WO2019239042A1 - Method for modifying a target nucleic acid of a host cell - Google Patents

Abstract

The invention relates to a method for modifying, by cutting and/or total or partial degradation, a target nucleic acid sequence of a host cell, comprising bringing said host cell into contact in vitro or ex vivo with a biological system comprising: a first component comprising at least one protein selected from the proteins R350 and R354 of giant virus group A Mimivirus or at least one expression vector of at least one nucleic acid sequence of the genes R350 and/or R354 respectively encoding for at least one of said proteins R350 and/or R354; and a second component comprising at least one nucleic acid sequence, called an affinity sequence, said affinity sequence comprising at least one complementary sequence of a specific sequence of said target sequence, said specific sequence comprising at least 10 nucleotides, preferably 15 nucleotides, of said target sequence, said specific sequence being flanked at 3' and 5' by adjacent sequences surrounding a sequence of 15 nucleotides of the Zamilon virophage contained and repeated four times in the R349 gene of giant virus Mimivirus.

Description

 Method for modifying a target nucleic acid sequence of a host cell.

The present invention relates to a method of modifying a target nucleic acid sequence of a host cell comprising contacting said host cell in vitro or ex vivo with a biological system.

Prokaryotes are able to acquire adaptive immunity against the invasion of genetic elements, such as plasmids or phages, by incorporating into their genome short sequences of foreign DNA (from 21 to 72 nucleotides [nt] ), a system called CRISPR- Cas (short grouped and regularly spaced palindromic repetitions) [1, 2]. The acquisition of these foreign DNA sequences occurs adjacent to the Cas genes which have the role of cleaving the foreign DNA into small fragments. CRISPR-Cas thus confers resistance to a second infection [14, 15]

To date, the CRISPR-Cas system has only been observed in bacteria and archaea [16], but not in viruses [1, 2].

The discovery of giant amoeba viruses, living in competition with other microbes, has challenged the classical definition of a virus [4, 5]. Since their discovery, giant viruses have revealed unique phenotypic and genotypic characteristics that go against the classical definition of a virus, placing them close to certain microbes. Mimiviruses with a diameter greater than 0.5 micrometer are easily visible with a simple optical microscope. They have a large and complex genome that contains sequences from other organisms [6]. In addition, their possible infection with viruses, virophages, which multiply in the Mimivirus virus factory, and the presence of mobile elements (transpovirons, polintons) have sparked lively debate on the origin of these viruses [7 ]. In addition, virophages, like bacteriophages, can integrate into the Mimivirus genome in the form of provirophagus [9]. One of virophages which parasitizes the family of Mimiviruses was baptized zamilon

[10].

In this study published in the journal Nature [8], the researchers observed that a group among the Mimiviruses (called lines A) developed resistance against infection by a virophage named zamilon, while the giant viruses of lines B and C are susceptible to infection by this virophage. The researchers found the presence of a repeated sequence of zamilon DNA which is used to hook the virophage only in line A. This complex was thus named MIMIVIRE (Mimivirus Virophage Résistance Element) and presents functional similarities with the system of defense CRISPR-Cas existing until then only in bacteria and archaea and comprising an enzyme unwinding the DNA (helicase R350) and another cutting it (nuclease R354). Inactivation of the MIMIVIRE complex has made it possible to restore the susceptibility of Mimivirus to virophageal infection. The partner proteins included in this MIMIVIRE complex are involved in the specific degradation of foreign DNA. The viral defense system, MIMIVIRE, thus confers immunity to giant viruses which have been able to integrate the DNA of the infecting virophage into their genome.

More specifically, 59 strains from the collection of giant viruses of the Mimiviridae family of Acanthamoeba polyphaga (including 28 from line A, 8 from line B and 23 from line C), were infected by the zamilon or Sputnik virophages , another virophage [11]. After 24 hours, an increase in Sputnik DNA was observed in all three lines of Mimiviridae. In contrast, zamilon was able to replicate only with strains of lines B and C of Mimiviridae, but no strain of line A. A sequence of 28 nucleotides (nt), specific to zamilon, and three repetitions of 15 nt were were found in all of the genomes of line A, unlike the other two lines (B and C). These sequences are located in a gene encoding a transposase-A in zamilon, and are integrated into the R349 gene of Mimivirus and into the orthologous genes, in all the Mimiviruses of line A. These repeated sequences are separated by sequences of 9.48 and 63 nt respectively. The study of the genomic environment in the vicinity of repeated zamilon sequences revealed ORFs (open reading frames) conserved in Mimiviruses. These ORFs have distant similarity to the protein Cas which are of the helicase and nuclease type. Silenced by RNA interference, the inactivation of ORFs containing the repeated DNA sequence of zamilon, or of ORFs close to the protein cases, made it possible to restore the susceptibility of the A line of Mimiviruses to infection by zamilon. In addition, gene expression has confirmed that they encode an R350 helicase and an R354 nuclease.

From now on, the giant viruses of line B and C are called respectively Momovirus for line B and Megavirus for line C, Mimivirus being reserved for line A. In the remainder of this patent application when it is indicated Mimivirus, it it is therefore a giant Mimivirus virus of line A, the genome sequence of which is deposited in Genebank under the reference AY653733.1 and / or which has been deposited in the National Collection of Cultures of Microorganisms of the Institut Pasteur (Paris) according to the Budapest Treaty under the registration number CNCM 1-5322 on May 31, 2018 under the reference APBC1.

In a recent article [17], the nuclease structure and activity of the R354 protein were studied.

The Mimivirus proteins R350 and R354 are known to those skilled in the art and have been described [8, 17] and reproduced in the sequences SEQ. ID. N °: 1 and 2 of the sequence listing.

This viral defense system thus confers adaptive immunity to giant viruses which have been able to integrate the DNA of the infecting virophage into their genome. This is why this complex was named Mimivire (Mimivirus virophage resistance element) [11]. Following the study of Nature 2016 (8), it was however disputed that Mimivirus could also be capable of developing a tool of the biological scissor type similar to that of CRISPR-Cas [12].

However, according to the present invention, it has been demonstrated that the MIMIVIRE system can be adapted - but differently than the CRISPR-Cas system as described for example in EP 2 800 811 - to offer a new biological tool of the comparable “biological scissors” type. to the CRISPR-Cas system for foreign genes other than zamilon.

The aim of the present invention is therefore to research the methods according to which the Mimivire system can be adapted to provide a “biological scissors” system analogous to the CRISPR-Cas system for targeting genes other than the zamilon gene in Mimivirus and thus for modifying non-coding genes or sequences of bacteria or eukaryotic cells in particular.

To do this, the present invention provides a method of modifying by cut and / or total or partial degradation of a target nucleic acid sequence of a host cell comprising contacting said host cell in vitro or ex vivo. with a biological system comprising:

a first component comprising at least one protein chosen from the proteins R350 and R354 of giant virus Mimivirus of line A or at least one expression vector of at least one nucleic acid sequence of the R350 and / or R354 genes coding for respectively at least one said protein R350 and / or R354, and

a second component comprising at least one nucleic acid sequence, called affinity sequence, said affinity sequence comprising at least one sequence complementary to a specific sequence of said target sequence, said specific sequence of said target sequence comprising at least 10 nucleotides, preferably 15 nucleotides. The target sequence can be a DNA sequence or an RNA sequence transcribed from the genome of a said host cell. It is understood that said specific sequence of said target sequence is other than a repeated sequence of the zamilon virophage contained in said R349 gene.

The host cell can be a bacterium or archaeal cell or a unicellular or multicellular eukaryotic cell, in particular of a plant or animal.

The Mimivirus proteins R350 and R354 are known to those skilled in the art and have been described below in the sequences SEQ. ID. N °: 1 and 2 of the sequence listing.

These proteins R350 and R354 are known to have helicase and nuclease properties respectively comprising exonuclease and endonuclease activities so that the modification of said target sequence can comprise a cleavage at a specific site and / or a degradation total or partial of said target sequence.

The term “sequence complementary to a specific sequence of 15 nucleotides of said target sequence” means a DNA or RNA sequence which hybridizes with said target sequence and which does not hybridize with any other sequence of a said host cell and said R349, R350 or R354 genes.

On the other hand, it is understood that the said flanking sequences are taken from said R349 gene.

The Mimivirus R349 gene is known to those skilled in the art and has been described below in the sequence SEQ. ID. N °: 5 of the sequence listing.

Sequences of the R350 and 354 genes coding for the Mimivirus proteins 350 and 354 are known to those skilled in the art and have been described in the SEQ sequences. ID. N °: 3 and 4 of the sequence listing.

More particularly, said second component comprises said affinity sequence in the form of DNA or preferably in the form of RNA.

Preferably, said affinity sequence comprises at least 2 same so-called complementary sequences of the same specific sequence, preferably from 2 to 4, more preferably 4 same so-called complementary sequences of the same specific sequence, repeated spaced apart.

More preferably still, the said or each said complementary sequence of the same said specific sequence of the affinity sequence comprises is flanked in 3 'and 5' by adjacent sequences flanking a sequence of 15 nucleotides of the zamilon virophage contained and repeated 4 times in the giant Mimivirus R349 gene.

More preferably, the same complementary sequences of the same specific sequence of the target sequence are flanked in 3 ′ and 5 ′ of flanking sequences which are different from each other between the same same complementary sequences and the said flanking sequences corresponding respectively to the flanking sequences of the same repeat sequences of the zamilon virophage contained in the said R349 gene from Mimivirus.

It is understood that said flanking sequences are taken from the 28 nucleotide sequence (SEQ.ID.N 24) including the said 15 nucleotide repeat sequence from the zamilon virophage (SEQ.ID.N6) in the R349 gene or taken from the sequences of 9, 48 and respectively 63 nucleotides (SEQ. ID. N ° 26-28) which separate the repeated sequences of the zamilon virophage in the R349 gene, or a flanking sequence 3 ′ of the last specific specific sequence repeated 3 ′ of 15 nucleotides of the zamilon virophage in the R349 gene. More particularly, said affinity sequence comprises the modified R349 gene in which only the 4 repeated sequences of 15 nucleotides of the zamilon virophage are replaced at the same locations by the said 4 same sequences complementary to a said specific sequence of the said target sequence.

Such a modified R349 gene is illustrated by the sequence SEQ.ID No. 7 including a specific sequence of 15 nucleotides of the tetracycline resistance gene of the sequence SEQ.ID No. 23.

More particularly still, said affinity sequence is integrated into a cloning vector of said affinity sequence capable of transforming or transfecting a said host cell and replicating said said affinity sequence therein.

More particularly still, the said affinity sequence is integrated in the form of DNA placed under the control of a transcription promoter capable of transcribing the said DNA affinity sequence into RNA in the said host cell.

More particularly still, said host cell is a bacterium or a eukaryotic cell.

More particularly still, said first component comprises an expression vector of at least one R350 and / or R354 nucleic acid sequence coding for and capable of expressing at least one said R350 and / or R354 protein respectively in a said host cell. .

More particularly, the nucleic acid sequences coding for the proteins R350 and / or R354 are integrated into a plasmid, under the control of an expression promoter, for example the T7 promoter for a bacterium and the CMV promoter for a cell. eukaryotic, and framed nuclear localization sequences (NLS) in order to address proteins in the nucleus of the eukaryotic cell if necessary. More particularly still, the said first and second components are integrated into the same said vector, preferably a plasmid.

According to the present invention, it has in particular been discovered that the protein R350 is not a simple helicase but exhibits a specific endonuclease activity which specifically cuts the said specific specific sequences repeated within the target sequence of the host cell.

In a preferred embodiment, the present invention provides a method for at least cutting said target sequence at a specific site, characterized in that said first component comprises at least the R350 protein or at least one expression vector for at least one nucleic acid sequence of the R350 gene coding for respectively at least one said R350 protein.

More particularly, the present invention provides a method for at least cutting at a specific site and at least partially degrading said target sequence, characterized in that said first component comprises at least the R350 protein and the R354 protein or at least one vector of expression of at least nucleic acid sequences of the R350 and R354 genes coding for at least the said proteins R350 and R354, respectively.

More particularly still, said first and second components are integrated into a same said vector comprising the sequences of Mimivirus of line A including:

at least one modified part of the R349 gene, preferably the complete modified R349 gene, said modified part of the R349 gene including a part of the 349 gene including at least one of the 4 repeated sequences of 15 nucleotides of the zamilon virophage of the R349 gene, preferably the 4 repeated sequences, the said modification of the said modified part of the R349 gene consisting of the so-called repeated sequence (s) of 15 nucleotides of the zamilon virophage of the gene R349 is (or are) replaced by the same said sequence complementary to a specific sequence of said target sequence, and

a sequence allowing transcription into RNA in a said host cell of said modified part of the R349 gene, preferably of the complete modified R349 gene, and

- the R350 and 354 genes, as well as sequences allowing the expression of the R350 and R354 genes in a said host cell.

More particularly still, for a host cell of bacteria, said first and second components are integrated into the same said vector comprising the sequences of Mimivirus of line A including one of the sequences SEQ.ID. N ° 22 or SEQ.IDN ° 25 in which the sequence repeated 4 times specific for the gene for resistance to tetracycline SEQ. ID. No. 23 is replaced by a said sequence complementary to a specific sequence of 15 nucleotides of the target sequence.

More particularly still, for a host cell of bacteria, said first and second components are integrated into the same said vector comprising the sequences of Mimivirus of line A including one of the sequences SEQ. ID. N ° 37 or SEQ.ID N ° 40 in which the sequence repeated 4 times specific for the gene for resistance to tetracycline SEQ. ID. No. 39 is replaced by a said sequence complementary to a specific sequence of 15 nucleotides of the target sequence.

The present invention also provides a biological system useful for the implementation of a method according to the invention of modification by cutting and / or total or partial degradation of a target nucleic acid sequence of a host cell comprising the implementation in contact with said host cell in vitro or ex vivo, as defined above comprising: a first component comprising at least one protein chosen from proteins R350 and R354 of giant virus Mimivirus of line A or at least one expression vector of at least one nucleic acid sequence of gene (s) R350 and / or R354 coding for respectively at least one said protein R350 and / or R354, and

a second component comprising a nucleic acid sequence, called the affinity sequence, the said affinity sequence comprising at least one sequence complementary to a specific sequence of the said target sequence, the said specific sequence of the said target sequence comprising at least minus 10 nucleotides, preferably 15 nucleotides, preferably the or each said sequence complementary to a specific sequence of said target sequence being flanked in 3 ′ and 5 ′ by adjacent sequences flanking a sequence of 15 nucleotides of the zamilon virophage contained and repeated 4 times in the giant Mimivirus R349 gene.

More particularly, said or each said complementary sequence of the same said specific sequence of the affinity sequence is flanked in 3 'and 5' by adjacent sequences flanking a sequence of 15 nucleotides of the zamilon virophage contained and repeated 4 times in the gene R349 from giant Mimivirus virus.

The biological system according to the present invention obviously includes all of the characteristics stated upstream for said system when describing the method according to the invention.

The present invention also covers said biological methods and systems according to the invention implemented with said sequences of acids or proteins which are homologous or even orthologous to those of said sequences, and / or having similarities in particular with at least 90 % identity of said sequences.

Other characteristics and advantages of the present invention will emerge more clearly on reading the description which follows, made of by way of illustration and without limitation, with reference to the appended drawings in which:

FIGS. 1A-1 B and 2A-2B represent agarose electrophoresis gels of various nucleic acid sequences in the presence or not of the proteins R350 and / or R354.

Figure 3 is a diagram of the hypothetical mode of action of helicase R350 and nuclease R354.

Figure 4 shows a polyacrylamide electrophoresis gel (15%) showing the effect of RNA.

FIG. 5 is a diagram illustrating the mode of action of RNA on DNA replication in the presence of DNA polymerase.

FIG. 6 shows the constructs of the plasmids PP20 and PP21 for expression of the proteins R350 and R354.

FIGS. 7A-7B represent SDS-PAGE gels and Western-blot analysis of the proteins extracted from eukaryotic cells transfected or not by the plasmids PP20 and / or PP21 on a nitrocellulose membrane hybridized with an anti-FLAG antibody.

FIG. 8A represents the vector PP14 comprising the sequence SEQ.ID. N ° 25 including the modified Mimivire system of the sequence SEQ. ID. No. 22 comprising a sequence directed against the tetracycline resistance gene of the sequence SEQ.ID. # 23.

FIG. 8B represents the vector pACYC184 carrying a specific sequence of the gene for resistance to tetracycline (SEQ.ID. No. 23).

FIGS. 9 to 11 and 13 show comparative results of culture of bacteria with abolition of resistance to tetracycline for bacteria transformed by the vector PP14. FIG. 12 shows the results of the action of the Mimivire system tested using an amplification (PCR) of the tetracycline resistance gene from the bacterial culture.

