processes efficiently convert it into a recombinant plasmid.

UDG cloning vectors that enable cloning of any PCR product in all three reading frames were prepared from pEZC3201 (Figure 8K) as follows. Eight oligonucleotides were obtained from Life Technologies, Inc. (all written 5' 3': rfl top (GGCC GAT TAC GAT ATC CCA ACG ACC GAA AAC CTG TAT TTT CAG GGT) (SEQ. ID NO:19), rfl bottom (CAG GTT TTC GGT CGT TGG GAT ATC GTA ATC)(SEQ. ID NO:20), rf2 top (GGCCA GAT TAC GAT ATC CCA ACG ACC GAA AAC CTG TAT TIT CAG GGT)(SEQ. ID NO:21), rf2 bottom (CAG GTT TTC GGT CGT TGG GAT ATC GTA ATC T)(SEQ. ID NO:22), rf3 top (GGCCAA GAT TAC GAT ATC CCA ACG ACC GAA AAC CTG TAT TTT CAG GGT)(SEQ. ID NO:23), rf3 bottom (CAG GTT TTC GGT CGT TGG GAT ATC GTA ATC TT)(SEQ. ID NO:24), carboxy top (ACC GTT TAC GTG GACXSEQ. ID NO:25) and carboxy bottom (TCGA GTC CAC GTA AAC GGT TCC CAC TTA TTA)(SEQ. ID NO:26). The rfl, 2, and 3 top strands and the carboxy bottom strand were phosphorylated on their 5' ends with T4 polynucleotide kinase, and then the complementary strands of each pair were hybridized. Plasmid pEZC3201 -49- (Figure 8K) was cut with Not I and Sal I, and aliquots of cut plasmid were mixed with the carboxy-oligo duplex (Sal I end) and either the rfl, rf2, or rf3 duplexes (Not I ends) (10 pg cut plasmid (about 5 pmol) mixed with 250 pmol carboxy oligo duplex, split into three 20 l1 volumes, added 5 tl (250 pmol) ofrfl, rf2, or rf3 duplex and 2 l 2 units T4 DNA ligase to each reaction). After 90 minutes of ligation at room temperature, each reaction was applied to a preparative agarose gel and the 2.1 kb vector bands were eluted and dissolved in 50 ul of TE.

Part IH: PCR of CAT andphoA Genes Primers were obtained from Life Technologies, Inc., to amplify the chloramphenicol acetyl transferase (CAT) gene from plasmid pACYC184, and phoA, the alkaline phosphatase gene from E coli. The primers had 12-base extensions containing uracil bases, so that treatment of PCR products with uracil DNA glycosylase (UDG) would weaken base pairing at each end of the DNAs and allow the 3' strands to anneal with the protruding 3' ends of the rfl, 2, and 3 vectors described above. The sequences of the primers (all written 5' 3') were: CAT left, UAU UUU CAG GGU ATG GAG AAA AAA ATC ACT GGA TAT ACC (SEQ. ID NO:27); CAT right, UCC CAC UUA UUA CGC CCC GCC CTG CCA CTC ATC (SEQ. ID NO:28); phoA left, UAU UUU CAG GGU ATG CCT GTT CTG GAA AAC CGG (SEQ. ID NO:29); and phoA right, UCC CAC UUA UUA TIT CAG CCC CAG GGC GGC TTT C (SEQ. ID The primers were then used for PCR reactions using known method steps (see, U.S. patent No. 5,334,515, entirely incorporated herein by reference), and the polymerase chain reaction amplification products obtained with these primers comprised the CAT or phoA genes with the initiating ATGs but without any transcriptional signals. In addition, the uracil-containing sequences on the amino termini encoded the cleavage site for TEV protease (Life Technologies, Inc.), and those on the carboxy terminal encoded consecutive TAA nonsense codons.

Unpurified PCR products (about 30 ng) were mixed with the gel purified, linear rfl, rf2, or rf3 cloning vectors (about 50 ng) in a 10 pI reaction containing 1X REact 4 buffer (LTI) and 1 unit UDG (LTI). After 30 minutes at 37°C, 1 l1 aliquots of each reaction were transformed into competent E. coli DH5a cells (LTI) and plated on agar containing 50 tg/ml kanamycin. Colonies were picked and analysis of miniprep DNA showed that the CAT gene had been cloned in reading frame 1 (pEZC3601)(Figure 8C), reading frame 2 (pEZC3609)(Figure 8D) and reading frame 3 (pEZC3617)(Figure 8E), and that the phoA gene had been cloned in reading frame 1 (pEZC3606)(Figure 8F), reading frame 2 (pEZC3613)(Figure 8G) and reading frame 3 (pEZC3621)(Figure 8H).

PaIt IV: Subcloning of CATorphoAfrom UDG Cloning Vectors into a GST Fusion Vector Plasmids encoding fusions between GST and either CAT or phoA in all three reading frames were constructed by recombinational cloning as follows.

Miniprep DNA of GST vector donor pEZC3501(Figure 8B) (derived from Pharmacia plasmid pGEX-2TK as described above) was linearized with CIa I.

About 5 ng of vector donor were mixed with about 10 ng each of the appropriate circular gene donor vectors containing CAT or phoA in 8 pi reactions containing buffer and recombination proteins Int, Xis, and IHF (above). After incubation, 1 pl of each reaction was transformed into E. coli strain DH5a and plated on ampicillin, as presented in Table 7.

Table 7 DNA Colonies (10% of each transformation) Linear vector donor (pEZC3501/CIa) 0 Vector donor CAT rfl 110 Vector donor CAT rf2 71 Vector donor CAT rf3 148 Vector donor phoA rfl 121 Vector donor phoA rf2 128 Vector donor phoA rf3 31 -51- Part V: Expression of Fusion Proteins Two colonies from each transformation were picked into 2 ml of rich medium (CircleGrow, BiolOl Inc.) in 17 x 100 nun plastic tubes (Falcon 2059, Becton Dickinson) containing 100 ig/ml ampicillin and shaken vigorously for about 4 hours at 37°C, at which time the cultures were visibly turbid. One ml of each culture was transferred to a new tube containing 10 pi of 10% IPTG to induce expression of GST. After 2 hours additional incubation, all cultures had about the same turbidity; the A600 of one culture was 1.5. Cells from 0.35 ml each culture were harvested and treated with sample buffer (containing SDS and P-mercaptoethanol) and aliquots equivalent to about 0.15 A600 units of cells were applied to a Novex 4-20% gradient polyacrylamide gel. Following electrophoresis the gel was stained with Coomassie blue.

Results: Enhanced expression of single protein bands was seen for all 12 cultures. The observed sizes of these proteins correlated well with the sizes predicted for GST being fused (through attB recombination sites without stop codons) to CAT or phoA in three reading frames: CAT rfl 269 amino acids; CAT rf2 303 amino acids; CAT rf3 478 amino acids; phoA rfl 282 amino acids; phoA rf2 280 amino acids; and phoA rf3 705 amino acids.

Analysis: Both CAT and phoA genes were subcloned into a GST fusion vector in all three reading frames, and expression of the six fusion proteins was demonstrated.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims. All patents and publications cited herein are entirely incorporated herein by reference.

51.12- Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

3111 2/7GV9 634.SPE 1

Claims (617)

1. An isolated nucleic acid molecule comprising at least a first all recombination site comprising at least one mutation that enhances recombination specificity.

2. The isolated nucleic acid molecule of claim 1, further comprising at least a second recombination site selected from the group consisting of an atll site and a lox site. o

3. A nucleic acid molecule comprising at least a first lox site flanked by at least one promoter and at least one antibiotic resistance gene.

4. The nucleic acid molecule of claim 3, wherein said lox site is a loxP site.

5. The nucleic acid molecule of claim 3, wherein said lox site is a loxP site and wherein said promoter and said antibiotic resistance gene are operably linked.

6. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule is a vector.

7. A nucleic acid molecule comprising at least one promoter operably linked to at least one antibiotic resistance gene, wherein said promoter and said antibiotic resistance gene are separated by at least one recombination site.

8. The nucleic acid molecule of claim 7, wherein said first recombination site is selected from the group consisting of a lox site, an atll site, and mutants thereof.

9. is a lox site.

The nucleic acid molecule of claim 7, wherein said first recombination site The nucleic acid molecule of claim 10, wherein said lox site is a loxP site.

11. The nucleic acid molecule of claim 7, wherein said nucleic acid molecule further comprises at least one additional recombination site. 047/03.sw I I9lsp..dc,52 53

12. The nucleic acid molecule of claim 11, wherein said at least one additional. recombination site is selected from the group consisting oflox sites and at! sites.

13. The nucleic acid molecule of claim 11, wherein said at least one additional recombination site is a lox site.

14. The nucleic acid molecule of claim 13, wherein said lox site is a loxP site.

15. The nucleic acid molecule of claim 7, wherein said nucleic acid molecule comprises at least one cloning site.

16. The nucleic acid molecule of claim 7, wherein said nucleic acid molecule is a vector.

17. The nucleic acid molecule of claim 16, wherein said vector is an expression vector.

18. The nucleic acid molecule of claim 7, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

19. The nucleic acid molecule of claim 7, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

A nucleic acid molecule comprising a functional antibiotic resistance gene, wherein a first portion of said antibiotic resistance gene and a second portion of said antibiotic resistance gene are separated by at least a first recombination site.

21. The nucleic acid molecule of claim 20, wherein said first and second portions of said antibiotic resistance gene are operably linked. 04/07/O3.sw I 1797sp.doc.53 -54-

22. The nucleic acid molecule of claim 20, wherein said first portion of said antibiotic resistance gene is a promoter.

23. The nucleic acid molecule of claim 20, wherein said first recombination site is selected from the group consisting ofa lox site, an att site, and mutants thereof.

24. is a lox site.