FIGS. 14 and 15 show comparative results of culture of bacteria with abolition of resistance to tetracycline for bacteria transformed by the vector PP37.

FIG. 16 shows comparative results relating to the capacity of the Mimivire system to abolish the resistance of bacteria to tetracycline for bacteria transformed by the vector PP37, in a liquid medium.

FIG. 17 shows the results showing that the action of the Mimivire system is not due to the expression of lethal proteins but to the abolition of the gene for resistance to tetracycline.

The results of the examples of experiments carried out are reported below (paragraphs I.A, I. B, I.C) and the materials and methods are explained at the end of the description (Paragraph II). A sequence listing includes SEQ sequences. ID. N ° 1 to 40 mentioned below.

Two series of experiments were carried out below. The first series of experiments (IA) aims to clarify the understanding of the mechanism of action of the Mimivire system. The second series of experiments (I. B, IC) aims to confirm the results highlighted previously by using the MIMIVIRE system to modify a reporter gene in eukaryotic cells (I. B) by inactivating a gene easily identifiable reporter to modify a specific sequence of the tetracycline resistance gene in a procrayote system (E.coli) (IC l), to verify the ability of the Mimivire system to abolish the resistance of bacteria to tetracycline in the medium liquid (IC2), to check whether the death of a prokaryotic system (E.coli) in the presence of tetracycline is due to the modification of a specific sequence of the tetracycline resistance gene in said system by the Mimivire system (IC3 ) without protein mediation. IA) First series of experiments aims to clarify the understanding of the mechanism of action of the Mimivire system.

1) The 2 recombinant proteins R354 (Nuclease) and R350 (Helicase) were used on various nucleic substrates in particular (a) IORF4 of the zamilon virophage of 594 nucleotides (product amplified by the primers SEQ. ID. N ° 12 and 13 of the table 1 below) which carries the repeated sequence of 15 nucleotides of the zamilon virophage (SEQ. ID. No. 6) as well as (b) the R349 gene (SEQ. ID. No. 5), both obtained by PCR with the primers of sequences SEQ.ID.N ⁰ 10 to 13 of Table 1). Figures IA and IB represent agarose electrophoresis gels (1%) of DNA sequences of genes amplified by PCR of the ORF4 sequence of zamilon (fig.lA) and of the R349 gene of Mimivire (fig.lB) treated with a protein R354 (N), a protein R350 (N), the two proteins (N + H) and a control without protein (C).

Only an exonuclease activity of the protein R354 was observed with total non-specific degradation of the DNA (Figures IA and IB).

2) We investigated whether the action of the Mimivire system would involve the assistance of the RNA corresponding to the repeated sequence, which would thus form a heteroduplex with the single strand of IORF4, in particular during the replication of DNA from the virophage and would cause thus stopping the replication of virophagous DNA.

To do this, in vitro tests were carried out using a synthetic DNA template corresponding to (a) an area of 130 nucleotides of zamilon IORF4 including the repeated sequence (sense or "forward" sequence SEQ. ID. N ° 8 and antisense sequence or "reverse SEQ. ID. No. 9) and (b) an RNA of 15 nucleotides corresponding to the repeated sequence (sense or" forward "sequence SEQ. ID. No. 18 and antisense sequence or" reverse "SEQ. ID. N ° 19). After hybridization, the action of nuclease R354, helicase R350 or nuclease + helicase was tested. FIG. 2A represents polyacrylamide electrophoresis gels (15%) of nucleic acid sequences comprising the single-stranded DNA of the ORF4 gene of zamilon and an RNA sequence of 15 nucleotides complementary to the repeated sequence of the zamilon virophage, with a protein R354 (N), a protein R350 (N), the two proteins (N + H) and a control without protein (C).

FIG. 2B represents polyacrylamide electrophoresis gels (15%) of nucleic acid sequences comprising the double stranded DNA of the ORF4 gene of zamilon and an RNA sequence of 15 nucleotides complementary to the repeated sequence of the zamilon virophage, with a protein R354 (columns N), a protein R350 (N), the two proteins (N + H) and a control without protein (C).

The result surprisingly shows that the he I case cuts at the heteroduplex level while the nuclease completely degrades the DNA matrix (Figure 2A). At the same time, no action of the two proteins was observed on the double strand (FIG. 2B).

These results suggest that the protein encoded by the R350 gene has an endonuclease function contrary to bioinformatic (helicase) predictions. In order to determine the cleavage site exactly, the fragments resulting from this digestion were sequenced. The sequencing allowed us to locate between the 8th and 9th nucleotides of the repeated sequence the cleavage site (TGATAATG vAATCTGA).

This suggests that its action occurs only on the DNA / RNA heteroduplex found in particular during DNA replication. The mechanism of resistance of Mimivirus towards zamilon could operate through the introduction of this cut at the level of IORF4 of zamilon by the protein coded by the R350 gene followed by the degradation by the nuclease R354 as illustrated in the diagram of the figure. 3 on which the RNA is referenced by A, the protein R350 by B, and the protein R354 by C. FIG. 3 illustrates the mode of action of helicase and nuclease on IORF4 of zamilon in vivo.

In addition, it has been observed that the presence of this RNA of repeated sequence blocks the polymerization of an area of IORF4 of zamilon including the repeated sequence and generates truncated forms (FIG. 4). We can therefore infer that at the scale of zamilon replication, replication is stopped as illustrated in Figure 5 at IORF4.

FIG. 4 represents a polyacrylamide electrophoresis gel (15%) showing the effect of the RNA of complementary sequence (SEQ. ID. Nos. 18 and 19) at the repeated sequence unit on the replication of a oligonucleotide of 130 bases (SEQ. ID. N ° 8 and 9) corresponding to the sequence of IORF4. Electrophoresis was carried out on denaturing polyacrylamide gel (15% PAGE) of a synthetic DNA (sense (SEQ. ID. N ° 8) or antisense (SEQ. ID. N ° 9) used as matrix by l DNA dependent DNA polymerase (Klenow) after prior hybridization with a complementary RNA corresponding to the repeated sequence unit (SEQ. ID. Nos. 18 and 19). The controls were carried out under the same conditions in the absence of Klenow ( C = without Klenow) or RNA (+ = with RNA or - = without RNA).

Figure 5 is a diagram illustrating the mode of action of RNA complementary to the repeated sequence unit (1) on DNA replication (2) in the presence of DNA polymerase (3) showing the importance crucial of this RNA in the action process of the Mimivire system.

I.B) Use of the MIMIVIRE system in eukaryotic cells.

We sought to confirm the results previously highlighted using the MIMIVIRE system in a eukaryotic system by effecting the inactivation of an easily identifiable reporter gene. We chose to inactivate the Green Fluorescent Protein (GFP) gene in the human cell line HEK293. Initially, the R350 and R354 genes labeled FLAG and flanked by NLS sequences were cloned respectively in the plasmids pcDNA3.1 / Hygro or pcDNA3.1 / Zeo as illustrated in FIG. 6.

Figure 6 shows the constructs of the transfection plasmids PP20 and PP21 used in this study to validate the MIMIVIRE system in which the MIMI_R354 (nuclease) and MIMI_R350 (helicase) genes are under the control of the CMV promoter and framed by nuclear localization sequences ( NLS) to address helicase and nuclease in the cell nucleus.

293GFP cells were transfected with these plasmids to establish a new HEK293 GFP cell line which expresses the proteins R350 and R354. The expression of nuclease and helicase was confirmed by Western blotting with an anti-FLAG monoclonal antibody (FIGS. 7A-7B). This anti-FLAG antibody recognizes the bands at 101.6 KDa for helicase and 70.8 KDa for nuclease.

Figures 7A-7B show SDS-PAGE gels and Western blot analysis of proteins extracted from 293GFP cells. Electrophoresis was carried out on 10% SDS-PAGE gel of 50 μg of a protein extract originating from 293GFP cells transfected or not by the plasmids PP20 and / or PP21 (FIG. 7A) on a nitrocellulose membrane hybridized with an anti -FLAG at 1: 1000 (Fig.7B). The sizes are indicated on the left in KDa.

Secondly, an RNA of 45 nucleotides was synthesized (sense and antisense sequences SEQ.ID.No 20-21, Table 1) comprising 2 repeat specific sequence units of 15 nucleotides from GFP (sense sequences and antisense SEQ.ID.N ° 29-30, table 1) and framed with the same sequence as can be found in the R349 gene of Mimivirus (table 1, sense sequences SEQ.ID.No.20 including the sequence SEQ. ID. N ° 29 and antisense sequences SEQ.ID.N ° 21 including the sequence SEQ.ID. # 30). This 15 nucleotide sequence is part of the RNA guide used to inactivate the GFP gene with the CRISPR / Cas9 system as described in the article Science. 2014 Jan 3; 343: 84-87. This RNA was transfected into HEK293 GFP cells which express the R350 and R354 genes. Reading these experiments made it possible to follow the relative extinction of the fluorescence of GFP by FACS.

I.C) Use of the MIMIVIRE system in prokaryotic cells.

I.C.l. Modification of a specific sequence of the tetracycline resistance gene in E. coli

The system was vectorized in an expression vector PP14 (SEQ. ID. NO. 25) for prokaryote under the control of the T7 promoter inducible by IPTG. The sequence of the R349 gene has been modified (SEQ.

ID. N ° 7) in order to act against the tetracycline resistance gene by replacing the 15 nucleotides of the repeated zamilon sequence with 15 nucleotides specific for the tetracycline resistance gene (SEQ. ID. N ° 23: CACCCTGGATGCTGT) . The R350 and R354 genes have been added following R349 and are organized into operons under the control of the inducible T7 promoter (Annex 1, SEQ. ID. NO. 22).

Figures 8A-8B represent the Mimivire vector (PP14-Figure 8A) carrying the sequence SEQ. ID. N ° 25 including the sequence SEQ.ID.N ° 22 with the R349 gene modified (SEQ. ID. N ° 7) by a sequence directed against the tetracycline resistance gene (SEQ. ID. N ° 23) as well as genes R350 (SEQ. ID. N ° 3) and R354 (SEQ. ID. N ° 4) under the control of the same T7 promoter (SEQ. ID. N ° 31) with an "operon" organization at l using Lac operon sequences (SEQ. ID. # 32), Shine-Dalgarno sequence and T7 terminator (SEQ. ID. # 33). The PP14 vector into which the R349, R350 and R354 genes are cloned is part of the family of pET vectors which are expression vectors in bacteria. These vectors embark among others, an inducible T7 promoter, that is to say that the expression is made only in the presence of a certain compound in the culture medium, namely IPTG (Isopropyl bDl-thiogalactopyranoside). This vector makes it possible to synthesize constitutively a protein called lacl which binds to the Lac operator sequence and thus blocks the transcription of the cloned gene downstream of the T7 promoter because the T7 RNA polymerase previously fixed on the T7 promoter cannot transcribe the gene because of the steric hindrance created by the presence of the lacl protein. However, in the presence of IPTG, the lacl protein complexes with this IPTG molecule, causing a change in conformation of the lacl protein making it unable to bind to the lac operator sequence, which allows the T7 RNA polymerase to transcribe the gene. cloned downstream of the T7 promoter. And, the T7 terminator stops the T7 RNA polymerase at the end of the transcription.

The Shine-Dalgarno sequence is recognized by the ribosome of the bacteria. In bacteria, it is possible to translate a messenger RNA known as “polycistronic”, that is to say which contains the transcription of several genes in succession according to an “operon” organization. To do this, each part of messenger RNA which contains the transcription of a gene must be preceded by a Shine-Dalgarno sequence recognized by the ribosome which can therefore bind to it and initiate the translation of the messenger RNA.

In the presence of IPTG in the medium, transcription into RNA of the modified R349 is carried out which carries the 4 repeat units corresponding to tetracycline on the one hand and on the other hand, transcription of another messenger RNA which is carried out carries the R350 (helicase) and R354 (nuclease) genes. The 2 transcriptions are independent of each other.

This PP14 vector is then transferred by electroporation into E.coli BL21 bacteria previously transformed by the vector. pACYC184 (FIG. 8B) allowing the expression of the tetracycline resistance gene. The bacteria are established according to different dilutions on an LB / agar dish containing either Ampicilin / Tetracycline / IPTG (50pg / ml; 12pg / ml; O.lmM respectively) or Ampicilin / IPTG (50pg / ml; ImM respectively).

FIGS. 9 to 11 and 13 show comparative results of culture of bacteria with abolition of resistance to tetracycline for bacteria transformed by the vector PP14.

Figure 9 shows the results of the first experiment. On the Amp / IPTG boxes there are (from left to right) 16; 3 and 48 bacteria for dilutions corresponding to 1/10 respectively; 1/50; and the rest of the transformation. On the boxes

Amp / Tet / IPTG, there are respectively 0; 0 and 8 bacteria.

Figure 10 shows the results of the second experiment. On the Amp / IPTG boxes there are (from left to right) 5 and 28 bacteria for spread dilutions corresponding respectively to 1/5 and to the rest of the culture. Starting volume of 1ml. On the boxes

Amp / Tet / IPTG, there are respectively 9 and 4 bacteria. These results show that the Mimivire system makes it possible to abolish resistance to tetracycline. These results have been confirmed by other experiments ensuring the presence or absence of the vector conferring resistance to tetracycline.

First, we tested the induction of expression of genes in the Mimivire system by IPTG on the growth of E. coli BL21- DE3 doubly transformed (pACYC184 and PP14).

Figure 11 shows the effect of the induction of the Mimivire system: The bacteria containing the vector pACYC184 are electroporated with the vector PP14 containing the Mimivire system and spread out at equal volume on LB / Agar Ampicillin / Tetracycline + or IPTG dishes. In FIG. 11, it is clearly seen that the induction of the expression of the genes of the Mimivire system results in the drastic reduction in the number of colonies regardless of the antibiotic.

Following these results, we wanted to characterize doubly transformed bacterial clones of the Mimivire system. To do this, the bacterial clones from the LB / Ampicillin / Tetracycline Agar boxes without IPTG were cultured in Ampicillin + IPTG medium in order to activate the Mimivire system and, in parallel, the same clones were cultured Ampicillin medium without IPTG as negative control. The action of the Mimivire system is tested using an amplification (PCR) of the Tetracycline resistance gene from the bacterial culture. In the hypothesis of an action of the Mimivire system, this amplification should be impossible.

A test of the action of the Mimivire system by PCR was carried out using the bacterial culture of 10 clones from the LB / Agar Ampicillin / Tetracycline box and pushed for 16 hours in Ampicillin alone or Ampicillin + IPTG.

The PCR result shows the amplification of a 1.3 kb fragment corresponding to the tetracycline resistance gene.

Under the experimental conditions used for the amplification of the tetracycline resistance gene, this result does not make it possible to visualize a total or partial absence of the vector pACYC184 carrying the tetracycline resistance gene. A weak presence of an intact vector can generate a fragment during amplification and thus explain the results obtained in FIG. 12. A real-time amplification (q PCR) allowed a better interpretation of the action of the Mimivire system.

It should be noted that out of the 10 clones, only 2 grew in Ampicillin / IPTG medium (clone 7 and 10) while the 10 clones grew in Ampicillin medium alone. This last result shows mainly that the induction of the Mimivire system has an effect on the growth of bacteria just like the result from Figure 11.

FIG. 13 shows the spread of clone 7 on a Tetracycline dish (284 bacteria) with IPTG and on a Tetracycline dish (402 bacteria) without IPTG at a dilution corresponding to 103 theoretical CFUs.

Clone 7 responds to the induction of the Mimivire system via IPTG because we go from 346 (Ampicillin / IPTG boxes) to 284 bacteria (Tetracycline / IPTG boxes), a decrease of about 18%, whereas we do not shows no significant difference in control without IPTG (passage from 393 to 402 bacteria).

The absence of colonies on tetracycline confirms an action of the Mimivire system on the inhibition of resistance to Tetracycline.

I.C.2. Study of the capacity of the Mimivire system to abolish the resistance of bacteria to tetracycline in liquid medium

E.coli bacteria were transformed with the plasmid pACYC184 containing the tetracycline resistance gene (SEQ. ID. N ° 35).

The vector Mimivire PP37 carries the sequence SEQ. ID. N ° 40 including the sequence SEQ.ID.N ° 37 with the R349 gene modified (SEQ. ID. N ° 38) by a sequence directed against the tetracycline resistance gene (SEQ. ID. N ° 39) as well as genes R350 (SEQ. ID. N ° 3) and R354 (SEQ. ID. N ° 4) under the control of the same T7 promoter (SEQ. ID. N ° 31) inducible by IPTG, with an organization in “operon” using Lac operon sequences (SEQ. ID. N ° 32), Shine-Dalgarno sequence and T7 terminator (SEQ. ID. N ° 33).

The vector PP37Vector (SEQ ID No. 40) into which the R349, R350 and R354 genes are cloned is part of the family of pET vectors which are expression vectors in bacteria. These vectors embark among others, an inducible T7 promoter, that is to say that expression is only done in the presence of a certain compound in the culture medium, namely IPTG (Isopropyl bDl-thiogalactopyranoside). This vector is the same as that of the PP14 vector but with the 15 nucleotides specific for the tetracycline resistance gene corresponding to the SEQ. ID. N ° 39: CGGCTCTTACCAGCC.

The E.coli BL21 bacteria previously transformed with the plasmid pACYC184 were transformed with the vector PP37Vector. The bacteria were spread on dishes containing 100 mg / L of ampicillin and 12 mg / L of tetracycline supplemented or not with ImM of IPTG.

Figure 14 shows the results of the first experiment. On the Amp / Tet boxes there are (from left to right) 1597 and 259 bacteria for spread dilutions corresponding respectively to 1/1 and 1/10. It is noted that on the Amp / Tet / IPTG boxes, no bacteria are obtained.

Figure 15 shows the results of the second experiment. On the Amp / Tet boxes there are (from left to right) 562 and 60 bacteria for spread dilutions corresponding respectively to 1/1 and 1/10. Starting volume of 1ml. It is noted that on the Amp / Tet / IPTG boxes, no bacteria are obtained.

The results obtained (FIGS. 14 and 15) show the absence of colonies on the agar containing tetracycline in the presence of IPTG which induces the expression of the proteins of the Mimivire system. These results highlight the ability of the Mimivire system to abolish the resistance of bacteria to tetracycline causing the death of bacteria.

The induction of the MIMIVIRE system was tested in a liquid medium. To do this, two colonies of Escherichia coli having the plasmids PP37Vector and pACYC184, were selected on dishes containing ampicillin (100 mg / L) and tetracycline (12 mg / l) and cultivated in 2 ml of medium containing ampicillin (100 µg / L) and (12 µg / mL) at 37 ° C with stirring at 200 rpm. After 2 hours, each culture was divided into two tubes in which one tube was supplemented with ImM from IPTG to induce expression of proteins from the Mimivire system. At regular time intervals, 10 µl was diluted in 1 ml from which 100 µL was spread on agar dishes containing 100 µg / L of ampicillin and 12 µg / mL of tetracycline.

As shown in Figure 16, the induction of expression of proteins in the Mimivire system by IPTG induces the death of bacteria 4 hours after induction. It is in fact observed that 30 and 20 times fewer colonies were obtained for clone 1 and clone 2 respectively after addition of IPTG. These results highlight the ability of the Mimivire system to abolish the resistance of bacteria to tetracycline in a liquid medium, causing the death of bacteria.

I.C.3. Verification of the involvement of mediator proteins in the activity of the Mimivire system

To exclude the expression of lethal proteins by the Mimivire system in E. coli bacteria, their viability was tested on various media. To do this, 4 colonies of E. coli having the plasmids PP37Vector and pACYC184 were collected and cultured in 1 ml of medium containing ampicillin and tetracycline at 37 ° C. with stirring at 200 rpm. After two hours, each culture was supplemented with 1 mM IPTG to induce expression of proteins from the Mimivire system. After 4 hours, 10 µL was diluted in 1 ml, then 100 µL was spread on agar dishes containing ampicillin and tetracycline or containing ampicillin but no tetracycline.

The results presented in FIG. 17 confirm that the death of the bacteria is not due to the expression of lethal proteins but to the abolition of resistance to tetracycline. These results make it possible to prove that the Mimivire activity can be transferred into E. coli to act against a target gene by including 15 specific repeat sequences of nucleotides of said sequence. The substitution of 15 nucleotides of the Zamilon sequence by 15 nucleotides specific for the tetracycline resistance gene did not create an abortive protein. Only the amino acids encoding the DN ES repeat sequence of Zamilon have been mutated. These results exclude the possibility that the Mimivire system is mediated by protein interactions.

II) Materials and Methods

1) Enzymatic treatment

The enzymatic reactions were carried out by incubating each PCR, in vitro transcription (IVT) or primer product with the nuclease (R354), the helicase (R350) or the two enzymes. The enzymatic reactions were carried out in CutSmart® buffer (Ref: B7204S) from New England Biolabs (potassium acetate 50 mM, trisium acetate 20 mM, magnesium acetate 10 mM, 100 ug ml-1 of BSA, pH 7, 9) at 32 ° C for 2 hours, using a protein concentration of 0.5 mg. ml-1 for each enzyme. After incubation, all reactions were treated with proteinase K to prevent DNA or RNA from spilling into the wells of the gels. After incubation, the products of the enzymatic reactions were subjected to electrophoresis on agarose gel (1.5%) or denaturing PolyAcrylamide (d PAGE) at 15%. The controls were carried out under the same conditions, in the absence of the enzyme R354 or R350.

2) Cultivation conditions.

2.1) In eukaryotic cells:

The Acanthamoeba castellanii amoebas (ATCC 30010) were seeded at 5 × 10 ⁶ in Page's amoebic saline medium (PAS) as described previously (7). The H line EK293 GFP (human embryonic kidney cells, Clinisciences) was cultured at 37 ° C. in a 5% CO ₂ incubator in Eagle medium modified by Dulbecco (DM EM) supplemented with 10% FBS (Gibco) , GlutaMAX 2 mM (Life Technologies), 100 U / ml of penicillin, 100 ug / ml. Streptomycin and 100 pg / ml of nonessential amino acid.

2.2) In prokaryotic cells:

The chemically competent Escherichia Coli One Shot ™ BL21 (DE3) bacteria (Thermo Fischer, Ref: C600003) were cultured in Luria-Bertani (LB) medium with an appropriate antibiotic. The bacteria were spread on LB-agar with an appropriate antibiotic (12pg / ml for tetracycline and 50pg / ml for ampicillin). The Mimivire system was induced by the addition of 0.2 mM isopropyl b-D-1-thigalactopyranoside (IPTG) on LB-agar plates.

3) Infection and transfection test.

The A. Castellanii amoebae were infected or co-infected with Mimivirus or Mimivirus and the zamilon virophage respectively using an MOI of 10. The cells were then incubated at 32 ° C and at regular points post-infection, the cells were collected for RNA extraction with the mirRena ™ miRNA Isolation Kit (Ambion). The 293GFP cells were seeded on 6-well plates (Corning) 48 h before transfection at a density of 300,000 cells / well. The cells were transfected at 80-90% confluence using Lipofectamine 2000 (Life Technologies) following the protocol recommended by the manufacturer. A total of 4 ug of PP20vector / PP21vector (hosting the sequence NLS-R350-NLS and N LS-R354-N LS respectively) were used for transfection. MIMI_R354 (Nuclease) and MIMI_R350 (Helicase) are labeled with 3 FLAG sequences at the N-terminal (3xDYKDDDDK) to confirm the expression of the corresponding proteins by Western Blot experiments with an anti-FLAG monoclonal antibody (Clone M2, Sigma). The next day, these cells are seeded in a T75 flask. Selection with hygromycin (100 µg / ml) and zeocin (100 µg / ml) was applied on day 3 after transfection. These cells were cultured under these conditions for 3 weeks in order to isolate stable doubly transfected cells (line 293GFP [PP20 / PP21]).

4) Transformation test.

The plasmids used in these experiments are the PP14 synthesized by Genscript hosting an inducible expression of the Mimivire system under the control of the T7 promoter (see appendix 1 and FIG. 8A), the vector PP37 hosting an inducible expression of the Mimivire system under the control of the T7 promoter (see Annex 2), and the vector pACYC184 (Mo Bi Tec company Ref: V32402). The Escherichia Coli One Shot ™ BL21 (DE3) bacteria were transformed by the plasmid pACYC184 according to the manufacturer's protocol and instructions. The bacteria were spread on a plate of LB-agar tetracycline (Tet) (day 1). The next day, a clone was removed and cultured in 10 ml of Tet selective LB medium overnight at 37 ° C. with stirring at 200 rpm. On day 3, 200 µl of the culture was inoculated into 20 ml of fresh LB + Tet medium until the OD at 600 nm reached 0.7. The bacteria were harvested by centrifugation at 7000 rpm for 1 minute at 4 ° C and washed three times with 10% glycerol. After the first centrifugation, all steps were carried out on ice and in a cold room. The bacteria were resuspended in 50 µl of 10% glycerol and 200 ng of PP14 vector were added. The mixture was transferred to a Gene Puiser® cuvette with a space of 0.1 cm (Bio-Rad, Ref 165-2089) and an electric pulse was produced with the MicroPulser Electroporator (Bio-Rad, Ref: 165-2100 ) using the Ecl program. The electroporation time indicated by the device after the pulse was 5.8 ms. After electroporation, 1 ml of LB was added immediately and incubated for 1 hour at 37 ° C with shaking at 200 rpm. Several dilutions were plated on TB / Amp / IPTG or Amp / IPTG selective LB-agar plates and incubated overnight at 37 ° C. The next day, the colonies were counted.

5) Cloning, expression and purification. The sequences of the genes coding for the protein R350 and R354 of Mimivirus have been optimized to maximize expression in E.coli and synthesized by GenScript. The protein R354 includes a polyhistidine tag (SEQ. ID. NO. 34) at the C-terminus. The sequence of R349 remains unchanged with the exception of the repeat unit of 15 nucleotides which corresponds to a tetracycline sequence (SEQ.ID. No. 7 including SEQ.ID. N ° 23 repeated 4 times or SEQ. ID. N ° 38 including SEQ. ID. N ° 39 repeated 4 times). These genes were inserted between the Xbal and Xhol cleavage sites of the plasmid pET22b (+) (annex 1, SEQ.ID. No. 22).

The recombinant proteins were expressed in E. coli BL21 (DE3) -pGro7 / GroEL (TaKaRa) using ZYP-5052 medium. Each culture was cultivated at 37 ° C. until an absorbance at 600 nm of 0.8 was obtained, followed by the addition of L-arabinose (0.2% w / v) and induction with a transition. temperature at 18 ° C for 20 h. The cells were harvested by centrifugation (5,000 g, 30 min, 4 ° C) and the resulting pellets were resuspended in loading buffer (20 mM M ES pH 6.0) and stored at -80 ° C overnight. The frozen cells were thawed and incubated on ice for 1 h after addition of lysozyme and phenylmethylsulfonyl fluoride (PMSF) at respective final concentrations of 0.25 mg ml-1 and 0.1 mM. The partially lysed cells were then disturbed by three consecutive sonication cycles (30 s, amplitude 45) carried out on a Q700 sonication system (QSonica). The cellular debris was discarded after a centrifugation step (11,000 g, 20 min, 4 ° C).

The recombinant protein was purified using a cation exchange column from 6 ml of RESOURCE S (GE Heathcare) using 20 mM MES buffer pH 6.0 and a linear gradient to 0.5 M NaCl in 10 volumes. column. The fractions were analyzed by SDS-PAGE stained with Coomassie blue (10%) and the helicase activities were evaluated on an electrophoresis on denaturing polyacrylamide gel as described above (15% of d PAGE). The active fractions (generating a cleavage at the level of the repeated unit of 15 nucleotides of zamilon) were pooled and further purified using an affinity chromatography on immobilized metal (loading and washing buffer: Tris 50 mM pH 8, 300 mM NaCI, 20 mM imidazole, elution buffer: Tris 50 mM pH 8, 300 mM NaCI, imidazole 250 and 500 mM) on a 5 ml column of raw HisTrap FF (GE Healthcare). The fractions were analyzed using 10% SDS-PAGE (Coomassie staining) and evaluated using helicase activity as previously described.

6) RNA extraction and characterization

The cultures obtained were treated with the mirVana ™ miRNA kit (Ambion). Enriched fractions of small RNA (<200 nt), as well as total RNA were obtained. The RNA samples were characterized using RNA 6000 Pico Total RNA chips (Agilent) combined with the Agilent 2100 Bioanalyzer or electrophoresis on denaturing polyacrylamide gel (d PAGE-15% acrylamide) for small RNAs.

7) Analysis by Northern Blot

To confirm the expression and size of the "repeat RNA", hybridizations by Northern Blot were carried out using 10 μg of total RNA or 2 μg of small RNA. The RNA samples were denatured for 5 minutes at 95 ° C in a loading buffer containing 50% formamide followed by immediate cooling on ice. RNA samples were run on denaturing Polyacrylamide gels with 15% urea (d PAGE), electrobiotized on Hybond N + membranes (GE Helathcare) at 20 V for 1 hour using a semi-dry transfer ( Hoefer TE 77, GE Healthcare). The membrane is then cross-linked to UV for 2 min.

Probes (LNA), e.g. 5'-TCA-GAT-TCA-TTA-TCA-G-3 'and 5'- CTG-ATA-ATG-AAT-CTG-A-3', with digoxigenin (DIG) at both ends, were purchased with Eurogentec. Pre-hybridization and hybridization were performed using an Easy Hybridization buffer (Roche Applied Science) at 37 ° C. After hybridization, the membranes were washed twice using a low stringency buffer solution (2X SSC, 0.1% SDS) and a high stringency buffer solution (0.1X SSC 0.1% SDS) for ten minutes at room temperature and fifteen minutes at 60 ° C, respectively.

The membranes were then hybridized with a probe marked with DIG as recommended by the manufacturer (DIG-System, Roche Diagnostics), with the exception of the detection of the hybridized probe which was carried out using a monoclonal anti-digoxin conjugated to horseradish peroxidase (Jackson Immunoresearch, 1: 5000). After several washes, transfers were revealed by chemiluminescence tests (ECL, GE Healthcare). The resulting signal was detected on Hyperfilm ™ ECL (GE Healthcare) using an automated film processor (Hyperprocessor ™, GE Healthcare).

8) In vitro transcription

All in vitro transcriptions were carried out according to the manufacturer's instructions and recommendations (Kit MegashortscriptTM by Thermofisher, ref: AM 1354). Briefly, the DNA templates were hybridized with primers including the T7 promoter and lac operon (SEQ.ID. Nos. 14-15 and 16-17) and mixed with the T7 reaction buffer (1 x), a T7 solution NTP (7.5 mM), the enzyme T7 (2 μl for a reaction of 20 μl) for 4 hours at 37 ° C. The DNA templates were degraded by TURBO DNase (2 U) for 15 minutes at 37 ° C. The RNA transcripts were precipitated with ethanol. The final pellets were resuspended in nuclease-free water.

9) aborted PCR

The DNA matrices were incubated with the complementary RNAs corresponding to the repeat unit of zamilon of 15 nucleotides (SEQ.ID. No. 18-19) as well as an elongation primer at 95 ° C for 5 minutes then by gradually cooling to 25 ° C. All mixtures were incubated with 5 U of Klenow exo- from ThermoFisher (ref: EP0422) for 1 hour at 37 ° C. The controls were carried out with the same conditions at without the RNA or Klenow.

10) Purification of RNAs or DNAs from Polyacrylamide Gel

The recovery of nucleic acid from polyacrylamide bands excised from gels was carried out by the crushing and soaking method as described above (18).

11) RNA and DNA sequencing

The single-stranded DNA samples of 65 nucleotides were sequenced using MiSeq technology (Illumina Inc., San Diego, CA, USA). The RNAseq approach was chosen and the banks were built using Truseq stranded mRNA strategy and were bar-coded with another RNAseq project.

The samples were measured on the Nanodrop and the maximum volume of 16.5 pL, between 36 and 160ng, was involved in synthesizing the second strand DNA. The Truseq strand mRNA procedure was followed from there and all of the purification steps of Ampure were replaced by alcoholic precipitation due to the small size of the DNA fragments. The library profiles were visualized by DNA1000 bioanalyzer (Agilent Technologies Inc, Santa Clara, CA, USA) at 71, 78, 130 base pairs. The final concentration of the banks was 143 and 333 nmol / l for the 6 samples.

Banks were normalized to 2 nM and pooled. After a denaturation step and a dilution to 15 μM, the pool was loaded onto the reagent cartridge, then onto the instrument with the flow cell. Automated cluster generation and sequencing execution were performed on the MiSeq Reagent V3 kit in 150 cycles. Total information of 5 Gb was obtained from a cluster density of 1618 K / mm2 with a quality control filter passing by 85.6% (18,644,000 readings of paired pairs). Within this series, the representations of the index were determined to be 0.69%, 0.47%, 0.84%, 0.65%, 0.76% to 0.94%, respectively. The minimum of 152,675 to the maximum of 302,220 paired readings according to the samples leads to an overall number of 1,407,155 paired readings which have been adjusted and then analyzed.