The nucleic acid molecule of claim 20, wherein said first recombination site The nucleic acid molecule of claim 24, wherein said lox site is a loxP site.

26. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule further comprises at least one additional recombination site.

27. The nucleic acid molecule of claim 26, wherein said at least one additional recombination site is selected from the group consisting of lox sites and atl sites.

28. The nucleic acid molecule of claim 26, wherein said at least one additional recombination site is a lox site.

29. The nucleic acid molecule of claim 28, wherein said lox site is a loxP site.

The nucleic acid molecule of claim 20, wherein said nucleic acid molecule comprises at least one cloning site.

31. is a vector. The nucleic acid molecule of claim 20, wherein said nucleic acid molecule

32. The nucleic acid molecule of claim 31, wherein said vector is an expression vector.

33. The nucleic acid molecule of claim 20, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an 04/07/0 jwl 1797spa.do, 54 ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

34. The nucleic acid molecule of claim 20, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

The nucleic acid molecule of claim 20, wherein said first portion of said gene is located adjacent to said recombination site.

36. The nucleic acid molecule of claim 20, wherein said second portion of said gene is located adjacent to said recombination site.

37. A nucleic acid molecule comprising at least one promoter operably linked to at least one antibiotic resistance gene, wherein- said promoter and said antibiotic resistance gene are separated by at least one loxP site.

38. The nucleic acid molecule of claim 37, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

39. The nucleic acid molecule of claim 37, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

40. A nucleic acid molecule comprising at least one functional antibiotic resistance gene, wherein said functional gene comprises a promoter and an antibiotic resistance gene separated from each other by at least one loxP site.

41. The nucleic acid molecule of claim 40, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene. 04Mj7/03.l IlI 9 spa.doc. S -56-

42. The nucleic acid molecule of claim 40, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

43. A host cell comprising the nucleic acid molecule according to claim 3 or claim

44. The host cell according to claim 43, wherein said host cell is an Escherichia coli cell.

45. A host cell comprising the nucleic acid molecule according to any one of claims 7, 10, 11, 14, 20, 21, 25, 26, 29, 37 or

46. The host cell according to claim 45, wherein said host cell is an Escherichia coli cell.

47. A polypeptide encoded by a nucleic acid molecule, wherein said nucleic acid molecule comprises at least a first recombination site comprising at least one mutation which enhances recombination specificity, and wherein said polypeptide comprises amino acids encoded by said first recombination site.

48. The polypeptide of claim 47, wherein said nucleic acid molecule further comprises at least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene.

49. The polypeptide of claim 47, wherein said nucleic acid molecule further comprises a second recombination site.

The polypeptide of claim 48, wherein said gene or partial gene comprises a nucleic acid sequence encoding a tag sequence.

51. The polypeptide of claim 50, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag. 04/07/03,sw I l797plp.doc,56 -57-

52. The polypeptide according to any one of claims 47-49, wherein said polypeptide is a fusion polypeptide.

53. The polypeptide of claim 52, wherein said fusion polypeptide comprises an amino terminal tag sequence.

54. The fusion polypeptide of claim 53, wherein said tag sequence is encoded by a gene or partial gene.

55. The fusion polypeptide of claim 53, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

56. The fusion polypeptide of claim 53, wherein said fusion polypeptide comprises a carboxy terminal tag sequence.

57. The fusion polypeptide of claim 56, wherein said tag sequence is encoded by a gene or partial gene.

58. The fusion polypeptide of claim 56, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

59. A polypeptide encoded by a nucleic acid molecule, wherein said nucleic acid molecule comprises at least a first nucleic acid sequence selected from the group consisting a DNA sequence having at least 80% homology to any one of SEQ ID NOs: 1- 16, a complementary DNA sequence thereto, and an RNA sequence corresponding any of these sequences, and wherein said polypeptide comprises amino acids encoded by said first nucleic acid sequence.

The polypeptide of claim 59, wherein said nucleic acid molecule further comprises at least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene. 107/03,swl 1797Isp.doc. 7 -58-

61. The polypeptide of claim 59, wherein said nucleic acid molecule further comprises at least a second nucleic acid sequence selected from the group consisting of SEQ ID NOs:1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

62. The polypeptide of claim 60, wherein said gene or partial gene comprises a nucleic acid sequence encoding a tag sequence.

63. The polypeptide of claim 62, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

64. The polypeptide according to any one of claims 59-61, wherein said polypeptide is a fusion polypeptide.

65. The fusion polypeptide of claim 64, wherein said fusion polypeptide comprises an amino terminal tag sequence.

66. The fusion polypeptide of claim 65, wherein said tag sequence is encoded by a gene or partial gene.

67. The fusion polypeptide of claim 65, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

68. The fusion polypeptide of claim 64, wherein said fusion protein comprises a carboxy terminal tag sequence.

69. The fusion polypeptide of claim 68, wherein said tag sequence is encoded by a gene or partial gene.

70. The fusion polypeptide of claim 68, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

71. A polypeptide encoded by a nucleic acid molecule, wherein said nucleic acid molecule comprises at least a first mutated recombination site wherein said mutation 04tOIO3swI 1797spa.doc.5S -59- removes one or more stop codons from said recombination site, and wherein said polypeptide comprises amino acids encoded by said first mutated recombination site.

72. The polypeptide of claim 71, wherein said nucleic acid molecule further comprises a least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene.

73. The polypeptide of claim 71, wherein said nucleic acid molecule further comprises a second recombination site.

74. The polypeptide of claim 72, wherein said gene or partial gene comprises a nucleic acid sequence encoding a tag sequence.

75. The polypeptide of claim 74, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

76. The polypeptide according to any one of claims 71-73, wherein said polypeptide is a fusion polypeptide.

77. The fusion polypeptide of claim 76, wherein said fusion polypeptide comprises an amino terminal tag sequence.

78. The fusion polypeptide of claim 77, wherein said tag sequence is encoded by a gene or partial gene.

79. The fusion polypeptide of claim 77, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

80. The fusion polypeptide of claim 76, wherein said fusion polypeptide comprises a carboxy terminal tag sequence.

81. The fusion polypeptide of claim 80, wherein said tag sequence is encoded by a gene or partial gene. 04/07/03.w I 197spadoc.59

82. The fusion polypeptide of claim 80, wherein.said tag sequence is selected from the group consisting of a GST tag and a His tag.

83. A polypeptide encoded by a nucleic acid molecule, wherein said nucleic acid molecule comprises at least a first mutated recombination site wherein said mutation avoids hairpin formation, and wherein said polypeptide comprises amino acids encoded by said first mutated recombination site.

84. The polypeptide of claim 83, wherein said nucleic acid molecule further comprises at least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene.

85. The polypeptide of claim 83, wherein said nucleic acid molecule further comprises a second recombination site.

86. The polypeptide of claim 84, wherein said gene or partial gene comprises a nucleic acid sequence encoding a tag sequence.

87. The polypeptide of claim 85, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

88. The polypeptide according to any one of claims 83-85, wherein said polypeptide is a fusion polypeptide.

89. The fusion polypeptide of claim 88, wherein said fusion polypeptide comprises an amino terminal tag sequence.

90. The fusion polypeptide of claim 89, wherein said tag sequence is encoded by a gene or partial gene.

91. The fusion polypeptide of claim 89, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag. 0407/03.sw I 1797 spa -61

92. The fusion polypeptide of claim 88, wherein said fusion polypeptide comprises a carboxy terminal tag sequence.

93. The fusion polypeptide of claim 92, wherein said tag sequence is encoded by a gene or partial gene.

94. The fusion polypeptide of claim 92, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

A polypeptide encoded by a nucleic acid molecule, wherein said nucleic acid molecule comprises at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:l-16, a complementary DNA sequence thereto, and an RNA sequence corresponding any of these sequences, and wherein said polypeptide comprises amino acids encoded by said first nucleic acid sequence.

96. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs: 1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

97. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs: 1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

98. A composition comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances 4 0 O.Lw I 1797sp.dC.61I 62 recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

99. The composition of claim 98, wherein said mutated ait site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

100. The composition of claim 98, wherein said mutated all site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

101. The composition of claim 98, wherein said mutated alt site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence asset forth in SEQ ID NOs.: 1-16.

102. A composition comprising: a first nucleic acid molecule comprising a first portion of a gene and at least a first recombination site; a second nucleic acid molecule comprising a second portion of said gene and at least a second recombination site; and at least one recombination protein capable of causing recombination between said first and second recombination sites.

103. A composition comprising: a first nucleic acid molecule comprising a first portion of an antibiotic resistance gene and at least a first recombination site; a second nucleic acid molecule comprising a second portion of said antibiotic resistance gene and at least a second recombination site; and at least one recombination protein capable of causing recombination between said first and second recombination sites.

104. A composition comprising: a first nucleic acid molecule comprising at least one promoter and at least a first recombination site; w I 17' 9 7 pl.doc.62 63 a second nucleic acid molecule comprising at least one antibiotic resistance gene and at least a second recombination site; and at least one recombination protein capable of causing recombination between said first and second recombination sites.

105. The composition of claim 102, wherein said first and second recombination sites are selected from the group consisting of lox sites, aft sites, and mutants thereof.

106. The composition of claim 102, wherein said first and second recombination sites are lox sites.

107. The composition of claim 106, wherein said fox sites are loxP sites.

108. The composition of claim 103, wherein said first and second recombination sites are selected from the group consisting of lox sites, art sites, and mutants thereof.

109. The composition of claim 103, wherein said first and second recombination sites are lox sites.

110. The composition of claim 109, wherein said lox sites are loxP sites.

111. The composition of claim 104, wherein said first and second recombination sites are selected from the group consisting of lox sites, aft sites, and mutants thereof

112. The composition of claim 104, wherein said first and second recombination sites are lox sites.

113. The composition of claim 112, wherein said lox sites are loxP sites.

114. The composition of claim 102, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site. 04/1073.w I 1797pA.dc,63 -64-

115. The composition of claim 114, wherein said at least one additional recombination site is selected from the group consisting oflox sites and alt sites.