12) Mimivire test

The 293GFP cells [PP20 / PP21] were seeded on 6-well plates at 150,000 cells / well 24 h before transfection. The cells were transfected using lipofectamine RNAiMAX (Life Technologies) following the protocol recommended by the manufacturer. A total of 30 pmol of RNA was used for the transfection. The transfected cells were analyzed by FACS three days after transfection.

13) SDS-PAGE and Western Blot

The transfected or non-transfected cells were lysed by sonication in a solubilization buffer (7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (w / v) and centrifuged at 10,000 g, 20 min, 4 ° C. Soluble proteins were collected and the concentration was determined using the Bio-Rad De Protein Assay (Bio-rad).

Fifty micrograms of soluble proteins were fractionated on a 10% polyacrylamide gel electrophoresis and then revealed by InstantBIue Protein Stain (Expedeon). In addition, resolved proteins were transferred to a nitrocellulose membrane (Trans-blot

Transfer Medium, Biorad, Hercules, CA, USA) at 100 V for 1 h using a semi-dry transfer unit (Hoefer TE 77, GE

Healthcare, Vél izy-Vi I la cou bl ay, France). The membranes were then blocked in PBS supplemented with 0.3% Tween-20 and 5% skimmed milk powder (PBS-Tween-Milk) for 1.5 h and incubated with mouse monoclonal anti-FLAG antibodies (1: 1000). The immunoreactive bands were detected using an anti-mouse goat immunoglobulin conjugated with peroxidase (GE Healthcare, Vélizy-Villacoublay, France) diluted 1: 5000 in the blocking buffer for 1 h at room temperature. Three washes of 15 min were applied between each step. The immunostained bands were visualized with the chemiluminescence-based kit, as described by the manufacturer (GE Healthcare, Vél izy-Vi I la co u bl ay, France). The resulting signal was captured by a Fusion FX7 imaging system (Vilber Lourmat, France).

14) Secondary structure prediction for RNA hosting two 15 nucleotide GFP repeats

Secondary structures were predicted using the Mfold web server. We have kept the secondary structure with the minimum free energy.

15) Gel digestion and MALDI-TOF spectrometry

The excised bands of the gels were discolored and subjected to digestion in the gel with trypsin (Proteomics grade trypsin, Agilent Technologies, Cedar Creek, TX). The tryptic peptides were then extracted from the gel by successive treatments with water and acetonitrile (H PLC quality). The extracts were combined and concentrated in a Speedvac evaporator. The peptide solution was then deposited on the matrix-assisted laser desorption ionization target (MALDI). Mass analyzes were carried out on a Brüker Autoflex Speed MALDI - time of flight (MALDI-TOF) speed spectrometer (Brüker Daltonique, Wissembourg, France). The mass spectra were calibrated externally using peptides from the tryptic digestion of bovine serum albumin (Brüker). Mass lists of tryptic peptides were used to identify proteins, using Mascot software (Matrixscience). Searches were performed against an internal database containing the labeled sequence of MIMI_R350 (Helicase like), including a polyhistidine tag at the amino-N-terminus.

Table 1:

Annexe 1 :

Annex 1 :

SEQ.ID. No. 22 = insert synthesized and cloned by genscript into pET-22b (+) at the Xba I / Xho I restriction sites successively comprising the sequences of: a) T7 promoter (SEQ.ID. No. 31) TAATACGACTCACTATAG; b) operonLac (SEQ.ID. N ° 32) G AA TTGTGA G CG G A T AA CAA TTCQ

 c) restriction site Xba 1 = TCTAGA;

d) Shine-Dalgarno sequence: AAGGAGA; e) first underlined sequence = SEQ.ID. N ° 7 of the modified 349 gene including the unit repeated 4 times drawn from the tetracycline resistance gene SEQ.ID. N ° 23 = CACCCTGGATGCTGT_with the 3 spacer sequences interspersed SEQ.ID. N ° 26,27 and 28, f) first spacer between the first and second underlined sequences including the terminator T7 SEQ.ID. N ° 33 CTA GCA T AA CCCCTT GGGGCCTCTAAA CGGGTCTTGA GGGG / / / / / IG, Promoter sequence T7, SEQ.ID. N ⁰ 31 TAATACGACTCACTATAG e. t the sequence of the Lac operator, SEQ.ID. # 32: GAATTGTGAGCGGATAACAATTCC and a Shine-Dalgarno Sequence; g) second underlined sequence = R350 gene sequence; h) second spacer between the second and third underlined sequences including a Shine-Dalgarno sequence, and i) third underlined sequence = R354 gene sequence; j) restriction site Xho I = CTCGAG; k) sequence of the poly histidine tag

SEQ.ID. N ° 34: CACCACCACCACCACCAQ i) the terminator T7 SEQ.ID.N ° 33:

CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG

SEQ.ID.N ° 22: sequences organized in operon including the modified R349 gene with specific sequence of 15 nucleotides of the tetracycline resistance gene (SEQ ID N ° 23) and the R350 and 354 genes.

TAA TACGACTCACTA 771 GGG GAA TTGTGA GCGGA T AA CAA TTCCCCX CT AG AAAT AATTTT GT TT AACTTT NKG AA GGA GAT AT AG AT ATGG AATT AATT AGT CGT GT CTTT ACT CAT GG AG AAAA T ATPT ACTT GTT AGTT CT ACAAAT AAGTT AT AT AT AT AT AAT G AAT ATGGTT CAT GT GGTTT CAAAAT AGGT ACAG AT AAAACTT AT ATT G AAAGT CCAGT AT AT AT ATT G ACATT AAATT A GAT GAT GAT GATT CT GTT AAAGCGTTTT ATT CTT GT AATTT ATT CACG AT G ATTCAT ACAT CCA AAGG AAAAATTT AT ATTT ATCA GT GGTGG AGGT G AAAT CG AT GCAT AT GAT A GTGAAAGTGATGTTGGAAGTGATGCTGAGTCTGATGCTGAGTCTGATGCTGAAT ATAG ACT CAAAAT AAT ACAAAT ACT CCC AT AAAT AAT AT CACACT AATT AATTT G GATT CAT CAAT G AAT CTG AT CAAT CAT GT AGT G ATPT G ACT GT GG ACCT CG AT CCACCCT GGATGCTGTTG AAGAAG IIIII GTTG ATCGT AAT GAT AAT AATT CAG AT AAT ATT GGT AATT CAAAT AGT AT CA CCCT GG ATGCTGTATCTAT G ACT G AAATAG GT AAATT AAAACC A TGGTAG AT G AAATPT GTCT CAAAAAAAT ATT AA TT CTGTT GG AATT AACG AT ATT GTT ATT C GT CCAAG CG G ACAGT ATGTG AGT AAT ATGG GATAT ATT ACAG AAT CAG GTAGTGTAT CATTT C AT ACCAAT AAAAAT GGTTT CTT ATT ATT CAAGT CCAAT GTT CAT G AAAT CCTTTT CGTT GT ATAG AAG AAAAAT AATTTT ATTT ATTTGGCCAT ACCATTT AAAAAAT ACCAGGCA TCTCT AAG AG AT ATT GCT CCTTTT CAT ACAATT AAT AAAACAAAAAAT GGT ATT AAAT GG AAGT ATTT CAAAAT AGT GTTT CCATTT G ACACT G AAAAAAT AG AATTTT GTG AATA ATTAT ATTAT ATTAT II ATTT CATT CT AC AAAAAT GT AAATT ATT AT CCT T CTT GG AT AT ATTT CAAAT CT G AG ATT GAT ATT AAT AGT AAAAAT AT GTT CTTTT CAAGT GATA CT AAT AGT GTGTAT GTT AAAG AT AAT AACAACGT GT AT AAAT ACCAT AATTT CAAT AATT CT CT TG AAAAAT ACAT CG AT AACAAACT CG ACTT GG ATGTCGT AATT GTG CCT G ATAGTT AT AG ACC AAT GG AAAT G AG ACT ACT ATT AAAGTT AGGTAT AACATT GT AT AGCG ATT ACAATTTT AAT GG AT GT G AGG AT GAT G AAG AACAT ATPT CG AAATT AT G AAAA CAAT ATGT ACCT CAT ATT AT CGGT CT G AATT ATPT G AAAGTTT CATT GT AGTT ATT GT CAAT AAT CCAAAT ATGTT G ACG AT AACAACTGACGATGGTAAAATTTTCTTTAACATTCATGATATTAC IIIII ACAAAAGATTTTAT AAT GG AAT CGTTT ATCTTG AT AATGGTT CGTT ATPT AT CT CACAG AT AGT G AAATTT CAG AT CAAAAT GT ATGG AAACT AACTGT AT AGCT GT T ACCT ATTT AATTT ACCGG ACAAAATT G AT G AAATTT ACT CTT CGTCT G AATTT ATT GTT CT AA AATT AATT GGA MCA AAT AIIIII ATTATCCGGTTGAAAATTTTGATACTGCTCAAGATTTTAA G ACAAG AT GT GGGG AAATTT CATT G AAAAAT AATTAT ATAT AATTC AT CAT ACT ACAGT AT CT ATT AAT ATT G AT ACGG ATT GT ACT ACT CAT AATT CTTT CG AG AG ATT GTTT AT CCT G ACACAAT CT CTT AGTT ATT CCGCAG AAT ATT CT ATT CG AATT GT AG ACG ACAAAAAT ATTGGTTTT GGT G ATGGGCCT AAAATT G AATPT GTG AAT CG GCT ATT AT G CAATTTT ATT AT AAGT ATTT AATT G CT CAT AATPT C ACACAG AGTTT AA TTT ACAAG A ATTT G CCAA ACT GA AACCCACCG A AATT AA AT ATTT G GG AT C AAT GTTT CAT AT GGTTATCTGT CAAAAT AATT CTT CGTTACCG ATT AG ATT ACCT CT AGCTTTT GCTGT AG AAAT TT AT GG AAAAG AACCCACAATT G AT G AATT GG AAT ATTTTGCTTGT AAT G AAG AT G AAACT GG ATT CAAACAT ATTT AT CCAGCCAAAT ACAAT CCCG AATT AGTT AAAG AATT CGGTTAT G AAT CT T AT AA GATT AATT AT G AAG AT G AT ACT G AT AAAAAT AT CT TG ACG AAAAAAT ATT GCG AGCA ATT AGCTGCCGGTTTT AAAAG AT ACGGCAAT AT CAAG AACA T AAAACAAAT G AATTT ACCCACATT GG ACT ATT ACATTT CT GGCCCAT ACAAAATT AAT AG AAC CAT ATT AATT AAT AAT CTT GTTTT AT CGGG AGGT AAGG AT AAAAAT AAT AATT ATTT ATTT GATT G AATT AAAAAT CTT GCT AAAAAATT GG ACA GCAT CAACTT GTGT AAG ACCAG AT AACAAAT AT AG AATT AT CATT ATTT CCAAAT CT AAAAACG CT AAAGC AG GT ATT CG ATTT GGT ACTT GT AATTT AG AAATT CAT AT CG ACG AAAAAAT GTTG G AT G AACAT AAT ATT GAT AT ACAGT CAAAG AGGTTTT AATT ACACCTGCTCAAGG ATT CAAAG ATT AAG AT CCGGCTGCTAACAAAGCCCG AAAGG AAGCT G AGTTGGCT GCT GCCACCGCT G AGCAA T AA CTA GCA TA A CCCCTTGGGGCCTCTAAA CGGGT ATGGTGA GTC CGA GTC CGA GTC GTA CGA GTC GTA CGA AA 7TÛXCACT AG AAAT AATPT GTTT AACTTT NKG AA GG AG AT AT AGCAAT G AACCGT CGT AACCGT AGCAACG ACCT G AACCCGGAGCCGAGCATCGAAAACCCGAACAACCAGATTGCGGGAATTACGGGTGTGTGTGTGTGTCT ACAAGCTGCCGG AG AT CATT CGT AAAG AG AACG AAG ACC CGTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCAGGAATTCGTGGGCCAATACCTG AACCCGCAGGGTCCGTATACCAGCATCCTGCTGTACCATGGTCTGGGTAGCGGCAAGACCGC G AGCGCG AT CAACCT G AT G AACATT CTGT ACAACT AT G ACAACGGCACCAACTTT AT CGTT CT GATTAAAGCGAGCCTGCACAACGACCCGTGGATGCAGGACCTGAAGGAGTGGCTGGGTCGTG AT CCGAGCG AACAG AACGT GG ACAACGTT ACCAAGCTGG AT CGTT ACAAAAACAT CCACTT CG T GCACT AT G ACAGCCCGTT CGCGG AT AGCAGCTTTAT G AGCGTT ATT AAG ACCCTGG ACCT GA GCAAACCG ACCAT GT ACAT CATT G AT G AGGCGCACAACTTT AT CCGT AACGT GT AT AGCAACA TT AACAGCAAACT GGGCAAGCGT GCG AAAGTT AT CT ACG AGT ACAT CAT G AAGG ACAAGCGT G AAAACAAG AACACCCGT AT CGTGCT GATT AGCGCG ACCCCGGCG AT CAACACCCCGTT CG AA CTGGCGCTGATGTTTAACCTGCTGCGTCCGGGTATTTTCCCGAGCAGCGAGCTGGATTTCAA CCGT ACCTTT GT G CAGC AAAGCAGCTACCCG AT CTC G AACCCG AT G AAG AAAAACAT CTWG GA GCGTCGTATCCTGGGCCTGGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCGCGTC AAG AACT G GTAA ACAT CAACCTGCCG AT G AGCGCGT ACCAGT AT G AT AT CT ACCGT ATPT CG AGAAACTGGAGGCGGAAATTCAAGAACGTGCGCGTCGTCGTGGCAAGCAGAGCCAACTGTAC CGT ACCT AT ACCCGT CAGGCGT GCAACTT CGT CTWG CCGT ACGTT AACAT G AACGT G AACGGT GAACTGCGTCCGCGTCCGGGCAAGTTCCGTCTGAGCGAAAAACTGGCGGACGATTTTAGCAA GGGCAAAAACCTGGACGTTCCGGATACCGAGAAAGAAATCCTGAACAAGTATACCAAAGCGA TT G AG AACT ACCT G AACG AG CAGC AACGTT ATPT CAG AACAT CAACAAG AAAG ACGCGG AG A ACGGTCGT CGTA CATT AACG ACCTGG AT G AATT CAAG AAAGGCTTT GGT ACCAAGTT CAACA GCTTT CT GCAGT ACT AT CAAAGCG AGGGT CCGCGT AGCAGCCT GCT G ACCG AAAT GT ACAAC T GCAGCCCG AAAAT GCTGGCG AT CGCGTT CAT G ACCT AT ATT AGCCCGGGCAAGGT G AT G AT CT ACAGCAACT AT GTGGTT ATGG AAGGCAT CG ACGTT AT G AAAATTT T CGTCTG ATCGG TTT CAACG ATPT ACG ATCGCGCGT G AGT ACATGGGCTATT GCG AAT ACCACGGT CGT AT CG A CCCG AAGG AT CGT GT GCGT AT CAAG AACAT GTT CAACG ACAAG AACAACGT GT ACGGCAACAA GTGCAAAGTT AT AGGCTG AGTG AGCAC GG AACCGT ATT GG CAGC AAGTT CGT AT CCAGCAAGT G ATTGGCCGTGGT GTT CGT CAAT GCAGCCACCGT G ACCTGCCG AT G AGCG AGCGT ATCGTGG AT ATTT ACCGTT A T AAGGTT AT CAAACCGG AAAACCT GG ACCCGG ACG AT ACCGTGCGT CAAAGCACCG CAG AGT ACGTTGAAGATCAGGCGAAGAGCAAAGCGAACCTGATTGAGAGCTTCCTGGGCGCTATGAAA GAAGCGGCGGTTGATTGCGAGCTGTTTAAGGAACACAACATGATGAGCCAGAGCTACTATTG CTTCAAATTTCCGGAGAGCGCGGTGACCAAGACCAACGTTGGCCCGGCGTACCGTGAAGACA TCAAGGACGATGTGAAATATGATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGCGTATTC GTGTGGTT AAGGT G AACGCGGTTT ACCAAAT CAACACCG ACAACAACAACCCGGT GT AT AGCA GCCCG ACCAAGT ACTGGT AT AACAAG AAAACCGGCAT GGTTT AT G ACTT CG AG ACCCACT ACC CGGTGGGT CAGGTT G AATTT AT CG AT AACCTGCCG AACAAGCT GG ACAAAG AT ACCT ACAT CA T GCGT ATT G AT GT G AT CATG CCG AG T AGCGTT AACACCT AACACT AG AAAT AAT TTT GTTT AACTTT AAG AAG G AG AT AT AGCG AT G ACCG ACATT AG CT ACT AT AACAACG AG AT CG AT AAAATT CT GTGG AACAT CCT GGGT G ACG ATT ATTT CACCCAAG ACG AATTT G ACG AT CT G GT G AACAGCGTTGCG AACACCATTT ACCAGT AT G G ACAACG GTAA AGCAT CG AT AAGCTG AAA GT CAT AT G AATT GC CGTT AT CTC G AACAAGTT CAAGCT GTGCT ACAT CT CAG CTC AT AT AACG ACAGC G G AACCAAGT STW ACG AG AAG GGT AAAAGCGTT AGCAAAACCAT CGGCAAG AACAG CACCAACGACGATGAGGACGATGACGAAGATATCGCGGTGATTAAGCTGAGCGATATTGAGG CGGGCGAAAACTGGTTCAAGAAAAGCCCGAAAATCAGCAGCAAGCAGTTTCAAAGCGTTGAC AAAGTT G AGGT GGCG ACCT AAG ACCT G AT CAGCCACAAGCACG ATT ACCCG AAAGGACAGAGAGAGAGGAGGAC ATGACGAC

GTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTTCACCACGGTAACAAGTATGAACAAATCG GCACCATGTTCTACAGCTTTCGTAACAACGTTGAGGTGGGTGAGTACGGCCTGCTGCAGCAC AGCGGTCACAAGTTTATCGCGGCGAGCCCGGATGGCATCTGCAGCAAGAAAGCGAACACCGG TGGCCTGAGCAAACTGGTGGGTCGTCTGCTGGAGATTAAGTTCCCGTTTAGCCGTGAAATCA ACAACAGCGGT G AT AT AT CT CTGG ACGGCG GCCCGCACT ACT ATPT CT GCAGGT GCAAACCC

AGCTGTATGTT CAGC AG AT GG ACG AATGCG ACTT CCTGCAGTGCAAAATT G CAG AGT ACG AT AGCTGGG AAG CADTC GTG AAGG AT AGCAACCCG AT CGTT CCGGGT CT G AGCAAAACCACCAA CTC GG AG AAGGGCT GCCT GATT CAGCT G AGCG ACAAAAACCT G AT CGGCAGCG ACG ACAAGG AAAAATGCCTGTATAACAGCAAATACATCTATCCGCCGAAGCTGCACATGACCAACGAGGAAA T CG AG AAGTGG ATT AGCAGCG STW CAT G AACT ACCACAACAACG ACCT G AGCG AG AACT AT A

T GATT AT CGT G GT G TC G AT CT ACTGGCGT AGCCAAGTT ACCTGCAACCT GATT AAGCTG AACA AAGAAGCGTTCGAGGAAAAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTTCTGTTTTATC GTCAGCACAGCGACAAGCTGGATAAACTGATTAAGTTTGTGGAGAAGGTTAAAGAAGATAAC AGCGCGGAGATTTTCAGCTACATCAACGAAGACTTTCTGAGCCTGAACAAAGATAGCAAGTAC G AGCCGCTGT AT ACGG AAG AG CAGC GGCGT AAT AAG AT STW AACCAAAT CAAGGCG AAG AA

AGCGCAGATGT ACAAG AACAAG AGCTACAACAAGT ACACCAAGTTCAGCAACCTCGAG CA CCA

CCA CCA CCA CCA CT G AG AT CCGGCT GCT AACAAAGCCCG AAAGG AAGCT G AGTTGGCT GCTG CCACCGCTG AGCAAT AA CTA GCA T AA CCCCTTGGGGCCT C

TG.

Annex 2:

SEQ.ID. No. 37 = insert synthesized and cloned by genscript in pET-22b (+) at the Xba I / Xho I restriction sites successively comprising the sequences of: a) T7 promoter (SEQ.ID. No. 31) TAATACGACTCACTATAG; b) operonLac (SEQ.ID.N ° 32) G AA TTGTGA G CG G A T AA CAA TTCC;

 c) restriction site Xba 1 = TCTAGA;

 d) Shine-Dalgarno sequence: AAGGAGA; e) first underlined sequence = SEQ.ID. N ° 38 of the modified 349 gene including the unit repeated 4 times drawn from the tetracycline resistance gene SEQ.ID. N ° 39 = CGGCTCTTACCAGCC_with the 3 spacer sequences interspersed SEQ.ID. N ° 26,27 and 28, f) first spacer between the first and second underlined sequences including the terminator T7 SEQ.ID. N ° 33 CTA GCA TAA CCCCTTGGGGCCTCTAAA CGGGTCTTGA GGGGTTTTTTG, the sequence of

Promoter T7, SEQ.ID. N ⁰ 31 TAATACGACTCACTATAG e. t the sequence of the Lac operator, SEQ.ID. # 32: GAATTGTGAGCGGATAACAATTCC and a Shine-Dalgarno Sequence; g) second underlined sequence = R350 gene sequence; h) second spacer between the second and third underlined sequences including a Shine-Dalgarno sequence, and i) third underlined sequence = R354 gene sequence; j) restriction site Xho I = CTCGAG; k) sequence of the poly histidine label SEQ.ID. N ° 34 \ CAC CAC CAC CAC CAC CAC; i) the terminator T7 SEQ.ID.N ° 33:

CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG

SEQ.ID.No.37: sequences organized in operon including the modified R349 gene with specific sequence of 15 nucleotides of the tetracycline resistance gene (SEQ ID No.39) and the R350 and 354 genes.

T AAT ACG ACT CACT AT AGGGG AATT GT G AGCGG AT AACAATT CCCCT CT AG AAAT AATPT GT TT AACTTT AAG AAGG AG AT AT AG AT ATGG AATT AATT AGT CGT GT CTTT ACT CAT GG AG AAAA T ATPT ACTT GTT AGTT CT ACAAAT AAGTT AT AT ATT ATGGGT AAT AAT G AAT ATGGTT CAT GT

GGTTT CAAAAT AGGT ACAG AT AAAACTT AT ATT G AAAGT CCAGT AT AT ATT G ACATT AAATT A

G AT G AT G AT GATT CT GTT AAAGCGTTTT ATT CTT GT AATTT ATT CACG AT G ATTCAT ACAT CCA

AAGG AAAAATTT AT CT ATCAAG AT CATTT ATTT GT GGTGG AGGT G AAAT CG AT GCAT AT G AT A

GTGAAAGTGATGTTGGAAGTGATGCTGAGTCTGATGCTGAGTCTGATGCTGAATCAGATTCA G A AA AT CAT ACT CAAAAT AAT ACAAAT ACT CCC AT AAAT AAT AT CACACT AATT AATTT G GATT

CAT CAAAT AATT CCACT CAAT CCGGCT CTT ACCAGCCT AAT G AAT CCGGCTCTT ACCAGCCCAA

T G AAT CT G AT CAAT CAT GT AGT G ATPT G ACT GT GG ACCT CG AT CCGGCT CTT ACCAGCCT GA

AGAAG I I I I GTT G AT CGT AAT G AT AAT AATT CAG AT AAT ATT GGT AATT CAAAT AGT AT CGG

CT CTT ACCAGCCAT CT AT G ACT G AAAAAGCTGG AAT AATTTT GTT AAACG AAATT AAAACCAT GGTAG AT G AAATTTT GT CT CAAAAAAAT ATT AATT CT GTTGG AATT AACG AT ATT GTT ATT CG

T CCAAGCGG ACAGT ATGTG AGT AAT ATGGG ATAT ATT ACAG AAT CAGGT AGT GTAT CATTT CA

T ACCAAT AAAAATGGTTTCTT ATT ATT CAAGTCCAAT GTTCAT G AAAT CCTTTT CGTT G AAG AA

AT GTTT AT GT ACT CT AAAAAT AATTTT ATTT ATTT GGCCAT ACCATTT AAAAAAT ACCAGGCAT

CT CT AAG AG AT ATT GCT CCTTTT CAT ACAATT AAT AAAACAAAAAAT GGT ATT AAAT GG AAGT A TTT CAAAAT AGT GTTT CCATTT G ACACT G AAAAAAT AG AATTTT GT G AT AATTTCTTTT AT ACT

T AT G AAT CCAAT ACTT GTT AT CAT CAT GTT ATTT CATT CT ACAAAAAT GT AAATT ATT AT CCTT

CTT GG AT AT ATTT CAAATCT G AG ATT G AT ATT AAT AGT AAAAAT AT GTT CTTTT CAAGT G AT AC

T AAT AGT GT GT AT GTT AAAG AT AAT AACAACGT GTAT AAAT ACCAT AATTT CAAT AATT CT CTT

G AAAAAT ACAT CG AT AACAAACT CG ACTT GG ATGT CGT AATT GTGCCT G AT AGTT AT AG ACCA AT GG AAAT G AG ACT ACT ATT AAAGTT AGGT AT AACATT GTAT AGCG ATT ACAATTTT AAT GG A

T GT G AGG AT G AT G AAG AACAT ATPT CG AAATT AT G AAAAAT CAAT AT GT ACCT CAT ATT AT C

GGTCT G AATT ATTTT G AAAGTTT CATT GT AGTT ATTGT CAAT AAT CCAAAT AT GTT G ACG AT A

ACAACTGACGATGGTAAAA I I I I CTTTAACATTCATGATATTAC I I I I ACAAAAGATTTTATA

AT GG AAT CGTTT AT CTT G AT AAT GGTT CGTT ATTTT AT CT CACAG AT AGT G AAATTT CAG AT C AAAAT GT AT GG AAACT AACTGG AT GT CAATT GTGT G AGCT AGCTG ATT CT ACCAT AT AT AG II ACCT ATTT A ATTT ACCG G AC AA AATT G AT GA AATTT ACT CTT CGT CT GA ATTT ATT GTTCT A AA ATT AATT GG AAACAAAT AIIIII ATTATCCGGTTGAAAATTTTGATACTGCTCAAGATTTTAAG ACAAG AT GTGGGG AAATTT CATT G AAAAAT AATT CAGT AATT CAGT ATT AGT AGT ATTAG AAAAGTT AT CAT ACT ACAGT AT CT ATT AAT ATT G AT ACGG ATT GT ACT A CT CAT AATT CTTT CG AG AG ATT GTTT AT CCT G ACACAAT CT CTT AGTT ATT CCGCAG AAT ATT CT ATT CG AATT GT AG ACG ACAAAAAT ATT GGTTTTGGT G AT GGGCCT AAAATT G AATPT GTG A ATCGG CT ATT AT GC AATPT ATT AT AAGT ATTT AATT G CT CAT AATTTT C ACACAG AGTTT AAT TT ACAAG AATTT GCCAAACT G AAACCCACCG AAATT AAAT ATTT GGG AT CAAT GTTT CAT AT G GTT AT CT GT CAAAAT AATT CTT CGTT ACC AG ATT ACCT CT AGCTTTT GCTGT AG AAATTT AT GG AAAAG AACCCACAATT G AT G AATTGG AAT ATTTT GCTT GT AAT G AAG AT G AAACTGG AT T CAAACAT ATTT AT CCAGCCAAAT ACAAT CCCG AATT AGTT AAAG AATT CGGTT AT G AAT CTT ATG ATAATT AT AATT AT G AAG AT G AT ACT G AT AAAAAT AT CTT G ACG AAAAAAT ATT GCG AGC AATT AGCT GCCGGTTTT AAAAG AT ACGGCAAT AT CAAG AACAT A AAACAAAT G AATTT ACCCAC ATT GG ACT ATT ACATTT CT G GCCC AT ACAAAATT AAT AG AACCA T ATT AATT AAT AAT CTT GTTTT AT CGGG AGGT AAGG AT AAAAAT AAT AAT ATATT ATTT GATT AACT CTTT GT CT G AAAAT G AATT AAAAAT CTTGCTAAAAAATT GG ACAGC AT CAACTT GTGT AAG ACCAG AT AACAAAT AT AG AATT AT CATT ATTT CCAAAT CT AAAAACG CT AAAGCAGGT ATT CG ATTT GGT ACTT GT AATTT AG AAATT CAT AT CG ACG AAAAAAT GATG G AT ACAGT CAAAG AGGTTTT AATT ACACCT GCT CAAGG ATT CAAAG ATT AA GATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGG IIIIIIG AG AT CT CG AT CCC G CG A AATT AAT ACG ACTCACTAT AG GG GA ATT GTG AG CG G AT AACA ATT CCCCACT AG AA AT A ATTTT CTWG AACTTT AAG AAGG AG AT AT AGCAAT G AACCGT CGT AACCGT AGCAACG ACCT GA ACCCGGAGCCGAGCATCGAAAACCCGAACAACCAGATTGCGGAGGAATTCCCGGGTAACAAC AGCGT GT AT AAAAGCG ACGGCT ACGTT G AT CT G AAG AACAACGGT CGT CT GTACT CTG ATG AAACAAT ACAAGCT GCCGG AG AT CATT CGT AAAG AG AACG AAG ACCC GTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCAGGAATTCGTGGGCCAATACCTGA ACCCGCAGGGTCCGTATACCAGCATCCTGCTGTACCATGGTCTGGGTAGCGGCAAGACCGCG AGCGCG AT CAACCT G AT G AACATT CT GT ACAACT AT G ACAACGGCACCAACTTT AT CGTT CT G ATTAAAGCGAGCCTGCACAACGACCCGTGGATGCAGGACCTGAAGGAGTGGCTGGGTCGTGA T CCGAGCG AACAG AACGT GG ACAACGTT ACCAAGCTGG AT CGTT ACAAAAACATCCACTT CGT GCACT ATG ACAGCCCGTTCGCGG AT AGCAGCTTTATG AGCGTT ATT AAG ACCCTGGACCTGA GCAAACCG ACCAT GT ACAT CATT G AT G AGGCGCACAACTTT AT CCGT AACGT GT AT AGCAACA TT AACAGCAAACT GGGCAAGCGT GCG AAAGTT AT CT ACG AGT ACAT CAT G AAGG ACAAGCGT G AAAACAAG AACACCCGT AT CGTGCT GATT AGCGCG ACCCCGGCG AT CAACACCCCGTT CG AA CTGGCGCTGATGTTTAACCTGCTGCGTCCGGGTATTTTCCCGAGCAGCGAGCTGGATTTCAA CCGT ACCTTT GT G CAGC AAAGCAGCTACCCG AT CTC G AACCCG AT G AAG AAAAACAT CTWG GA GCGTCGTATCCTGGGCCTGGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCGCGTC AAG AACT G GTAA ACAT CAACCTGCCG AT G AGCGCGT ACCAGT AT G AT AT CT ACCGT ATPT CG AGAAACTGGAGGCGGAAATTCAAGAACGTGCGCGTCGTCGTGGCAAGCAGAGCCAACTGTAC CGT ACCT AT ACCCGT CAGGCGT GCAACTT CGT CTWG CCGT ACGTT AACAT G AACGT G AACGGT GAACTGCGTCCGCGTCCGGGCAAGTTCCGTCTGAGCGAAAAACTGGCGGACGATTTTAGCAA GGGCAAAAACCTGGACGTTCCGGATACCGAGAAAGAAATCCTGAACAAGTATACCAAAGCGA TT G AG AACT ACCT G AACG AG CAGC AACGTT ATPT CAG AACAT CAACAAG AAAG ACGCGG AG A ACGGTCGT CGTA CATT AACG ACCTGG AT G AATT CAAG AAAGGCTTT GGT ACCAAGTT CAACA GCTTT CT GCAGT ACT AT CAAAGCG AGGGT CCGCGT AGCAGCCT GCT G ACCG AAAT GT ACAAC T GCAGCCCG AAAAT GCTGGCG AT CGCGTT CAT G ACCT AT ATT AGCCCGGGCAAGGT G AT G AT CT ACAGCAACT AT GTGGTT ATGG AAGGCAT CG ACGTT AT G AAAATTT T CGTCTG ATCGG TTT CAACG ATPT ACG ATCGCGCGT G AGT ACATGGGCTATT GCG AAT ACCACGGT CGT AT CG A CCCG AAGG AT CGT GT GCGT AT CAAG AACAT GTT CAACG ACAAG AACAACGT GT ACGGCAACAA GTGCAAAGTT AT AGGCTG AGTG AGCAC GG AACCGT ATT GG CAGC AAGTT CGT AT CCAGCAAGT G ATTGGCCGTGGT GTT CGT CAAT GCAGCCACCGT G ACCTGCCG AT G AGCG AGCGT ATCGTGG AT ATTT ACCGTT A T AAGGTT AT CAAACCGG AAAACCT GG ACCCGG ACG AT ACCGTGCGT CAAAGCACCG CAG AGT ACGTTGAAGATCAGGCGAAGAGCAAAGCGAACCTGATTGAGAGCTTCCTGGGCGCTATGAAA GAAGCGGCGGTTGATTGCGAGCTGTTTAAGGAACACAACATGATGAGCCAGAGCTACTATTG CTTCAAATTTCCGGAGAGCGCGGTGACCAAGACCAACGTTGGCCCGGCGTACCGTGAAGACA TCAAGGACGATGTGAAATATGATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGCGTATTC GTGTGGTT AAGGT G AACGCGGTTT ACCAAAT CAACACCG ACAACAACAACCCGGT GT AT AGCA GCCCG ACCAAGT ACTGGT AT AACAAG AAAACCGGCAT GGTTT AT G ACTT CG AG ACCCACT ACC CGGTGGGT CAGGTT G AATTT AT CG AT AACCTGCCG AACAAGCT GG ACAAAG AT ACCT ACAT CA T GCGT ATT G AT GT G AT CATG CCG AG T AGCGTT AACACCT AACACT AG AAAT AAT TTT GTTT AACTTT AAG AAG G AG AT AT AGCG AT G ACCG ACATT AG CT ACT AT AACAACG AG AT CG AT AAAATT CT GTGG AACAT CCT GGGT G ACG ATT ATTT CACCCAAG ACG AATTT G ACG AT CT G GT G AACAGCGTTGCG AACACCATTT ACCAGT AT G G ACAACG GTAA AGCAT CG AT AAGCTG AAA GT CAT AT G AATT GC CGTT AT CTC G AACAAGTT CAAGCT GTGCT ACAT CT CAG CTC AT AT AACG ACAGC G G AACCAAGT STW ACG AG AAG GGT AAAAGCGTT AGCAAAACCAT CGGCAAG AACAG CACCAACGACGATGAGGACGATGACGAAGATATCGCGGTGATTAAGCTGAGCGATATTGAGG CGGGCGAAAACTGGTTCAAGAAAAGCCCGAAAATCAGCAGCAAGCAGTTTCAAAGCGTTGAC AAAGTT G AGGT GGCG ACCT AAG ACCT G AT CAGCCACAAGCACG ATT ACCCG AAAGGACAGAGAGAGAGGAGGAC ATGACGAC

GTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTTCACCACGGTAACAAGTATGAACAAATCG GCACCATGTTCTACAGCTTTCGTAACAACGTTGAGGTGGGTGAGTACGGCCTGCTGCAGCAC AGCGGTCACAAGTTTATCGCGGCGAGCCCGGATGGCATCTGCAGCAAGAAAGCGAACACCGG TGGCCTGAGCAAACTGGTGGGTCGTCTGCTGGAGATTAAGTTCCCGTTTAGCCGTGAAATCA ACAACAGCGGT G AT AT AT CT CTGG ACGGCG GCCCGCACT ACT ATPT CT GCAGGT GCAAACCC

AGCTGTATGTT CAGC AG AT GG ACG AATGCG ACTT CCTGCAGTGCAAAATT G CAG AGT ACG AT AGCTGGG AAG CADTC GTG AAGG AT AGCAACCCG AT CGTT CCGGGT CT G AGCAAAACCACCAA CTC GG AG AAGGGCT GCCT GATT CAGCT G AGCG ACAAAAACCT G AT CGGCAGCG ACG ACAAGG AAAAATGCCTGTATAACAGCAAATACATCTATCCGCCGAAGCTGCACATGACCAACGAGGAAA T CG AG AAGTGG ATT AGCAGCG STW CAT G AACT ACCACAACAACG ACCT G AGCG AG AACT AT A

T GATT AT CGT G GT G TC G AT CT ACTGGCGT AGCCAAGTT ACCTGCAACCT GATT AAGCTG AACA AAGAAGCGTTCGAGGAAAAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTTCTGTTTTATC GTCAGCACAGCGACAAGCTGGATAAACTGATTAAGTTTGTGGAGAAGGTTAAAGAAGATAAC AGCGCGGAGATTTTCAGCTACATCAACGAAGACTTTCTGAGCCTGAACAAAGATAGCAAGTAC G AGCCGCTGT AT ACGG AAG AG CAGC GGCGT AAT AAG AT STW AACCAAAT CAAGGCG AAG AA

AGCGCAGATGTACAAGAACAAGAGCTACAACAAGTACACCAAGTTCAGCAACCTCGAGCACCA CCACCACCACCACT G AG AT CCGGCT GCT AACAAAGCCCG AAAGG AAGCT G AGTTGGCT GCTG CCACCGCTG AGCAAT AACT AGCAT AACCCCTTGGGGGTGTCT

SEQUENCE LISTING

SEQ. ID. N ° 1: protein R350 of Mimivirus

MNRRNRSNDLNPEPSIENPNNQIAEEFPGNNSVYKSDGYVDLKNNGRLFPIWILKN FKQYKLPEIIRKENEDPCNVQVKLELRKYQEFVGQYLNPQGPYTSILLYHGLGSGKT ASAINLMNILYNYDNGTNFIVLIKASLHNDPWMQDLKEWLGRDPSEQNVDNVTKL DRYKNIHFVHYDSPFADSSFMSVIKTLDLSKPTMYIIDEAHNFIRNVYSNINSKLGK RAKVIYEYIMKDKRENKNTRIVLISATPAINTPFELALMFNLLRPGIFPSSELDFNRT FVTESSYPILNPMKKNMFERRILGLVSYYIGATPDLYARQELKYINLPMSAYQYDIY RIFEKLEAEIQERARRRGKQSQLYRTYTRQACNFVFPYVNMNVNGELRPRPGKFRL SEKLADDFSKGKNLDVPDTEKEILNKYTKAIENYLNETERYFQNINKKDAENGRTII NDLDEFKKGFGTKFNSFLQYYQSEGPRSSLLTEMYNCSPKMLAIAFMTYISPGKVMI YSNYVVMEGIDVMKIYFRLIGFNDFTIAREYMGYCEYHGRIDPKDRVRIKNMFNDK NNVYGNKCKVIMLSPSATEGIQLLDIRQEHIMEPYWTEVRIQQVIGRGVRQCSH RD LPMSERIVDIYRYKVIKPENLDPDDTVRQSTDEYVEDQAKSKANLIESFLGAMKEAA VDCELFKEHNMMSQSYYCFKFPESAVTKTNVGPAYREDIKDDVKYDSGLNSKNSIV ERIRVVKVNAVYQINTDNNNPVYSSPTKYWYNKKTGMVYDFETHYPVGQVEFIDN LPNKLDKDTYIMRIDVIIPSITGSVNT

SEQ. ID. N ° 2: protein R354 from Mimivirus

MTDISYYNNEIDKILWNILGDDYFTQDEFDDLVNSVANTIYQYDNEVSIDKLKVIIE FVILNKFKLCYIYDNDSILNQVKYEKKSVGSKTIGKNSTNDDEDDDEDIAVIKLSDIE AGENWFKKSPKISSKQFQSVDKVEVATYEDLISHKH DYPKEIYKESHYIRRNTRLDV IKKIPQFEQKSKEWLKQRTESLTATAISVVFDEDPYKHPIVILLDKCGRGLPFVENKF VHHGNKYEQIGTMFYSFRNNVEVGEYGLLQHSGHKFIAASPDGICSKKANTGGLSK LVGRLLEIKFPFSREINNSGDLDGDICPHYYFLQVQTQLYVTEMDECDFLQCKIDEY DSWEDFVKDSNPIVPGLSKTTNLEKGCLIQLSDKNLIGSDDKEKCLYNSKYIYPPKL HMTNEEIEKWISSEIMNYHNNDLSENYMIDRVIYWRLSQVTCNLIKLNKEAFEEKI PLLQQFWDYVLFYRQHSDKLDKLIKFVEKVKEDNSAEIFSYINEDFLSLNKDSKYEP LYQEETEWRKKYNQIKAKKAQMYKNKSYNKYTKFSN

SEQ. ID. N ° 3: R350 gene from Mimivirus ATGAACCGTCGTAACCGTAGCAACGACCTGAACCCGGAGCCGAGCATCGAAAAC CCGAACAACCAGATTGCGGAGGAATTCCCGGGTAACAACAGCGTGTATAAAAGC GACGGCTACGTTGATCTGAAGAACAACGGTCGTCTGTTCCCGATCTGGATTCTG AAGAACTTTAAACAATACAAGCTGCCGGAGATCATTCGTAAAGAGAACGAAGACC CGTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCAGGAATTCGTGGGCC AATACCTGAACCCGCAGGGTCCGTATACCAGCATCCTGCTGTACCATGGTCTGG GTAGCGGCAAGACCGCGAGCGCGATCAACCTGATGAACATTCTGTACAACTATG ACAACGGCACCAACTTTATCGTTCTGATTAAAGCGAGCCTGCACAACGACCCGTG GATGCAGGACCTGAAGGAGTGGCTGGGTCGTGATCCGAGCGAACAGAACGTGG ACAACGTTACCAAGCTGGATCGTTACAAAAACATCCACTTCGTGCACTATGACAG CCCGTTCGCGGATAGCAGCTTTATGAGCGTTATTAAGACCCTGGACCTGAGCAA ACCGACCATGTACATCATTGATGAGGCGCACAACTTTATCCGTAACGTGTATAGC AACATTAACAGCAAACTGGGCAAGCGTGCGAAAGTTATCTACGAGTACATCATGA AGGACAAGCGTGAAAACAAGAACACCCGTATCGTGCTGATTAGCGCGACCCCGG CGATCAACACCCCGTTCGAACTGGCGCTGATGTTTAACCTGCTGCGTCCGGGTA TTTTCCCGAGCAGCGAGCTGGATTTCAACCGTACCTTTGTGACCGAAAGCAGCT ACCCGATCCTGAACCCGATGAAGAAAAACATGTTTGAGCGTCGTATCCTGGGCC TGGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCGCGTCAAGAACTGA AGTACA TCAACCTGCCGATGAGCGCGTACCAGTATGATATCTACCGTATTTTCGA GAAACTGGAGGCGGAAATTCAAGAACGTGCGCGTCGTCGTGGCAAGCAGAGCCA ACTGTACCGTACCTATACCCGTCAGGCGTGCAACTTCGTGTTTCCGTACGTTAAC ATGAACGTGAACGGTGAACTGCGTCCGCGTCCGGGCAAGTTCCGTCTGAGCGAA AAACTGGCGGACGATTTTAGCAAGGGCAAAAACCTGGACGTTCCGGATACCGAG AAAGAAATCCTGAACAAGTATACCAAAGCGATTGAGAACTACCTGAACGAGACCG AACGTTATTTTCAGAACATCAACAAGAAAGACGCGGAGAACGGTCGTACCATCAT TAACGACCTGGATGAATTCAAGAAAGGCTTTGGTACCAAGTTCAACAGCTTTCTG CAGTACTATCAAAGCGAGGGTCCGCGTAGCAGCCTGCTGACCGAAATGTACAAC TGCAGCCCGAAAATGCTGGCGATCGCGTTCATGACCTATATTAGCCCGGGCAAG GTGATGATCTACAGCAACTATGTGGTTATGGAAGGCATCGACGTTATGAAAATT TACTTTCGTCTGATCGGTTTCAACGATTTTACGATCGCGCGTGAGTACATGGGCT ATTGCGAATACCACGGTCGTATCGACCCGAAGGATCGTGTGCGTATCAAGAACA TGTT CAACG ACAAG AACAACGT GT ACGGCAACAAGTGCAAAGTT AT CATGCT GA GCCCGAGCGCGACCGAGGGTATTCAACTGCTGGATATCCGTCAGGAGCACATTA TGGAACCGTATTGGACCGAAGTTCGTATCCAGCAAGTGATTGGCCGTGGTGTTC GTCAATGCAGCCACCGTGACCTGCCGATGAGCGAGCGTATCGTGGATATTTACC GTTATAAGGTTATCAAACCGGAAAACCTGGACCCGGACGATACCGTGCGTCAAA GC ACCGACGAGTACGTTGAAGATCAGGCGAAGAGCAAAGCGAACCTGATTGAGA GCTTCCTGGGCGCTATGAAAGAAGCGGCGGTTGATTGCGAGCTGTTTAAGGAAC ACAACATGATGAGCCAGAGCTACTATTGCTTCAAATTTCCGGAGAGCGCGGTGA CCAAGACCAACGTTGGCCCGGCGTACCGTGAAGACATCAAGGACGATGTGAAAT ATGATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGCGTATTCGTGTGGTTA AGGTGAACGCGGTTTACCAAATCAACACCGACAACAACAACCCGGTGTATAGCA GCCCG ACCAAGTACTGGTAT AACAAG AAAACCGGCATGGTTT ATG ACTTCG AG A CCCACTACCCGGTGGGTCAGGTTGAATTTATCGATAACCTGCCGAACAAGCTGG ACAAAGATACCTACATCATGCGTATTGATGTGATCATTCCGAGCATTACCGGTAG CGTT AACACCT AA

SEQ. I D. N ° 4: gene R354 of M im ivi rus ATGACCGACATTAGCTACTATAACAACGAGATCGATAAAATTCTGTGGAACATCC TGGGTGACGATTATTTCACCCAAGACGAATTTGACGATCTGGTGAACAGCGTTG CGAACACCATTTACCAGTATGACAACGAAGTGAGCATCGATAAGCTGAAAGTGAT CATCGAATTCGTTATCCTGAACAAGTTCAAGCTGTGCTACATCTACGATAACGAC AGCAT CTC G AG AAG ACG AACCAAGTG STW AAAAGCGTTGGT AGCAAAACCAT C GGCAAGAACAGCACCAACGACGATGAGGACGATGACGAAGATATCGCGGTGATT AAGCTGAGCGATATTGAGGCGGGCGAAAACTGGTTCAAGAAAAGCCCGAAAATC AGCAGCAAGCAGTTTCAAAGCGTTGACAAAGTTGAGGTGGCGACCTACGAAGAC CT GATCAGCCACAAGCACGATT ACCCGAAAGAG ATTT AT AAGGAAAGCCACT ACA TCCGTCGTAACACCCGTCTGGATGTGATCAAGAAAATTCCGCAATTCGAGCAGAA GAGCAAAGAATGGCTGAAACAACGTACCGAGAGCCTGACCGCGACCGCGATTAG CGTGGTTTTTGATGAAGACCCGTATAAACACCCGATCGTTATTCTGCTGGACAAG TGCGGTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTTCACCACGGTAACAAG TATGAACAAATCGGCACCATGTTCTACAGCTTTCGTAACAACGTTGAGGTGGGT GAGTACGGCCTGCTGCAGCACAGCGGTCACAAGTTTATCGCGGCGAGCCCGGAT GGCATCTGCAGCAAGAAAGCGAACACCGGTGGCCTGAGCAAACTGGTGGGTCGT CTGCTGG AG ATT AAGTTCCCGTTT AGCCGTG STW CAACAACAGCGGTG AT CT G GACGGCGATATCTGCCCGCACTACTATTTTCTGC AGGTGCAAACCCAGCTGTAT GTTACCGAGATGGACGAATGCGACTTCCTGCAGTGCAAAATTGACGAGTACGAT AGCTGGGAAGACTTTGTGAAGGATAGCAACCCGATCGTTCCGGGTCTGAGCAAA ACCACCAACCTGGAGAAGGGCTGCCTGATTCAGCTGAGCGACAAAAACCTGATC GGCAGCGACGACAAGGAAAAATGCCTGTATAACAGCAAATACATCTATCCGCCG AAGCTGCACATGACCAACGAGGAAATCGAGAAGTGGATTAGCAGCGAAATCATG AACTACCACAACAACGACCTGAGCGAGAACTATATGATTGATCGTGTGATCTACT GGCGTCTGAGCCAAGTTACCTGCAACCTGATTAAGCTGAACAAAGAAGCGTTCG AGGAAAAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTTCTGTTTTATCGTCA GCACAGCGACAAGCTGGATAAACTGATTAAGTTTGTGGAGAAGGTTAAAGAAGA TAACAGCGCGGAGATTTTCAGCTACATCAACGAAGACTTTCTGAGCCTGAACAAA GATAGCAAGTACGAGCCGCTGTATCAGGAAGAGACCGAATGGCGTAAGAAATAT AACCAAATCAAGGCGAAGAAAGCGCAGATGTACAAGAACAAGAGCTACAACAAG TACACCAAGTTCAGCAAC