116. The composition of claim 105, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site.

117. The composition of claim 116, wherein said at least one additional recombination site is selected from the groupconsisting oflox sites and art sites.

118. The composition of claim 104, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site.

119. The composition of claim 118, wherein said at least one additional recombination site is selected from the group consisting oflox sites and alt sites.

120. The composition of claim 102, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y5, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

121. The composition of claim 102, wherein said at least one recombination protein is Cre.

122. The composition of claim 102, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

123. The composition of claim 103, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y6, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

124. The composition of claim 103, wherein said at least one recombination protein is Cre. 0407t3.,w II 797,p..doc.64

125. The composition of claim 103, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

126. The composition of claim 104, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y8, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

127. The composition of claim 104, wherein said at least one recombination protein is Cre.

128. The composition of claim 104, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and-Xis.

129. The composition of claim 102, wherein said first nucleic acid molecule and said second nucleic acid molecule is a vector.

130. The composition of claim 103, wherein said first nucleic acid molecule and said second nucleic acid molecule is a vector.

131. The composition of claim 104, wherein said first nucleic acid molecule and said second nucleic acid molecule is a vector.

132. The composition of claim 102, wherein said first or said second portions of said gene are PCR products.

133. The composition according to any one of claims 102, 103 or 104, further comprising at least one host cell.

134. The composition of claim 133, wherein said host cell is an Escherichia coli cell.

135. A composition comprising at least one isolated Int protein and at least one isolated IHF protein. 04107/03,.wl 179lspa.doc.6S 66

136. A composition comprising at least one isolated Int protein, at least one isolated IHF protein and at least one isolated Xis protein.

137. The composition according to any one of claims 135 or 136, further comprising at least one vector comprising at least one recombination site.

138. The composition of claim 137, wherein said at least one recombination site is at least one att recombination site or a mutant or variant thereof.

139. The composition of claim 137, wherein said at least one recombination site is at least one att recombination site.

140. The composition according to any one of claims 138 or 139, wherein said at least one att recombination site is selected from the group consisting of an attB site, an attP site, an attL site, an attR site, and a mutant or variant thereof.

141. The composition according to any one of claims 135 or 136, further comprising at least one vector comprising at least two recombination sites.

142. The composition of claim 141, wherein said at least one of said recombination sites is an aft recombination site or a mutant or variant thereof.

143. The composition of claim 141, wherein said at least one of said recombination sites is an att recombination site.

144. The composition according to any one of claims 142 or 143, wherein said artt recombination site is selected from the group consisting of an attB site, an attP site, an attL site, an attR site, and a mutant or variant thereof

145. The composition according to any one of claims 135 or 136, further comprising at least one isolated FIS protein.

146. The composition according to any one of claims 135 or 136, further comprising spermidine. 04/0703.sw I 1797spa.doc.66 -67-

147. The composition according to any one of claims 135 or 136, further comprising Tris-HCI.

148. The composition according to any one of claims 135 or claim 136, further comprising ethylenediamine tetraacetic acid (EDTA).

149. The composition according to any one of claims 135 or 136, further comprising bovine serum albumin (BSA).

150. The composition according to any one of claims 135 or 136, further comprising at least one additional isolated recombination protein selected from the group consisting of a Cre protein, an FLP protein, a y5 protein, a Tn3 rcsolvase protein, a Hin protein, a Gin protein, and a Cin protein.

151. The composition according to any one of claims 135 or 136, further comprising at least one isolated Cre recombination protein.

152. A composition comprising at least one isolated Int protein, at least one isolated IHF protein, spermidine, Tris-HC1, EDTA and BSA.

153. A composition comprising at least one isolated Int protein, at least one isolated IHF protein, at least one isolated Xis protein, spermidine, Tris-HCl, EDTA and BSA.

154. A composition comprising at least one isolated Int protein, at least one isolated IHF protein and spermidine.

155. A composition comprising at least one isolated Int protein, at least one isolated IHF protein, at least one isolated Xis protein and spermidine.

156. The composition according to any one of claims 152-155, further comprising at least one vector comprising at least one recombination site. 04/07/03.sw 11 797p.doc.67 -68-

157. The composition of claim 156, wherein said at least one recombination site is at least one att recombination site or a mutant or variant thereof.

158. The composition of claim 156, wherein said at least one recombination site is at least one att recombination site.

159. The composition according to any one of claims 157 or 158, wherein said at least one att recombination site is selected from the group consisting of an attB site, an attP site, an attL site, an attR site, and a mutant or variant thereof.

160. The composition according to any one of claims 152-155, further comprising at least one vector comprising at least two recombination sites.

161. The composition of claim 160, wherein said at least one of said recombination sites is an att recombination site or a mutant or variant thereof.

162. The composition of claim 160, wherein said at least one of said recombination sites is an att recombination site.

163. The composition according to any one of claims 161 or 162, wherein said att recombination site is selected from the group consisting of an attB site, an attP site, an attL site, an attR site, and a mutant or variant thereof.

164. The composition according to any one of claims 152-155, further comprising at least one isolated FIS protein.

165. The composition according to any one of claims 152-155, further comprising at least one additional isolated recombination protein selected from the group consisting of a Cre protein, an FLP protein, a y5 protein, a Tn3 resolvase protein, a Hin protein, a Gin protein, and a Cin protein.

166. The composition according to any one of claims 152-155, further comprising at least one isolated Cre recombination protein. 04107/(13,swI 1797ipa.doc,6 69

167. A kit comprising at least one container containing the composition according to any one of claims 135-137, 141, 145-156, 160, or 164-166.

168. A method for in vitro cloning of a nucleic acid of interest, comprising: mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site, wherein said first and/or second vector further comprises a nucleic acid of interest; incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites, thereby producing a chimeric nucleic acid molecule comprising said nucleic acid of interest; contacting one or more hosts with said mixture; and selecting for a host comprising said chimeric nucleic acid molecule, and selecting against a host comprising said first vector and against a host comprising said second vector, thereby cloning said nucleic acid of interest, wherein said first and/or said second recombination site contains one or more mutations.

169. The method of claim 168, wherein said first and/or second recombination site contains at least one mutation that removes one or more stop codons.

170. The method of claim 168, wherein said first and/or second recombination site contains at least one mutation that avoids hairpin formation.

171. The method of claim 168, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs:I-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

172. The method of claim 168, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a mutated al recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto. 04OD7O03.swl 1797spa.doc.69

173. The method of claim 172, wherein said mutated att site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

174. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1- 16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

175. A reaction mixture made by the method of claim 174.

176. A method for apposing an expression signal and a gene or partial gene comprising: mixing a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site; and contacting said mixture with at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene, wherein at least one of said recombination sites comprises at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, or a nucleic acid sequence complementary thereto.

177. A method for apposing an expression signal and a gene or partial gene comprising: mixing a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site; and contacting said mixture with at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene, wherein at least one of said recombination sites comprises a mutation which removes one or more stop codons from said recombination site. 04/07/03.i1 I 1l97pa.doc.O70 -71

178. A method for apposing an expression signal and a gene or partial gene comprising: mixing a first nucleic acid molecule comprising said expression signal and at least a first recombination site, and a second nucleic acid molecule comprising said gene or partial gene and at least a second recombination site; and contacting said mixture with at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites thereby apposing said expression signal and said gene or partial gene, wherein at least one of said recombination sites comprises a mutation which avoids hairpin formation.

179. The method of any one of claims 176-178, wherein said recombination occurs in vivo.

180. The method of any one of claims 176-178, wherein said recombination occurs in vitro.

181. An in vitro method for apposing an expression signal and a gene or partial gene comprising: mixing in vitro a first nucleic acid molecule comprising said expression signal, and at least a first recombination site and at least a second recombination site, vherein said first and second recombination sites do not recombine with each other; and (ii) a second nucleic acid molecule comprising said gene or partial gene, and at least a third recombination site and at least a fourth recombination site, wherein said third and fourth recombination sites do not recombine with each other; and contacting said mixture in vitro with at least one recombination protein under conditions sufficient to cause recombination of said first and third recombination sites and/or said second and fourth recombination sites, thereby apposing said expression signal and said gene or partial gene such that expression of said gene or partial gene can be controlled by said expression signal and such that a single protein is expressed upon expression of said gene or partial gene. 04/07/13,w I 1797spa d:.71 72-

182. An in vitro method for apposing an expression signal and a gene or partial gene comprising: mixing in vitro a first nucleic acid molecule comprising said expression signal, and at least a first recombination site and at least a second recombination site, wherein said first and second recomnbination sites do not recombine with each other; and (ii) a second nucleic acid molecule comprising said gene or partial gene, and at least a third recombination site and at least a fourth recombination site, wherein said third and fourth recombination sites do not recombine with each other; and contacting said mixture in vitro with at least one recombination protein under conditions sufficient to cause recombination of said first and third recombination sites and/or said second and fourth recombination sites, thereby apposing said expression signal and said gene or partial gene.

183. The method of any one of claims 176-178, 181, and 182, wherein said recombination produces a third nucleic acid molecule in which said expression signal and said gene or partial gene are apposed, and further comprisiig introducing said third nucleic acid molecule into a cell.

184. The method of any one of claims 176-178, 181, and 182, wherein said first nucleic acid molecule and/or said second nucleic acid molecule further comprises at least one Selectable marker.

185. The method of claim 184, wherein said at least one Selectable marker is a toxic gene.

186. The method of any one of claims 176-178, 181, and 182, wherein said expression signal is a promoter.

187. The method of claim 186, wherein said promoter is selected from the group consisting of a repressible promoter, an inducible promoter and a constitutive promoter.

0410703..1 I7I97spa.doc.72 73

188. The method of claim 186, wherein said promoter is selected from the group consisting of a CMV promoter and a tac promoter.

189. The method of any one of claims 176-178, 181, and 182, wherein said gene or partial gene comprises a nucleic acid sequence encoding a tag sequence.

190. The method of claim 189, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

191. The method of any one of claims 176-178, 181, and 182, wherein said first nucleic acid molecule and/or said second nucleic acid molecule further comprises at least one additional nucleic acid sequence selected from the group consisting of a cloning site, a restriction site, an operon, and an origin of replication.

192. The method of any one of claims 176-178, 181, and I 82, further comprising expressing said gene or partial gene to produce a protein.

193. The method of claim 192, wherein said protein is a fusion protein.

194. The method of claim 193, wherein said fusion protein comprises a tag sequence.

195. The method of claim 194, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

196. The method of claim 192, wherein said fusion protein comprises an amino terminal tag sequence.

197. The method of claim 192, wherein said fusion protein comprises a carboxy terminal tag sequence.

198. The method of any one of claims 176-178, 181, and 182, wherein said at least one recombination protein is encoded by a bacteriophage selected from the group consisting of bacteriophage lambda, phi80, P22, P2, 186, P4 and PI. 04 l703.,w I 17971p..do.73 -74-

199. The method of any one of claims 176-178, 181, and 182, wherein said at least one recombination protein is encoded by bacteriophage lambda.

200. The method of any one of claims 176-178, 181, and 182, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis, and Cre.

201. The method of any one of claims 176-178, 181, and 182, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, and Xis.

202. The method of any one of claims 176-178, 181, and 182, wherein said at least one recombination protein is selected from the group consisting of., Tn3 resolvase, Hin, Gin, Cin, and Flp.

203. The method of any one of claims 176-178, 181, and 182, wherein said at least one recombination protein is Cre.

204. The method of any one of claims 176-178, 181, and 182, wherein at least one of said recombination sites is selected from the group consisting of att sites and lox sites.

205. The method of any one of claims 176-178, 181, and 182, wherein at least one of said recombination sites is an att site.

206. The method of any one of claims 176-178, 181, and 182, wherein at least one of said recombination sites is a lox site.

207. The method of any one of claims 176-178, 181, and 182, wherein said first nucleic acid molecule is a vector.

208. The method of any one of claims 176-178, 181, and 182, wherein said second nucleic acid molecule is a vector. 0407103.r I 1797spa.doc.74

209. The method of any one of claims 176-178, 181, and 182, wherein said first and second nucleic acid molecules are vectors.

210. An in vitro method for apposing an expression signal and a gene or partial gene, comprising: inserting, by T:A cloning, a DNA segment comprising said gene or partial gene into a first nucleic acid molecule comprising at least a first recombination site, thereby producing a second nucleic acid molecule comprising said gene or partial gene and at least said first recombination site; mixing in vitro said second nucleic acid molecule with a third nucleic acid molecule comprising said expression signal and at least a second recombination site; and incubating said mixture in vitro in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and said second recombination sites, thereby producing a fourth nucleic acid molecule wherein said expression signal and said gene or partial gene have been apposed such that expression of said gene or partial gene can be controlled by said expression signal.

211. The method of claim 210, wherein said at least one recombination protein is selected from the group consisting of lnt, IHF, Xis, and Cre.

212. The method of claim 210, wherein said at least one recombination protein is Cre.

213. The method of claim 210, wherein said first or second recombination sites are selected from the group consisting ofatt sites and lox sites.

214. The method of claim 210, wherein said first or second recombination site is a lox site.

215. The method of claim 210, wherein said second nucleic acid molecule is a vector.

216. The method of claim 210, wherein said third nucleic acid molecule is a vector. 04/107/03.s. I 179"pa.doc.,5 -76-

217. The method of claim 210, wherein said second and third nucleic acid molecules are vectors.

218. The method of claim 210, wherein said expression signal is a promoter.

219. The method of claim 210, wherein said second nucleic acid molecule further comprises at least one additional nucleic acid sequence selected from the group consisting of a Selectable marker, a cloning site, a restriction site, an operon, and an origin of replication.

220. The method of claim 210, wherein said third nucleic acid molecule further comprises at least one additional nucleic acid sequence selected from the group consisting of a Selectable marker, a cloning site, a restriction site, an operon, and an origin of replication.

221. The method of claim 210, further comprising expressing said gene or partial gene to produce a protein.

222. The method of claim 221, wherein said protein is a fusion protein.

223. The method of claim 222, wherein said fusion protein comprises a tag sequence.

224. The method of claim 223, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

225. The method of claim 210, wherein at least one of said recombination sites comprises a mutation which removes one or more stop codons from said recombination site.

226. The method of claim 210, wherein at least one of said recombination sites comprises a mutation which avoids hairpin formation. 04/07I3.swI 1797spa.doc.76 77

227. The method of claim 210, further comprising introducing said fourth nucleic acid molecule into a cell.

228. The method of claim 227, further comprising expressing said gene or partial gene to produce a protein.

229. The method of claim 228, wherein said protein is a fusion protein.

230. The method of claim 229, wherein said fusion protein comprises a tag sequence.

231. The method of claim 230, wherein said tag sequence is selected from the group consisting of a GST tag and a His tag.

232. The method of claim 227, wherein at least one of said recombination sites comprises a mutation which removes one or more stop codons from said recombination site.

233. The method of claim 227, wherein at least one of said recombination sites comprises a mutation which avoids hairpin formation.

234. The method of claim 182, further comprising: contacting one or more hosts with said mixture; and selecting for a host comprising a nucleic acid molecule in which said expression signal and said gene or partial gene have been apposed, and selecting against a host comprising said first nucleic acid molecule and against a host comprising said second nucleic acid molecule.

235. The method of claim 234, wherein said selection is accomplished using at least one Selectable marker.

236. The method of claim 234, wherein said Selectable marker is selected from the group consisting of an antibiotic resistance gene and a toxic gene. 040710).tw I 1797tpadoc.77 78

237. The method of claim 234, wherein said host is E. coll.

238. The method of claim 234, further comprising expressing said gene or partial gene to produce a protein.

239. The method of claim 210, further comprising: contacting one or more hosts with said mixture; and selecting for a host comprising a nucleic acid molecule in which said expression signal and said gene or partial gene have been apposed, and selecting against a host comprising said second nucleic acid molecule and against a host comprising said third nucleic acid molecule.

240. The method of claim 239, wherein said selection is accomplished using at least one Selectable marker.

241. The method of claim 240, wherein said Selectable marker is selected from the group consisting of an antibiotic resistance gene and a toxic gene.

242. A method for in vitro cloning comprising: mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site; and incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs:1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

243. A method for in vitro cloning comprising: mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site; and M04/O71).sl 1797sp..dc.7I 79 incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs:1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

244. A method for in vitro cloning comprising: mixing in vitro a first vector comprising at least a first recombination site and a second vector comprising at least a second recombination site; and incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and second recombination sites, wherein said first and/or second recombination site comprises at least a first nucleic acid sequence selected from the group consisting of a mutated aft recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

245. The method of claim 244, wherein said mutated att site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

246. The method of claim 244, wherein said mutated aft site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

247. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at Least one mutation, wherein said mutation removes one or more stop codons from said recombination site.

248. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising 04107/0,lw I 1797tp* dc.79 at least a first recombination site containing at least one mutation, wherein said mutation avoids hairpin formation.

249. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs: 1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

250. A method of making a reaction mixture, comprising mixinrg at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs: 1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

251. A method of making a reaction mixture, comprising mixing at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first mutated aft recombination site containing at least one mutation that enhances recombination specificity.

252. The method of claim 251, wherein said mutated at site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

253. The method of claim 251, wherein said mutated aft site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

254. The method of claim 251, wherein said mutated at site comprises at least one nucleic acid sequence that hybridizes under stringent conditions to at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

255. A reaction mixture made by the method of any one of claims 247-251.

256. A method for in vitro cloning of a nucleic acid molecule, comprising: 04103.OJ. I 17gflpa doc.lo -81- mixing in vitro a first vector comprising at least a first and second recombination sites, and a second vector comprising at least a third and fourth recombination sites, wherein said first and/or second vector further comprises a nucleic acid molecule to be cloned, and wherein said first and second recombination sites do not recombine with each other and said third and fourth recombination sites do not recombine with each other; incubating said mixture in the presence of at least one recombination protein under conditions sufficient to cause recombination of at least said first and third and/or said second and fourth recombination sites, thereby producing a product molecule comprising said nucleic acid molecule; contacting one or more hosts with said mixture; and selecting for a host comprising said product molecule, and against a host comprising said first vector and against a host comprising said second vector.

257. The method of claim 256, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and Cre.

258. The method of claim 256, wherein said at least one recombination protein is selected from the group consisting of Int, IIF and Xis.

259. The method of claim 256, wherein said at least one recombination protein is selected from the group consisting of, Tn3, Tn7, resolvase, Hin, Gin, Gin and iFlp.

260. The method of claim 256, wherein said at least one recombination protein is Cre.

261. The method of claim 256, wherein said first, second, third and/or fourth recombination sites are selected from the group consisting of att and lox P sites.

262. The method of claim 256, wherein said first, second, third and/or fourth recombination sites are att sites.

263. The method of claim 256, wherein said first, second, third and/or fourth recombination sites are lox P sites. 4lO/).3,w I 1797spdoC.ll I 82-

264. The method of claim 256, wherein said selection is accomplished using at least one Selectable marker.

265. The method of claim 264, wherein said Selectable marker is selected from the group consisting of an antibiotic resistance gene and a toxic gene.