S EQ. I D. N ° 5: R349 gene from M i m ivi ru s

ATGG AATT AGTCGTCTTT ACT CATGG AG AAAAT ATTTT ACTTGTT AGAC ATAT ATAT ATAT ATAT ATT ATT ATT ATT ATT ATT ATT ATT ATT AT CATTT ATTTGTG GT AG GA AATCGATGCATATGATAGTGAAAGTGATGTTGGAAGTGATGCTGAGTCTGATGC TG AGT CT G ATGCTG AAT CAG ATT CAG AAAAT CAT ACT CAAAAT ACT CCCAT ATAT AAT ATAT AAT AAT CTG AT AAT G AAT CTG ACAATG AAT CTG AT CAA TCATGTAGTGATTTTGACTGTGGACCTCGATCTGATAATGAATCTGATGAAGAAG TTTTTGTT G AT CGT AATG AT AAT AATT CAG AT AAT ATTGG AGT ATG ATG ATT AT AAT AT AAT AT AAT AT AAT AT CAAAAAAAT ATT AATT CTGTTGG AA TT AACG AT ATT GTT ATTCGTCCAAGCGG ACAGT ATGTG AGT AAT ATGG GATAT AT T ACAG AAT CAG GT AGTGTAT CATTT CAT ACC AAT AAAAAT G GTTT CTT ATT ATT C AAGT CCAAT GTT CAT G AAAT CCTTTT CGTTG AAG AAATGTTT ATGTACT CT AAAA AT AATTTT ATTT ATTTGGCCAT ACCATTT AAAAAAT ACCAGGCAT CT CT AAG AG A T ATTGCTCCTTTT C AT ACAATT AAT AAAAC AAAAAAT G GT ATT AAAT GG AAGT AT TT CAAAAT AGTGTTTCCATTTG ACACT G AAAAAAT AG AATTTTGT ATATAT ATAT AT ATTT ATATTT ATATAT ATAT ATTAT ATTAT ATTAT ATTAT ATTAT CAT ACAAAAAT GT AAATT ATCCTT CTTGG AT AT ATTT CAAAT CT ATT AAT AGT A AAAAT AT GTT CTTTT C AAGT G AT ACT AAT AGTGTGTATGTT AAAG AT AAT AACAA CGTGT AT AAATGACATGATATATAT ATT AAAGTT AGGTAT AACATTGT AT AGCG ATT ACAATTTT AATGG AT GTG AGG ATG AT G AAG AACAT ATTTT CG AAATT AT G AAAAAT CAAT ATGT ACCT CAT AT T AT CGGT CTG AATT ATTTTG AAAGTTT CATTGTAGTT ATTGTCAAT AATCCG ATATGAT ATT A CTTTTT ACAAAAG ATTTT AT AATGG AATCGTTT AT CTTG AT AATGGTTCGTT ATT TT AT CT CACAG AT AGTG AAATTT CAG AT CAAAATGT ATGG AAACT AACTGG AT GT CAATT GT GTG AGCT AGCTG ATT CT ACCAT AT AT AGTT ACCT ATTT AATTT ACCGG ACAAAATT G AT G AAATTT ACT CTTCGT CTG AATTT ATTGTT CT AAAATT AATTGG AAACAAAT ATTTTT ATT AT CCG GTTG AAAATTTTG AT ACT G CT C AAG ATTTT AAG ACAAG AT GTG G GG AAATTT C ATTG AAAAT AT ATTT C AACAG ACAAT CT AAAAGTT AT CAT ACT ACAGT AT CT ATT AAT ATT GATACGGATTGTACTACTCATAATTCTTTCGAGAGATTGTTTATCCTGACACAAT CT CTT AGTT ATTCCGCAG AAT ATT CT ATTCG AATTGT AG ACG ACAAAAAT ATTGG TTTTGGTGATGGGCCTAAAATTGAATTTTGTGAATCGGCTATTATGCAATTTTAT T AT GTAA ATTT AATT G CT CAT AATTTT C ACAC AG AGTTT AATTT AC AAG AATTT G CCAAACT G AAACCCACCG AAATT STW ATTTGGG AT CAATGTTT CAT ATGGTT AT CTGTCAAAATAATTCTTCGTTACCGATTAGATTACCTCTAGCTTTTGCTGTAGAA ATTTATGGAAAAGAACCCACAATTGATGAATTGGAATATTTTGCTTGTAATGAAG AT G AAACTGG ATT CAAACAT ATTT AT CCAGCCAAAT ACAATCCCG AATT AGTT AA AG AATTCG GTT ATG AAT CTT ATG AACATTGTCT AAAAACTTTGT GT STW AT AAT T AT G AAG ATG AT ACTG AT AAAAAT AT CTTG ACG AAAAAAT ATTGCG AGCAATT AG CTGCCGGTTTT AAAAG AT ACGGCAAT AT CAAG AACAT AAAACAAATG AATTT ACC CACATTGG ACT ATT ACATTT CTGGCCCAT ACAAAATT AAT AG AACCAT ATT AATT AATAATCTTGTTTTATCGGGAGGTAAGGATAAAAATAATAATTATTTGGAAATGT T CAAAG AATTT ATT AACT CTTTGTCTG AAAATG AATT AAAAAT CTTGCT AAAAAA TTGG ACAGCAT CAACTTGTGTAAG ACCAG AT AACAAAT AT AG AATT AT CATT ATT TCCAAAT CT AAAAACGCT AAAGCAGGTATTCG ATTTGGTACTTGTAATTT AG AAA TT CAT AT GTC ACG AAAAAATGTTGG ATG AACAT AAT ATTG AT ACAGTCAAAG AGGT TTT AATT AC ACCT G CT CAAG G ATT CAAAG ATT AA

SEQ. I D. N ° 6: repeated sequence of the za m i lon virophage

 TGATAATGAATCTGA SEQ. I D. N ° 7: Gene R349 modified with repeated sequences of the tetracycline resistance gene (SEQ I D N ° 23)

ATGG AATT AGTCGTCTTT ACT CATGG AG AAAAT ATTTT ACTTGTT AGTT CTATAA ATG ATAT ATAT ATAT ATT ATT ATT ATT ATT ATT ATT ATT ATT CATTT ATTTGTG GTG G AG GT GA AATCGATGCATATGATAGTGAAAGTGATGTTGGAAGTGATGCTGAGTCTGATGC TG AGTCTG ATGCTG AAT AGC ATT CAG AAAAT CAT ACT CAAAAT AAT ACAAAT ACT CCCAT STW AAT AT CAC ACT AATT AATTT GG ATT CAT CAAAT AATTCC ACT CAAT C CACCCTGGATGCTGTTAATGAATCCACCCTGGATGCTGTCAATGAATCTGATCAA TCATGTAGTGATTTTGACTGTGGACCTCGATCCACCCTGGATGCTGTTGAAGAA GTTTTT GTTG ATCGT AATG AT AAT AATT AGC AT AAT ATT G GT AATT CAAAT AGTA T CACCCT GG ATG CTGTATCT ATG ACTG AAAAAGCT GG AAT AATTTTGTT AAACG A AATT AAAACCATGGTAG ATG AAATTTTGTCT CAAAAAAAT ATT AATT CTGTTGG A ATTAACGATATTGTTATTCGTCCAAGCGGACAGTATGTGAGTAATATGGGATATA TT ACAG AAT CAGGTAGTGT AT CATTT CAT ACCAAT AAAAATGGTTT CTT ATT ATT CAAGTCCAATGTT C ATG AAATCCTTTTCGTTG AAG AAATGTTT ATGT ACT CT AAA AAT AATTTT ATTT ATTTGGCCAT ACCATTT AAAAAAT ACCAGGCATCT CT AAGAG AT ATT G CTCCTTTT CAT AC AATT AAT AAAACAAAAAAT G GT ATT AAAT GG AAGT A TTT CAAAAT AGTGTTT CCATTTGACACTG AAAAAAT AG AATTTTGT G AT AATTT C TTTT AT ACTT ATG AAT CCAAT ACTTGTT AT CAT ATTT AA AT T ATTT CAAAT ATG AT ATT AAT AGT AAAAAT ATGTT CTTTT CAAGTG AT ACT AAT AGTGTG ATAT AATA ATAT ATAT AATTT CAAT AATT CT CTTG AAAAAT ACATCAT ATTAGATGATGATGATAT AATGG AT GT GAGGATGATGAAGAACATATTTTCGAAATTATGAAAAATCAATATGTACCTCATA TT AT CGGTCTG AATT ATTTTG AAAGTTT CATTGT AGTT ATTGTCAAT AATCCAAA T AT GTTG ACG AT AACAACTG ACG ATGGTAAAATTTT CTTT AACATT CATG AT ATT ACTTTTTACAAAAGATTTTATAATGGAATCGTTTATCTTGATAATGGTTCGTTAT TTTATCTCACAGATAGTGAAATTTCAGATCAAAATGTATGGAAACTAACTGGATG T CAATTGTGTG AG CT AG CT GATT CT CGTA AT AT AGTT ACCT ATTT AATTT CAGC G ACAAAATTG ATG AAATTT ACT CTTCGTCTG AATTT ATTGTT CT AAAATT AATT GG AAAC AAAT ATTTTT ATT AT CCG GTTG AAAATTTTG AT ACT G CT C AAG ATTTT AA G ACAAGATGTGGGG AAATTT CATTG AAAAAT AATT CAGTT CTTG AACT CGTT AAT ACT AGT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ATT ACT AT TGATACGGATTGTACTACTCATAATTCTTTCGAGAGATTGTTTATCCTG ACACAA T CT CTT AGTT ATTCCG CAG AAT ATT CT ATTCGAATTGTAG ACG AC AAAAAT ATT G GTTTTGGTGATGGGCCTAAAATTGAATTTTGTGAATCGGCTATTATGCAATTTTA TT AT AAGTATTT AATTGCT CAT AATTTT CACACAG AGTTT AATTT ACAAG AATTT GCCAAACTGAAACCCACCG AAATT STW ATTTGGG AT CAATGTTT CAT ATGGTT A T CT GTCAAAAT AATT CTTCGTT CAGC ATT AG ATT ACCT CT AGCTTTT G CTGTAG A AATTT ATGG AAAAG AACCCACAATTG ATG AATTGG AAT ATTTTGCTT GT AAT G AA G ATG AAACT GG ATT C AAAC AT ATTT ATCC AG CCAAAT AC AATCCCG AATT AGTT A AAG AATT CGGTT ATG AAT CTT ATG AACATTGTCT AAAAACTTTGT GT STW AT AA TTATGAAGATGATACTGATAAAAATATCTTGACGAAAAAATATTGCGAGCAATTA GCTGCCGGTTTT AAAAG AT ACGGCAAT ATCAAGAACAT AAAACAAATG AATTT AC CC AC ATT GG ACT ATT ACATTT CTG G CCCAT AC AAAATT AAT AG AACC AT ATT AAT T AAT AAT CTTG TTTT ATCG GG AG GT AAG G AT AAAAAT AAT AATT ATTT GG AAAT G TT CAAAG AATTT ATT AACT CTTTGTCTG AAA AAAAA ATTGG ACAGCAT CAACTTGTGT AAG ACCAG AT AACAAAT AT AG AATT AT CATT AT TT CCAAAT CT AAAAACG CT AAAG C AG GTATT CG ATTT G GTACTTGTAA TTT AG AA ATTCATATCGACGAAAAAATGTTGGATGAACATAATATTGATACAGTCAAAGAGG TTTT AATT AC ACCTGCT C AAG G ATT CAAAG ATT AA

SEQ. ID. N ° 8 direction sequence of 130 nu cleotides of IO RF4 from za mi lon AT AG AACAACCAAAAAAAT AT CAAAAT CT AG AG ATG AAT CAAGT G AAT CAG AAG A ATCTGATAATGAATCTGATAATGAATCCGATGAGGAAGTTGAATCAGAAACTGAG ATAGAACCAGTCAAATCTAA

SEQ. ID. N ° 9 anti sense sequence of 130 nucleotides of zamilon IORF4

TT AG ATTTG ACTGGTT CT AT CT CAGTTT CTG ATT CAACTT CCT CATCGG ATT CAT T AT CAG ATT CATT AT CAG ATT CTT CTG ATT CACTTG ATT CAT CT CT AG ATTTTG A T ATTTTTTTGGTTGTT CT AT

SEQ.ID.No.10: primer of the mimivirus R349 gene

 ATGGAATTAATTAGTCGTGTC

SEQ.ID.N ° 11: primer of the mimivirus R349 gene

TT AAT CTTTG AATCCTTG AG C

SEQ.ID.N ° 12: primer of the zamilon virophage IORF4 gene

ATGTCTGTTCTAGAAAACCT

SEQ.ID.N ° 13: primer of the IORF4 gene of the zamilon virophage TT AAGTT AT AATTTTGTAT A

SEQ.ID.N ° 14: primer of a sense sequence including the T7 promoter and Lac operon sequences

 T AAT ACG ACT CACT AT AGGGT ACT CCCAT AAAT AAT AT CAC

SEQ.ID.No.15: primer of a sense sequence including the T7 promoter and Lac operon sequences

 G AG AC AAAATTT CAT CT ACC

SEQ.ID.No.16: primer of an antisense sequence including the T7 promoter and Lac operon sequences

 T AAT ACG ACT CACT AT AG G G AG AC AAAATTT CAT CT ACC

SEQ.ID.N ° 17: primer of an antisense sequence including the T7 promoter and Lac operon sequences

 T ACT CCCAT AAAT AAT AT CAC

SEQ.ID.N ° 18: sense sequence of the RNA of the repeated sequence of the zamilon virophage

 UGAUAAUGAAUCUGA

SEQ.ID.N ° 19: RNA anti-sense sequence of the repeated sequence of the zamilon virophage UCAGAU UCAU UAUCA

 SEQ. I D. N ° 20: RNA sense sequence of the repeated G FP gene sequence

AAUCGCUGGACGGCGACG U UAAUGAAUCGCUGGACGGCGACGUCA SEQ. I D. N ° 21: one-way sequence of the RNA of the repeated sequence of the G FP gene

 UGACG UCGCCGUCCAGCGAU UCAU UAACG UCGCCG UCCAGCGAU U

SE D. I D. N ⁰ 22: sequences organized in operon with the modified R349 gene with specific sequence of 15 nucleotides of the tetracycline resistance gene (SEQ IDN 23 ) and the R350 and 354 genes.