266. The method of claim 256, wherein said host is E. coli.

267. An in vitro method of cloning a PCR product comprising: obtaining a PCR product comprising at least a first recombination site and at least a second recombination site which do not recombine with each other; and combining said PCR product in vitro with a vector comprising at least a third recombination site and at least a fourth recombination site which do not recombine with each other, under conditions such that recombination occurs between said first and third and/or said second and fourth recombination sites, thereby producing a product vector.

268. The method of claim 267, further comprising inserting said product vector into a host cell.

269. The method of claim 267, wherein said vector is an expression vector.

270. The method of claim 267, wherein said vector comprises at least one additional nucleic acid sequence selected from the group consisting of a selectable marker, a cloning site, a restriction site, a promoter, an operon, an origin of replication, and a gene or partial gene.

271. The method of claim 267, wherein said vector comprises at least one origin of replication.

272. The method of claim 267, wherein said vector comprises at least one promoter. 04/07/03.,w I 1l797spa.doc. 2 83

273. The method of claim 267, wherein said vector comprises at least one selectable marker.

274. The method of claim 267, wherein said PCR product is linear.

275. The method of claim 267, wherein said first, second, third or fourth recombination sites are lox sites or mutants thereof.

276. The method of claim 275, wherein said lox sites are selected from the group consisting of loxP sites and loxP51 I sites.

277. The method of claim 267, wherein said first, second, third or fourth recombination sites are att sites or mutants thereof.

278. The method of claim 277, wherein said aft sites are selected from the group consisting of atB sites, attP sites, attL sites and atR sites.

279. The method of claim 267, wherein said first, second, third or fourth recombination sites are selected from the group consisting of a lox site, an art site, an FRT site, and mutants thereof

280. The method of claim 267, wherein said product nucleic acid molecule and said vector are combined in the presence of at least one recombination protein.

281. The method ofclaim 280, wherein said recombination protein is Cre.

282. The method of claim 280, wherein said recombination protein is selected from the group consisting of Int, Xis and IHF.

283. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising at least one portion of a first gene and at least a first recombination site; providing a second nucleic acid molecule comprising at least one portion of a second gene and at least a second recombination site; and 04?O 0. lw t 797spa,do,.I3 -84- forming a mixture between said first and second nucleic acid molecules and at least one recombination protein, under conditions sufficient to cause recombination between said first and second recombination sites, thereby producing a third nucleic acid molecule in which said portions of said first and second genes are operably linked to form a functional gene.

284. The method of claim 283, wherein said first gene and said second gene are the same.

285. The method of claim 283, wherein said first gene or said second gene encodes a selectable marker.

286. The method of claim 283, wherein said first gene or said second gene is an antibiotic resistance gene.._

287. The method of claim 286, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

288. The method of claim 286, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

289. The method of claim 283, wherein said at least one portion of said first gene or said at least one portion of said second gene comprises a promoter.

290. The method of claim 283, wherein said at least one portion of said first gene and said at least one portion of said second gene are fragments of one or more structural genes.

291. The method of claim 283, wherein said first gene or said second gene encodes a heterodimeric gene product. O4/O7/0,sw I 1797,pA.doc.I4

292. The method of claim 283, wherein said first and second recombination sites are selected from the group consisting oflox sites, att sites, and mutants thereof.

293. The method of claim 283, wherein said first and second recombination sites are selected from the group consisting oflox sites and att sites.

294. The method of claim 283, wherein said first and second recombination sites are lox sites.

295. The method of claim 294, wherein said lox sites are loxP sites.

296. The method of claim 283, wherein said first and second recombination sites are att sites.

297. The method of claim 296, wherein said art sites are selected from the group consisting ofattB sites, attP sites, attL sites and attR sites.

298. The method of claim 283, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site.

299. The method of claim 298, wherein said at least one additional recombination site is selected from the group consisting of lox sites and att sites.

300. The method of claim 298, wherein said at least one additional recombination site is at least one lox site or a mutant thereof.

301. The method of claim 298, wherein said at least one additional recombination site is a lox site.

302. The method of claim 301, wherein said lox site is a loxP site.

303. The method of claim 298, wherein said at least one additional recombination site is at least one att site or a mutant thereof. 0410703.tw1 I1797.pl.do.l S -86-

304. The method of claim 298, wherein said at least one additional recombination site is an at site.

305. The method of claim 304, wherein said ant site is selected from the group consisting of an attB site, an attP site, an attL site and an attR site.

306. The method of claim 283, wherein said at least one portion of said first gene is located adjacent to said first recombination site.

307. The method of claim 283, wherein said at least one portion of said second gene is located adjacent to said second recombination site.

308. The method of claim 283, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises at least one cloning site.

309. The method of claim 283, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y5, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

310. The method of claim 283, wherein said at least one recombination protein is Cre.

311. The method of claim 283, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

312. The method of claim 283, wherein said at least one recombination protein is Int.

313. The method of claim 283, wherein said at least one recombination protein is IHF.

314. The method of claim 283, wherein said at least one recombination protein is Xis. 07/03..w i 1797 spa.doc,6 87

315. The method of claim 283, wherein said first nucleic acid molecule or said second nucleic acid molecule or said third nucleic acid molecule is a vector.

316. The method of claim 315, wherein said vector is an expression vector.

317. The method of claim 283, wherein said first nucleic acid molecule or said second nucleic acid molecule is linear.

318. The method of claim 283, wherein said at least one portion of said first gene or of said second gene is a PCR product.

319. The method of claim 283, further comprising expressing said functional gene.

320. The method of claim 283, further comprising contacting at least one host cell with said mixture, and selecting for a host cell comprising said third nucleic acid molecule.

321. The method of claim 320, further comprising selecting against a host cell comprising said first or said second nucleic acid molecule.

322. The method of claim 320, further comprising selecting against a host cell comprising said first and said second nucleic acid molecules.

323. The method of claim 320, further comprising expressing said functional gene in said selected host cell.

324. The method of claim 320, wherein said host cell is a prokaryotic cell.

325. The method of claim 320, wherein said host cell is a bacterial cell.

326. The method of claim 320, wherein said host cell is an Escherichia coli cell.

327. A method of producing a nucleic acid molecule comprising: 0407/03.sw I 1797sp.,doc.I 7 88 providing a first nucleic acid molecule comprising a first portion of an antibiotic resistance gene and at least a first recombination site; providing a second nucleic acid molecule comprising a second portion of said antibiotic resistance gene and at least a second recombination site; and forming a mixture between said first and second nucleic acid molecules and at least one recombination protein, under conditions sufficient to cause recombination between said first and second recombination sites, thereby producing a third nucleic acid molecule in which said first and second portions of said gene are operably linked to form a functional antibiotic resistance gene.

328. The method of claim 327, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an. ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

329. The method of claim 327, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

330. The method of claim 327, wherein said first or second portion of said gene comprises a promoter.

331. The method of claim 327, wherein said first and second recombination sites are selected from the group consisting of lox sites, att sites, and mutants thereof.

332. The method of claim 327, wherein said first and second recombination sites are selected from the group consisting of lox sites and att sites.

333. The method of claim 327, wherein said first and second recombination sites are lox sites.

334. The method of claim 333, wherein said lox sites are loxP sites.

335. The method of claim 327, wherein said first and second recombination sites are att sites. O4/7/O J s II 797sp.docS& -89-

336. The method of claim 335, wherein said aft sites are selected from the group consisting ofattB sites, attP sites, attL sites and attR sites.

337. The method of claim 327, wherein said first nucleic acid molecule or said second nucleic acid molecule furthercomprises at least one additional recombination site.

338. The method of claim 337, wherein said at least one additional recombination site is selected from the group consisting of lox sites and att sites.

339. The method of claim 337, wherein said at least one additional recombination site is at least one lox site or a mutant thereof.

340. The method of claim 337, wherein said at least one additional recombination site is a lox site.

341. The method of claim 340, wherein said lox site is a loxP site.

342. The method of claim 337, wherein said at least one additional recombination site is at least one att site or a mutant thereof.

343. The method of claim 337, wherein said at least one additional recombination site is an att site.

344. The method of claim 343, wherein said att site is selected from the group consisting of an attB site, an attP site, an attL site and an attR site.

345. The method of claim 327, wherein said first portion of said gene is located adjacent to said first recombination site.

346. The method of claim 327, wherein said second portion of said gene is located adjacent to said second recombination site. o04I 3.sw I 1797lsp.doc.9 90

347. The method of claim 327, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises at least one cloning site.

348. The method of claim 327, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y5, Tn3 resolvase, Hin, Gin, Cin and combinations thereof

349. The method of claim 327, wherein said at least one recombination protein is Cre.

350. The method of claim 327, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

351. The method of claim 327, wherein said first nucleic acid molecule or said second nucleic acid molecule or said third nucleic acid molecule is a vector.

352. The method of claim 351, wherein said vector is an expression vector.

353. The method of claim 327, wherein said first nucleic acid molecule or said second nucleic acid molecule is linear.

354. The method of claim 327, wherein said first or said second portions of said gene are PCR products.

355. The method of claim 327, further comprising contacting at least one host cell with said mixture, and selecting for a host cell comprising said third nucleic acid molecule.

356. The method of claim 355, further comprising selecting against a host cell comprising said first or said second nucleic acid molecule.

357. The method of claim 355, further comprising selecting against a host cell comprising said first and said second nucleic acid molecule. 04/07103.,w I 1797sp.doc.90 -91

358. The method of claim 355, wherein said host cell is a prokaryotic cell.

359. The method of claim 355, wherein said host cell is a bacterial cell.

360. The method of claim 355, wherein said host cell is an Escherichia coli cell.

361. The method of claim 327, further comprising introducing said third nucleic acid molecule into a host cell.

362. The method of claim 327, further comprising introducing said third nucleic acid molecule into a host cell and expressing said antibiotic resistance gene.

363. The method of claim 362, wherein said host cell is an Escherichia coli cell.

364. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising at least one promoter and at least a first recombination site; providing a second nucleic acid molecule comprising at least one antibiotic resistance gene and at least a second recombination site; and forming a mixture between said first and second nucleic acid molecules and at least one recombination protein, under conditions sufficient to cause recombination between said first and second recombination sites, thereby producing a third nucleic acid molecule in which said promoter and said antibiotic resistance gene are operably linked.

365. The method of claim 364, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

366. The method of claim 364, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

367. The method of claim 364, wherein said first and second recombination sites are selected from the group consisting of lox sites, att sites, and mutants thereof. 04l07103.SwI 1797 spa.ds. .91 -92-

368. The method of claim 364, wherein said first and second recombination sites are selected from the group consisting of lox sites and att sites.

369. The method of claim 364, wherein said first and second recombination sites are lox sites.

370. The method of claim 369, wherein said lox sites are loxP sites.

371. The method of claim 364, wherein said first and second recombination sites are att sites.

372. The method of claim 371, wherein said att sites are selected from the group consisting of atnB sites, attP sites, attL sites and attR sites.

373. The method of claim 364, wherein said first nucleicacid molecule or said second nucleic acid molecule further comprises at least one additional recomnbination site.

374. The method of claim 373, wherein said at least one additional recombination site is selected from the group consisting oflox sites and att sites.

375. The method of claim 373, wherein said at least one additional recombination site is at least one lox site or a mutant thereof.

376. The method of claim 373, wherein said at least one additional recombination site is a lox site.

377. The method of claim 376, wherein said lox site is a loxP site.

378. The method of claim 373, wherein said at least one additional recombination site is at least one atll site or a mutant thereof.

379. The method of claim 373, wherein said at least one additional recombination site is an attll site. O4tO7/03.w I 1797spa.d..92 -93

380. The method of claim 379, wherein said att site is selected from the group consisting of an attB site, an attP site, an attL site and an attR site.

381. The method of claim 364, wherein said promoter is located adjacent to said first recombination site.

382. The method of claim 364, wherein said antibiotic resistance gene is located adjacent to said second recombination site.

383. The method of claim 364, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises at least one cloning-site.

384. The method of claim 364, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, 6 Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

385. The method of claim 364, wherein said at least one recombination protein is Cre.

386. The method of claim 364, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

387. The method of claim 364, wherein said first nucleic acid molecule or said second nucleic acid molecule or said third nucleic acid molecule is a vector.

388. The method of claim 387, wherein said vector is an expression vector.

389. The method of claim 364, wherein said first nucleic acid molecule or said second nucleic acid molecule is linear.

390. The method of claim 364, wherein said first or said second portions of said gene are PCR products. I04K07/1. wI 1797spa.do.93 -94-

391. The method of claim 364, further comprising contacting at least one host cell with said mixture, and selecting for a host cell comprising said third nucleic acid molecule.

392. The method of claim 391, further comprising selecting against a host cell comprising said first or said second nucleic acid molecule.

393. The method of claim 391, further comprising selecting against a host cell comprising said first and said second nucleic acid molecule. I0

394. The method of claim 391, wherein said host cell is a prokaryotic cell.

395. The method of claim 391, wherein said host cell is a bacterial cell.

396. The method of claim 391, wherein said host cell is an Escherichia coli cell.

397. The method of claim 364, further comprising introducing said third nucleic acid molecule into a host cell.

398. The method of claim 364, further comprising introducing said nucleic acid molecule into a host cell and expressing said antibiotic resistance gene.

399. The method of claim 398, wherein said host cell is an Escherichia coli cell.

400. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising at least one promoter and at least a first loxP site; providing a second nucleic acid molecule comprising at least one antibiotic resistance gene and at least a second loxP site; and forming a mixture between said first and second nucleic acid molecules and at least one Cre recombination protein, under conditions sufficient to cause recombination between said first and second loxP sites, thereby producing a third nucleic acid molecule in which said promoter and said antibiotic resistance gene are operably linked. O407103,w I1797spt.doc,94 95

401. The method of claim 400, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

402. The method of 400, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

403. The method of claim 400, further comprising iitroducing said third nucleic acid molecule into a host cell.

404. The method of claim 400, further comprising introducing said third nucleic acid molecule into a host cell and expressing said antibiotic resistance gene.

405. The method according to any one of claims 403 or 404, wherein said host cell is an Escherichia coli cell.

406. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising at least one portion of a first gene and at least a first recombination site; providing a second nucleic acid molecule comprising at least one portion of a second gene and at least a second recombination site; and forming a mixture in vitro between said first and second nucleic acid molecules and at least one recombination protein, under conditions sufficient to cause recombination in vitro between said first and second recombination sites, thereby producing a third nucleic acid molecule in which said portions of said first and second genes are operably linked to form a functional gene.

407. The method of claim 406, wherein said first gene and said second gene are the same.

408. The method of claim 406, wherein said first gene or said second gene encodes a selectable marker. 04107/03.sw I 17971pm.doc.95 -96-

409. The method of claim 406, wherein said first gene or said second gene is an antibiotic resistance gene.

410. The method of claim 409, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

411. The method of claim 409, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

412. The method of claim 406, wherein said at least one portion of said first gene or of said second gene comprises a promoter.

413. The method of claim 406, wherein said at least one portion of said first gene or of said second gene is a fragment of a structural gene.

414. The method of claim 406, wherein said first gene or said second gene encodes a heterodimeric gene product.

415. The method of claim 406, wherein said first and second recombination sites are selected from the group consisting of lox sites, att sites, and mutants thereof.

416. The method of claim 406, wherein said first and second recombination sites are selected from the group consisting oflox sites and att sites.

417. The method of claim 406, wherein said first and second recombination sites are lox sites.

418. The method of claim 417, wherein said lox sites are loxP sites.

419. The method of claim 406, wherein said first and second recombination sites are att sites. 047/03.lw I 1797lpp DC.96 -97-

420. The method of claim 419, wherein said all sites are selected from the group consisting of attB sites, atP sites, autL sites and attR sites.

421. The method of claim 406, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site.

422. The method of claim 421, wherein said at least one additional recombination site is selected from the group consisting of lox sites and Ott sites.

423. The method of claim 421, wherein said at least one additional recombination site is at least one lox site or a mutant thereof.

424. The method of claim 421, wherein said at least one additional recombination site is a lox site.

425. The method of claim 424, wherein said lox site is a loxP site.

426. The method of claim 421, wherein said at least one additional recombination site is at least one att site or a mutant thereof.

427. The method of claim 421, wherein said at least one additional recombination site is an att site.

428. The method of claim 427, wherein said alt site is selected from the group consisting of an autB site, an atrP site, an attL site and an attR site.

429. The method of claim 406, wherein said at least one portion of said first gene is located adjacent to said first recombination site.

430. The method of claim 406, wherein said at least one portion of said second gene is located adjacent to said second recombination site.

431. The method of claim 406, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises at least one cloning site. 041/07/ 1.sw I 1797spa.dtc.97 -98-

432. The method of claim 406, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP,_, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

433. The method of claim 406, wherein said at least one recombination protein is Cre.

434. The method of claim 406, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

435. The method of claim 406, wherein said at least one recombination protein is

436. The method of claim 406, wherein said at least one recombination protein is IHF.

437. The method of claim 406, wherein said at least one recombination protein is Xis.

438. The method of claim 406, wherein said first nucleic acid molecule or said second nucleic acid molecule or said third nucleic acid molecule is a vector.

439. The method of claim 438, wherein said vector is an expression vector.

440. The method of claim 406, wherein said first nucleic acid molecule or said second nucleic acid molecule is linear.

441. The method of claim 406, wherein said at least one portion of said first gene or of said second gene is a PCR product.

442. The method of claim 406, further comprising expressing said functional gene. 04107/03,wIl 1 797sps.do.95 -99-

443. The method of claim 406, further comprising contacting at least one. host cell with said mixture, and selecting for a host cell comprising said third nucleic acid molecule.

444. The method of claim 443, further comprising selecting against a host cell comprising said first or said second'nucleic acid molecule.

445. The method of claim 443, further comprising selecting against a host cell comprising said first and said second nucleic acid molecules.

446. The method of claim 443, further comprising expressing said functional gene in said selected host cell.

447. The method of claim 443, wherein said host cell is a prokaryotic cell.

448. The method of claim 443, wherein said host cell is a bacterial cell.

449. The method of claim 443, wherein said host cell is an Escherichia coli cell.

450. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising a first portion of an antibiotic resistance gene and at least a first recombination site; providing a second nucleic acid molecule comprising a second portion of said antibiotic resistance gene and at least a second recombination site; and forming a mixture in vitro between said first and second nucleic acid molecules and at least one recombination protein, under conditions sufficient to cause recombination in vitro between said first and second recombination sites, thereby producing a third nucleic acid molecule in which said first and second portions of said gene are operably linked to form a functional antibiotic resistance gene.

451. The method of claim 450, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicoI resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene. Q4/O7O3.lswI I797pa doc99 100-

452. The method of claim 450, wherein said antibiotic resistance gene is a chloramphenicoI resistance gene. s

453. The method of claim 450, wherein said first or second portion of said gene comprises a promoter.

454. The method of claim 450, wherein said 'first and second recombination sites are selected from the group consisting of lox sites, att sites, and mutants thereof.

455. The method of claim 450, wherein said first and second recombination sites are selected from the group consisting of lox sites and att sites.

456. The method of claim 450, wherein said first and second recombination sites are lox sites.

457. The method of claim 456, wherein said lox sites are loxP sites.

458. The method of claim 450, wherein said first and second recombination sites are att sites.

459. The method of claim 458, wherein said att sites are selected from the group consisting ofattB sites, attP sites, attL sites and attR sites.

460. The method of claim 450, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site.

461. The method of claim 460, wherein said at least one additional recombination site is selected from the group consisting of lox sites and att sites.

462. The method of claim 460, wherein said at least one additional recombination site is at least one lox site or a mutant thereof. 04l7/O3,sw I 1797spa.doc.I O0 101

463. The method of claim 460, wherein said at least one additional recombination site is a lox site.

464. The method of claim 463, wherein said lox site is a loxP site.

465. The method of claim 460, wherein said at least one additional recombination site is at least one att site or a mutant thereof.

466. The method of claim 460, wherein said at least one additional recombination site is an aft site.

467. The method of claim 466, wherein said att site is selected from the group consisting of an attB site, an attP site, an attL site and an attR site.

468. The method of claim 450, wherein said first portion of said gene is located adjacent to said first recombination site.

469. The method of claim 450, wherein said second portion of said gene is located adjacent to said second recombination site.

470. The method of claim 450, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises at least one cloning site.

471. The method of claim 450, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

472. The method of claim 450, wherein said at least one recombination protein is Cre.

473. The method of claim 450, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis. 04/07/03,wI 1797spa.doc.l01 102-

474. The method of claim 450, wherein said first nucleic acid molecule or said second nucleic acid molecule or said third nucleic acid molecule is a vector.

475. The method of claim 474, wherein said vector is an expression vector.

476. The method of claim 450, wherein said first nucleic acid molecule or said second nucleic acid molecule is linear.

477. The method of claim 450, wherein said first or said second portions of said gene are PCR products.

478. The method of claim 450, further comprising contacting at least one host cell with said mixture, and selecting for a host cell comprising said third nucleic acid molecule.

479. The method of claim 478, further comprising selecting against a host cell comprising said first or said second nucleic acid molecule.

480. The method of claim 478, further comprising selecting against a host cell comprising said first and said second nucleic acid molecule.

481. The method of claim 478, wherein said host cell is a prokaryotic cell.

482. The method of claim 478, wherein said host cell is a bacterial cell.

483. The method of claim 478, wherein said host cell is an Escherichia coli cell.

484. The method of claim 450, further comprising introducing said third nucleic acid molecule into a host cell.

485. The method of claim 450, further comprising introducing said third nucleic acid molecule into a host cell and expressing said antibiotic resistance gene.

486. The method of claim 485, wherein said host cell is an Escherichia coli cell. 04/O)3.swI I17Ql 7p..dac.102 103

487. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising at least one promoter and at least a first recombination site; providing a second nucleic acid molecule comprising at least one antibiotic resistance gene and at least a second recombination site; and forming a mixture in vitro between said first and second nucleic acid molecules and at least one recombination protein, under conditions sufficient to cause recombination in vitro between said first and second recombination sites, thereby producing a third nucleic acid molecule in which said promoter and said antibiotic resistance gene are operably linked.

488. The method of claim 487, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

489. The method of claim 487, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

490. The method of claim 487, wherein said first and second recombination sites are selected from the group consisting of lox sites, att sites, and mutants thereof.

491. The method of claim 487, wherein said first andsecond recombination sites are selected from the group consisting of lox sites and at sites.

492. The method of claim 487, wherein said first and second recombination sites are lox sites.

493. The method of claim 492, wherein said lox sites are loxP sites.

494. The method of claim 487, wherein said first and second recombination sites are att sites. 04071/31.wI 797spa.doc, 103 104-

495. The method of claim 494, wherein said att sites are selected from the group consisting of attB sites, attP sites, attL sites and attR sites.

496. The method of claim 487, wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises at least one additional recombination site.

497. The method of claim 496, wherein said at least one additional recombination site is selected from the group consisting of lox sites and att sites.

498. The method of claim 496, wherein said at least one additional recombination site is at least one lox site or a mutant thereof.

499. The method of claim 496, wherein said at least one additional recombination site is a lox site.

500. The method of claim 499, wherein said lox site is a loxP site.

501. The method of claim 496, wherein said at least one additional recombination site is at least one att site or a mutant thereof.

502. The method of claim 496, wherein said at least one additional recombination site is an aft site.

503. The method of claim 502, wherein said aft site is selected from the group consisting of an attB site, an attP site, an attL site and an attR site.

504. The method of claim 487, wherein said promoter is located adjacent to said first recombination site.

505. The method of claim 487, wherein said antibiotic resistance gene is located adjacent to said second recombination site.

506. The method of claim 487, wherein said first nucleic acid molecule or said second nucleic acid molecule comprises at least one cloning site. 0407t03.lw I 1797pt.doc.10 4 105-

507. The method of claim 487, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP,_, Tn3 resolvase, Hin, Gin, Cin and combinations thereof.

508. The method of claim 487, wherein said at least one recombination protein is Cre.

509. The method of claim 487, wherein said at least one recombination protein is selected from the group consisting of Int, IHF and Xis.

510. The method of claim 487, wherein said first nucleic acid molecule or said second nucleic acid molecule or said third nucleic acid molecule is a vector.

511. The method of claim 510, wherein said vector is an expression vector.

512. The method of claim 487, wherein said first nucleic acid molecule or said second nucleic acid molecule is linear.

513. The method of claim 487, wherein said first or said second portions of said gene are PCR products.

514. The method of claim 487, further comprising contacting at least one host cell with said mixture, and selecting for a host cell comprising said third nucleic acid molecule.

515. The method of claim 514, further comprising selecting against a host cell comprising said first or said second nucleic acid molecule.

516. The method of claim 514, further comprising selecting against a host cell comprising said first and said second nucleic acid molecule.

517. The method of claim 514, wherein said host cell is a prokaryotic cell.

04107103.sw I 1797sp.doc.105 106-

518. The method of claim 514, wherein said host cell is a bacterial cell.

519. The method of claim 514, wherein said host cell is an Escherichia coli cell.

520. The method of claim 487, further comprising introducing said third nucleic acid molecule into a host cell.

521. The method of claim 487, further comprising introducing said nucleic acid molecule into a host cell and expressing said antibiotic resistance gene.

522. The method of claim 521, wherein said host cell is an Escherichia coli cell.

523. A method of producing a nucleic acid molecule comprising: providing a first nucleic acid molecule comprising at least one promoter and at least a first loxP site; providing a second nucleic acid molecule comprising at least one antibiotic resistance gene and at least a second loxP site; and forming a mixture in vitro between said first and second nucleic acid molecules and at least one Cre recombination protein, under conditions sufficient to cause recombination in vitro between said first and second loxP sites, thereby producing a third nucleic acid molecule in which said promoter and said antibiotic resistance gene are operably linked.

524. The method of claim 523, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

525. The method of claim 523, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

526. The method of claim 523, further comprising introducing said third nucleic acid molecule into a host cell. 04/Ol03.wl I 17971pa.doc.106 -107-

527. The method of claim 523, further comprising introducing said third nucleic acid molecule into a host cell and expressing said antibiotic resistance gene.

528. The method of claim 527, wherein said host cell is an Escherichia coli cell.

529. The method of claim 528, wherein said host cell is an Escherichia coli cell.

530. A kit for in vitro cloning of nucleic acid segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that is 80-99% homologous to one or more of SEQ ID NOs:1-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

531. A kit for in vitro cloning of nucleic acid segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a nucleic acid sequence that hybridizes under stringent conditions to one or more of SEQ ID NOs:l-16, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

532. A kit for in vitro cloning of DNA segments comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule comprising at least a first recombination site containing at least one nucleic acid sequence selected from the group consisting of a mutated att recombination site containing at least one mutation that enhances recombinational specificity, a complementary DNA sequence thereto, and an RNA sequence corresponding thereto.

533. The kit according to any one of claims 530-532, wherein the nucleic acid molecule comprises at least a first and a second recombination site.

534. The kit of claim 532, wherein said mutated att site comprises at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16. 0407/JM.swl I 791pAdoc. 107 108-

535. The kit of claim 532, wherein said mutated att site comprises at least one nucleic acid sequence that is 80-99% homologous to at least one nucleic acid sequence as set forth in SEQ ID NOs.: 1-16.

536. The kit according to any one of claims 530-533, wherein said recombination proteins are selected from the group consisting of y6, Tn3 resolvase, Hin, Gin, Cin, and Flp.

537. The kit according to any one of claims 530-533, wherein said recombination proteins are selected from the group consisting of Int, IHF, Xis and Cre.

538. The kit according to any one of claims 530-533, wherein said recombination proteins are selected from the group consisting of Int, IHF and Xis.

539. The kit according to any one of claims 530-533, wherein at least one of said recombination proteins is Int.

540. The kit according to any one of claims 530-533, wherein at least one of said recombination proteins is encoded by an organism selected from the group consisting of bacteriophage lambda, phi 80, P22, P2, 186, P4 and P 1.

541. The kit according to any one of claims 530-533, wherein at least one of said recombination proteins is encoded by bacteriophage lambda.

542. The kit according to any one of claims 530-533, wherein at least one of said recombination proteins is encoded by Bacillus thuringiensis.

543. The kit according to any one of claims 530-533, wherein said kit comprises Int and IHF.

544. The kit of claim 543, wherein said kit further comprises Xis.

545. The kit of any one of claims 530-533, wherein said kit comprises at least two recombination proteins that are different from each other. 0407/03.lw I 1797sps.doc.10 109-

546. The kit of any one of claims 530-533, wherein said kit comprises at least three recombination proteins that are different from each other.

547. The kit according to claim 533, wherein said first and second recombination sites have been engineered to enhance recombination efficiency.

548. A kit comprising at least one nucleic acid molecule, wherein said nucleic acid molecule comprises at least a first lox site flanked by at least one promoter and at least one antibiotic resistance gene.

549. The kit of claim 548, wherein said lox site is a loxP site.

550. The kit of claim 548, wherein said lox site is a loxP site and wherein said promoter and said antibiotic resistance gene are operably linked.

551. The kit of claim 548, wherein said nucleic acid molecule is a vector.

552. The kit of claim 548, wherein said kit further comprises one or more components selected from the group consisting of at least one recombination protein and at least one host cell.

553. The kit of claim 552, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, yS, TN3 resolvase, Hin, Gin, Cin and combinations thereof.

554. The kit of claim 552, wherein said at least one recombination protein is Cre.

555. The kit of claim 552, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and combinations thereof.

556. The kit of claim 552, wherein said host cell is an Escherichia coli cell. 0407/103.lwl 17971p.doac.109 -110-

557. A kit comprising at least one nucleic acid molecule, wherein said nucleic acid molecule comprises at least one promoter operably linked to at least one antibiotic resistance gene, wherein said promoter and said antibiotic resistance gene are separated by at least one recombination site.

558. The kit of claim 557, wherein said first recombination site is selected from the group consisting of a lox site, an antt site, and mutants thereof.

559. The kit of claim 557. wherein said first recombination site is a lox site:'

560. The kit of claim 559, wherein said lox'site is a loxP site.

561. The kit of claim 557, wherein said nucleic acid mblecule further comprises at least one additional recombination site.

562. The kit of claim 561, wherein said at least one additional recombination site is selected from the group consisting of lox sites and att sites.

563. The kit of claim 561, wherein said at least one additional recombination site is a lox site.

564. The kit of claim 563, wherein said lox site is a loxP site.

565. The kit of claim 557, wherein said nucleic acid molecule comprises at least one cloning site.

566. The kit of claim 557, wherein said nucleic acid molecule is a vector.

567. The kit of claim 566, wherein said vector is an expression vector.

568. The kit of claim 557, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene. 0407103/Ol.w I 1797sp..dc.I -I11-

569. The kit of claim 557, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

570. The kit of claim 557, wherein said kit further comprises one or more components selected from the group consisting of at least one recombination protein and at least one host cell.

571. The kit of claim 570, wherein said at least one recombination protein is selected from the group consisting of Cre, int, IHF, Xis, FLP, yS, TN3 resolvase, Hin, Gin, Cin and combinations thereof.

572. The kit of claim 570, wherein said at least one recombination protein is Cre.

573. The kit of claim 570, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and combinations thereof.

574. The kit of claim 570, wherein said host cell is an Escherichia coli cell.

575. A kit comprising at least one nucleic acid molecule, wherein said nucleic acid molecule comprises a functional antibiotic resistance gene, wherein a first portion of said antibiotic resistance gene and a second portion of said antibiotic resistance gene are separated by at least a first recombination site.

576. The kit of claim 575, wherein said first and second portions of said antibiotic resistance gene are operably linked.

577. The kit of claim 575, wherein said first portion of said antibiotic resistance gene is a promoter.

578. The kit of claim 575, wherein said first recombination site is selected from the group consisting of a lox site, an att site, and mutants thereof.

579. The kit of claim 575, wherein said first recombination site is a lox site. 041071,sw l 17971padoc.l I 112-

580. The kit of claim 579, wherein said lox site is a loxP site.

581. The kit of claim 575, wherein said nucleic acid molecule further comprises at least one additional recombination site.

582. The kit of claim 581, wherein said at least one additional recombination site is selected from the group consisting of lox sites and att sites.

583. The kit of claim 581, wherein said at least one additional recombination site is a lox site.

584. The kit of claim 583, wherein said lox site is a loxP site.

585. The kit of claim 575, wherein said nucleic acid molecule comprises at least one cloning site.

586. The kit of claim 575, wherein said nucleic acid molecule is a vector.

587. The kit of claim 586, wherein said vector is an expression vector.

588. The kit of claim 575, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

589. The kit of claim 575, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

590. The kit of claim 575, wherein said first portion of said gene is located adjacent to said recombination site.

591. The kit of claim 575, wherein said second portion of said gene is located adjacent to said recombination site. 04/07/03,swI 1797spa.doc. 112 113-

592. The kit of claim 575, wherein said kit further comprises one or more components selected from the group consisting of at least one recombination protein and at least one host cell.

593. The kit of claim 592, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y6, TN3 resolvase, Hin, Gin, Cin and combinations thereof.

594. The kit of claim 592, wherein said at least one recombination protein is Cre.

595. The kit of claim 592, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and combinations thereof.

596. The kit of claim 592, wherein said host cell is an Escherichia coli cell.

597. A kit comprising at least one nucleic acid molecule, wherein said nucleic acid molecule comprises at least one promoter operably linked to at least one antibiotic resistance gene, wherein said promoter and said antibiotic resistance gene are separated by at least one loxP site.

598. The kit of claim 597, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

599. The kit of claim 597, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

600. The kit of claim 597, wherein said kit further comprises one or more components selected from the group consisting of at least one recombination protein and at least one host cell. 04'07,/0,s*l 1797 pa.doc,I I -114-

601. The kit of claim 600, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y5, TN3 resolvase, Hin, Gin, Cin and combinations thereof.

602. The kit of claim 600, wherein said at least one recombination protein is Cre.

603. The kit of claim 600, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and combinations thereof.

604. The kit of claim 600, wherein said host cell is an Escherichia coli cell.

605. A kit comprising at least one nucleic acid molecule, wherein said nucleic acid molecule comprises at least one functional antibiotic resistance gene, wherein said functional gene comprises a promoter and an antibiotic resistance gene separated from each other by at least one loxP site.

606. The kit of claim 605, wherein said antibiotic resistance gene is selected from the group consisting of a chloramphenicol resistance gene, an ampicillin resistance gene, a methicillin resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

607. The kit of claim 605, wherein said antibiotic resistance gene is a chloramphenicol resistance gene.

608. The kit of claim 605, wherein said kit further comprises one or more components selected from the group consisting of at least one recombination protein and at least one host cell.

609. The kit of claim 608, wherein said at least one recombination protein is selected from the group consisting of Cre, Int, IHF, Xis, FLP, y7, TN3 resolvase, Hin, Gin, Cin and combinations thereof.

610. The kit of claim 608, wherein said at least one recombination protein is Cre. 04/07/3.sw l 1797pa.doc I 14 -115-

611. The kit of claim 608, wherein said at least one recombination protein is selected from the group consisting of Int, IHF, Xis and combinations thereof

612. The kit of claim 608, wherein said host cell is an Escherichia coli cell.

613. The kit according to any one of claims 552, 570, 592, or 600, wherein said nucleic acid molecule is a vector.

614. A kit comprising at least one isolated recombination protein and at least one isolated nucleic acid molecule, said nucleic acid molecule comprising at least a first att recombination site which comprises a core region having at least one mutation that enhances recombination efficiency or specificity in vitro in-the formation of a cointegrate DNA or a product DNA molecule.

615. The kit of claim 614, wherein said recombination site confers at least one enhancement selected from the group consisting of enhancing excisive recombination; (ii) enhancing integrative recombination; (iii) decreasing the requirement for host factors; (iv) increasing the efficiency of the formation reaction by recombination of said cointegrate DNA or of said product DNA; increasing the specificity of the formation reaction by recombination of said cointegrate DNA or of said product DNA; and (vi) increasing the specificity or yield of a subsequent recombination reaction of, or subsequent isolation of, the product DNA.

616. The kit of claim 614, wherein said core region comprises a nucleic acid sequence selected from the group consisting of: a) RKYCWGCTTTYKTRTACNAASTSGB (m-att) (SEQ ID NO: 1); b) AGCCWGCTTTYKTRTACNAACTSGB (m-attB) (SEQ ID NO:2); c) GTTCAGCTTTCKTRTACNAACTSGB (m-attR) (SEQ ID NO:3); d) AGCCWGCTTTCKTRTACNAAGTSGB (m-attL) (SEQ ID NO:4); c) GTTCAGCTTTYKTRTACNAAGTSGB (m-attP 1) (SEQ ID and a corresponding or complementary DNA or RNA sequence, wherein R=A or G; K=G or T/U; Y=C or T/U; W=A or T/U; N=A or C or G or T/U; S=C or G; and B=C or G or T/U. 04t7/03,swI 1797.pa.doc. -116-

617. The kit of claim 614, wherein said core region comprises a nucleic acid sequence selected from the group consisting of: a) AGCCTGCTTTTTTGTACAAACTTGT (attBl) (SEQ ID NO:6); b) AGCCTGCTTTCTTGTACAAACTTGT (attB2) (SEQ ID NO:7); c) ACCCAGCTTTCTTGTACAAACTTGT (attB3) (SEQ ID NO:8); d) GTTCAGCTTTTTTGTACAAACTTGT (attRl) (SEQ ID NO:9); e) GTTCAGCTTTCTTGTACAAACTTGT (attR2) (SEQ ID NO: f) GTTCAGCTTTCTTGTACAAAGTTGG (attR3) (SEQ ID NO:11); g) AGCCTGCTTTTTTGTACAAAGTTGG (attL1) (SEQ ID NO:12); h) AGCCTGCTTTCTTGTACAAAGTTGG (attL2) (SEQ ID NO:13); i) ACCCAGCTTTCTTGTACAAAGTTGG (attL3) (SEQ ID NO:14); j) GTTCAGCTTTTTTGTACAAAGTTGG (attPl) (SEQ ID k) GTTCAGCTTTCTTGTACAAAGTTGG (attP2, P3) (SEQ ID NO:16); and a corresponding or complementary DNA or RNA sequence. DATED this 8 th day o April, 2004 INVITROGEN CORPORATION By their Patent Attorneys: CALLINAN LAWRIE 07/04/04,swl 1797sp.116