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAAT AATTTTGTTT AACTTT AAG AAGG AG AT AT AG AT ATGG AATT AATT AGTCGTGT CT TT ACT CATGG AG AAAAT ATTTT ACTTGTT AGTT CT ACAAAT AAGTT AT AT ATT AT GGGTAATAATGAATATGGTTCATGTGGTTTCAAAATAGGTACAGATAAAACTTAT ATTGAAAGTCCAGTATATATTGACATTAAATTAGATGATGATGATTCTGTTAAAG CGTTTT ATT CTTGT AATTT ATT CACG ATG ATT CAT ACATCCAAAGG AAAAATTT A TCTATCAAGATCATTTATTTGTGGTGGAGGTGAAATCGATGCATATGATAGTGAA AGTGATGTTGGAAGTGATGCTGAGTCTGATGCTGAGTCTGATGCTGAATCAGAT T CAG AAAAT CAT ACT C AAAAT AAT ACAAAT ACTCCC AT STW AAT AT C ACACT AAT T AATTTGG ATT CAT CAAAT AATTCCACT CAATCCACCCTGG ATGCTGTT AATG AA TCCACCCTGGATGCTGTCAATGAATCTGATCAATCATGTAGTGATTTTGACTGTG GACCTCGATCCACCCTGGATGCTGTTGAAGAAGTTTTTGTTGATCGTAATGATAA T AATT CAG AT AAT ATTGGT AATT CAAAT AGT AT CACCCTGG ATGCTGTAT CT AT G ACTG AAAAAGCTGG AAT AATTTTGTT AAACG AAATT AAAACCATGGTAG ATG AAA TTTTGTCT CAAAAAAAT ATT AATT CTGTTGG AATT AACG AT ATTGTT ATT CGTCC AAGCGGACAGTATGTGAGTAATATGGGATATATTACAGAATCAGGTAGTGTATC ATTT CAT A CCAAT AAAAAT G GTTT ATT ATT CAAGTCCAATGTT C ATG AAAT C CTTTTCGTTG AAG AAATGTTT ATGTACT CT AAAAAT AATTTT ATTT ATTTGGCCA T ACCATTT AAAAAAT ACCAGGCAT CT AAG AG AT ATGCTCCTTTT CAT ACAAT AAAAT AAA AATTTTGT G AT AATTT CTTTT AT ACTT ATG AAT CCAAT A CTTGTT AT CAT CATGTT ATTT CATT CT ACAAAAATGTAAATT ATT ATCCTT CTTGG AT AT ATTT CAAAT CTG AG ATTG AT ATT AAT AGTAAAAAT ATGTT CTTTT CAAGTG AT ACT AATAT ATTAT CAAT AATT CT CTTG AAAAAT ACATCG AT AACAAACTCG ACTTGG ATGTCGT AATT GTGCCTG AT AGTT AT AG ACCAATGG AAATG AG ACT ACT ATT AAAGTT AGGT AT AA CATTGTATAGCGATTACAATTTTAATGGATGTGAGGATGATGAAGAACATATTTT CG AAATT ATG AAAAAT CAAT ATGTACCT CAT ATT ATCGGTCTG AATT ATTTTG AA AGTTT CATTGT AGTT ATTGTCAAT AATCCAAAT ATGTTG ACG AT AACAACTG ACG ATGGT AAAATTTT CTTT AACATT CATG AT ATT ACTTTTT ACAAAAG ATTTT AT AAT GG AATCGTTT AT CTTG AT AATGGTTCGTT ATTTT AT CT CACAG AT AGTG AAATTT CAGATCA AAATGTATGGAAACTAACTGGATGTCAATTGTGTGAGCTAGCTGATTC T ACCAT AT AT AGTT ACCT ATTT AATTT ACCGGACAAAATTG ATG AAATTT ACT CT T CGTCTG AATTT ATTGTT CT AAAATT AATTGG AAACAAAT ATTTTT ATT ATCCGG TTG ATA ATCCTT G AAAAAT AATT CAGTT CTTG AACT CGTT AAT ACT AGTATT ATT AACAG ACAAT CT AAAAGTT AT CAT ACT ACAGT AT CT ATT AAT ATTG AT ACGG ATTGT ACT ACT CAT A ATT CTTT CG AG ATT GTTT AT CCTG ACACGATT AGT AGT AGT ATT AGT AGT ATT AGT AATCGGCT ATT ATGCAATTTT ATT AT GTAA ATTT AATTGCT CAT A ATTTTCACACAGAGTTTAATTTACAAGAATTTGCCAAACTGAAACCCACCGAAAT T STW ATTTGGG AT CAATGTTT CAT ATGGTT AT CTGTCAAAAT AATT CTT CGTT A GCC ATT AG ATT ACCT CT AGCTTTTGCTGTAG AAATTT ATGG AAAAG AACCCACAA TTG ATG AATTGG AAT ATTTTGCTTGTAATG AAG AT G AAACTGG ATT CAAACAT AT TT ATCCAGCCAAAT ACAAT CCCG AATT AGTT AAAG AATT CGGTT ATG AAT CTT AT G AACATTGTCT AAAAACTTTGTGT AAAT AT AATT ATG AAG ATG AT ACT G AT AAAA AT AT CTTG ACG AAAAAAT ATTGCG AGCAATT AGCTGCCGGTTTT AAAAG AT ACGG CAAT AT CAAG AACATAT ACAATAT ACAATAT ACAATAT AG AACCAT ATT AATT AAT AAT CTTGTTTT ATCGGG AG GT AAGG AT AAAAAT AAT AATT ATTTGG AAATGTT CAAAG AATTT ATT AACT CTTT GTC TG AAAATG AATT AAAAAT CTTGCT AAAAAATTGG ACAGCAT CAACTTGTGT A AG ACCAG AT AACAAAT AT AG AATT AT CATT ATTT CCAAAT CT AAAAACGCT AAAG CAGGT ATTCG ATTTGGT ACTTGTAATTT AG AAATT CAT ATCG ACG AAAAAATGTT GG AT G AACAT AAT ATTG AT ACAGTCAAAG AGGTTTT AATT ACACCTGCT CAAGG A TTCAAAGATTAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCT GCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTC TTG AGGGGTTTTTTG AG AT AT CT CG AAT ACG ACT CCCGCG AAATT CACT AT AGG GG AATTGTG AGCGG AT AACAATTCCCCACT AG STW AATTTTGTTT AACTTT AAG AAGGAGATATAGCAATGAACCGTCGTAACCGTAGCAACGACCTGAACCCGGAGC CGAGCATCGAAAACCCGAACAACCAGATTGCGGAGGAATTCCCGGGTAACAACA GCGTGTATAAAAGCGACGGCTACGTTGATCTGAAGAACAACGGTCGTCTGTTCC CGATCTGGATTCTGAAGAACTTTAAACAATACAAGCTGCCGGAGATCATTCGTAA AGAGAACGAAGACCCGTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCA GGAATTCGTGGGCCAATACCTGAACCCGCAGGGTCCGTATACCAGCATCCTGCT GTACCATGGTCTGGGTAGCGGCAAGACCGCGAGCGCGATCAACCTG ATG AACAT TCTGTACAACTATGACAACGGCACCAACTTTATCGTTCTGATTAAAGCGAGCCTG CACAACGACCCGTGGATGCAGGACCTGAAGGAGTGGCTGGGTCGTGATC CGAGC GAACAGAACGTGGACAACGTTACCAAGCTGGATCGTTACAAAAACATCCACTTCG TGCACT ATG ACAGCCCGTTCGCGG AT AGCAGCTTT AGCGTT ATG ATT AAG CCALC GGACCTGAGCAAACCGACCATGTACATCATTGATGAGGCGCACAACTTTATCCGT AACGTGTAT AGCAACATT AACAGCAAACTGGGCAAGCGTGCGAAAGTT ATCT AC GAGTACATCATGAAGGACAAGCGTGAAAACAAGAACACCCGTATCGTGCTGATT AGCGCGACCCCGGCGATCAACACCCCGTTCGAACTGGCGCTGATGTTTAACCTG CTGCGTCCGGGTATTTTCCCGAGCAGCGAGCTGGATTTCAACCGTACCTTTGTG ACCGAAAGCAGCTACCCGATCCTGAACCCGATGAAGAAAAACATGTTTGAGCGT CGTATCCTGGGCCTGGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCG CGTCAAGAACTGAAGTACATCAACCTGCCGATGAGCGCGTACCAGTATGATATCT ACCGTATTTTCG AG AAACTGG AGGCGGAAATTCAAGAACGTGCGCGTCGTCGTG GCAAGCAGAGCCAACTGTACCGTACCTATACCCGTCAGGCGTGCAACTTCGTGT TTCCGTACGTTAACATGAACGTGAACGGTGAACTGCGTCCGCGTCCGGGCAAGT TCCGTCTGAGCGAAAAACTGGCGGACGATTTTAGCAAGGGCAAAAACCTGGACG TTCCGGATACCGAGAAAGAAATCCTGAACAAGTATACCAAAGCGATTGAGAACTA CCTGAACGAGACCGAACGTTATTTTCAGAACATCAACAAGAAAGACGCGGAGAA CGGT CGTACCAT CATT AACGACCTGG AATT ATG CAAG AAAGGCTTTGGTACCAA GTTCAACAGCTTTCTGCAGTACTATCAAAGC GAGGGTCCGCGTAGCAGCCTGCT GACCGAAATGTACAACTGCAGCCCGAAAATGCTGGCGATCGCGTTCATGACCTA TATTAGCCCGGGCAAGGTGATGATCTACAGCAACTATGTGGTTATGGAAGGCAT CGACGTTATGAAAATTTACTTTCGTCTGATCGGTTTCAACGATTTTACGATCGCG CGTGAGTACATGGGCTATTGCGAATACCACGGTCGTATCGACCCGAAGGATCGT GTGCGTATCAAGAACATGTTCAACGACAAGAACAACGTGTACGGCAACAAGTGC AAAGTTATCATGCTGAGCCCGAGCGCGACCGAGGGTATTCAACTGCTGGATATC CGTCAGGAGCACATTATGGAACCGTATTGGACCGAAGTTCGTATCCAGCAAGTG ATTGGCCGTGGTGTTCGTCAATGCAGCCACCGTGACCTGCCGATGAGCGAGCGT ATCGTGG ATTT ACCGTT AT AT AT AAGGTT CAAACCGG AAAACCTGG ACCCGG AC GATACCGTGCGTCAAAGCACCGACGAGTACGTTGAAGATCAGGCGAAGAGCAAA GCGAACCTGATTGAGAGCTTCCTGGGCGCTATGAAAGAAGCGGCGGTTGATTGC G AGCTGTTT AACACAACAT AAGG G ATG ACT AGCCAG AGCT ATTGCTT CAAATTT C CGGAGAGCGCGGTGACCAAGACCAACGTTGGCCCGGCGTACCGTGAAGACATCA AGGACGATGTGAAATATGATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGC GTATTCGTGTGGTTAAGGTGAACGCGGTTTACCAAATCAACACCGACAACAACAA CCCGGTGTATAGCAGCCCGACCAAGTACTGGTATAACAAGAAAACCGGCATGGT TTATGACTTCGAGACCCACTACCCGGTGGGTCAGGTTGAATTTATCGATAACCTG CCGAACAAGCTGGACAAAGATACCTACATCATGCGTATTG ATGTGATCATTCCGA GCATT ACCGGTAGCGTT AACACCT AACACT AG STW AATTTTGTTT AACTTT AAGG AAG AG AT AT ACCG AGCG ATG ACT AT AACAACG ACATT AGCT AG AT AT CG AAAAT TCTGTGGAACATCCTGGGTGACGATTATTTCACCCAAGACGAATTTGACGATCTG GTGAACAGCGTTGCGAACACCATTTACCAGTATGACAACGAAGTGAGCATCGAT AAGCTGAAAGTGATCATCGAATTCGTTATCCTGAACAAGTTCAAGCTGTGCTACA TCTACGATAACGACAGCATCCTGAACCAAGTGAAATACGAGAAGAAAAGCGTTG GTAGCAAAACCATCGGCAAGAACAGCACCAACGACGATGAGGACGATGACGAAG ATATCGCGGTGATTAAGCTGAGCGATATTGAGGCGGGCGAAAACTGGTTCAAGA AAAGCCCGAAAATCAGCAGCAAGCAGTTTCAAAGCGTTGACAAAGTTGAGGTGG CGACCTACGAAGACCTGATCAGCCACAAGCACGATTACCCGAAAGAGATTTATAA GGAAAGCCACTACATCCGTCGTAACACCCGTCTGGATGTGATCAAGAAAATTCCG CAATTCGAGCAGAAGAGCAAAGAATGGCTGAAACAACGTACCGAGAGCCTGACC GCGACCGCGATTAGCGTGGTTTTTGATGAAGACCCGTATAAACACCCGATCGTT ATTCTGCTGGACAAGTGCGGTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTT CACCACGGTAACAAGTATG AACAAATCGGCACCAT GTT CT ACAGCTTT CGTAACA ACGTTGAGGTGGGTGAGTACGGCCTGCTGCAGCACAGCGGTCACAAGTTTATCG CGGCGAGCCCGGATGGCATCTGCAGCAAGAAAGCGAACACCGGTGGCCTGAGCA AACTGGTGGGTCG TCTGCTGGAGATTAAGTTCCCGTTTAGCCGTGAAATCAACA ACAGCGGTGATCTGGACGGCGATATCTGCCCGCACTACTATTTTCTGCAGGTGC AAACCCAGCTGTATGTTACCGAGATGGACGAATGCGACTTCCTGCAGTGCAAAA TTGACGAGTACGATAGCTGGGAAGACTTTGTGAAGGATAGCAACCCGATCGTTC CGGGTCTGAGCAAAACCACCAACCTGGAGAAGGGCTGCCTGATTCAGCTGAGCG ACAAAAACCTGATCGGCAGCGACGACAAGGAAAAATGCCTGTATAACAGCAAATA CATCTATCCGCCGAAGCTGCACATGACCAACGAGGAAATCGAGAAGTGGATTAG CAGCGAAATCATGAACTACCACAACAACGACCTGAGCGAGAACTATATGATTGAT CGTGTGATCTACTGGCGTCTGAGCCAAGTTACCTGCAACCTGATTAAGCTGAAC AAAGAAGCGTTCGAGGAAAAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTT CT GTTTT ATCGT CAGCACAGCG ACAAGCTGG AT AAACT GATT AAGTTTGTGG AG AAGGTTAAAGAAGATAACAGCGCGGAGATTTTCAGCTACATCAACGAAGACTTTC TGAGCCTGAACAAAGATAGCAAGTACGAGCCGCTGTATCAGGAAGAGACCGAAT GGCGTAAGAAATATAACCAAATCAAGGCGAAGAAAGCGCAGATGTACAAGAACA AGAGCTACAACAAGTACACCAAGTTCAGCAACCTCGAGCACCACCACCACCACCA CTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCAC

CGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGG

TTTTTTG.

 SEQ. I D. N ° 23: specific sequence of 15 n keyotides of the tetracycline resistance gene

 CACCCTGGATGCTGT

SEQ. I D. N ° 24: 28 nt sequence of the za m i lon virophage

AAT CT G AT AAT G AAT CTG AT AAT G AAT C SEQ. I D. N ° 25: PP 14 vector

CCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGT CGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACC GAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTAC AATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTG ACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGA GCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGC GGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCAT CCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAA GCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTA AGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCT CACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGG TAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCA ATGCCAG CGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCA TCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTC CAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCA GACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTC TGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGC ACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCG CCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAA GATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCG GTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCAT GATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCG GAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGC CTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA GGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGG CAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGG GATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCA CCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGA TCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATG GTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCG AGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGA CCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTT GATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGC AGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCG CTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATT TAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAA CGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAA TGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATA CTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTC TCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTG TCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAG TAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGA GATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAA CBAA GCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTC GGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGAT GCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACT AT AGGGG AATTGT G AGCGG AT AACAATT CCCCT CT AG STW AATTTTGTTT AACT TTAAGAAGGAGATATAGATATGGAATTAATTAGTCGTGTCTTTACTCATGGAGAA AAT ATTTT ACTTGTT AGTT CT AC STW AAGTT AT AT ATT ATG G GT AAT AATG AAT ATG GTT C ATGTG CTWG C AAAAT AG GT AC AG AT AAAACTT AT ATTG AAAGTCC AGT AT AT ATTG ACATT AAATT AG ATG ATG AT GATT CTGTT AAAGCGTTTT ATT CTTGT AATTT ATT CACG AT GATT CAT ACATCCAAAGG AAAAATTT AT CT AT CAAG AT CAT TTATTTGTGGTGGAGGTGAAATCGATGCATATGATAGTGAAAGTGATGTTGGAA GTGATGCTGAGTCTGATGCTGAGTCTGATGCTGAATCAGATTCAGAAAATCATA CT CAAAAT AAT ACAAAT ACTCCCAT STW AAT AT CACACT AATT AATTTGG ATT CA T CAAAT AATTCCACT C AAT CC ACCCT GG ATG CTGTT AATCCACCCT GG ATG CTGTCAATGAATCTGATCAATCATGTAGTGATCTTGACTGTGGACCTCGATCCAC CCTGGATGCTGTTGAAGAAGTTTTGTTGATCGTAATATATA ATTGAT AGTAT ATATT AATTTTGTT AAACG AAATT AAAACCATGGT AG ATG AAATTTTGTCT CAAAA AAATATTAATTCTGTTGGAATTAACGATATTGTTATTCGTCCAAGCGGACAGTAT GTG AGT AAT ATGGG AT AT ATT ACAG AAT CAGGTAGTGTAT CATTT CAT ACCAAT A AAAATGGTTT CTT ATT ATT CAAGTCCAATGTT CATG AAATCCTTTTCGTTG AAG A AATGTTT ATGT ACT CT AAAAAT AATTTT ATTT ATTTGG CCAT ACC ATTT AAAAAAT ACCAG G CAT CT CT AAG AG AT ATTGCTCCTTTT CAT AC AATT AAT AAAAC AAAAAA TGGTATTAAATGGAAGTATTTCAAAATAGTGTTTCCATTTGACACTGAAAAAATA G AATTTTGTG AT AATTT CTTTT AT ACTT ATG AATCCAAT ACTTGTT AT CAT CAT G TT ATTT C ATT CT AC AAAAATGTAAATT ATT ATCCTT CTT G GATAT ATTT CAAAT CT GAGATTGATATTAATAGTAAAAATATGTTCTTTTCAAGTGATACTAATAGTGTGT ATGTT AAAG AT AAT AACAACGTGT AT STW CGTA AATTT CAAT AATT CT CTTG A AAAATACATCGATAACAAACTCGACTTGGATGTCGTAATTGTGCCTGATAGTTAT AG ACCAATGG AAATG AG ACT ACT ATT AAAGTT AGGT AT AT AACATTGT AGCG ATT ACAATTTT AATGG ATGTG AGG ATG ATG AAG ATG AAATT ATTTTCG AACAT CAAT AAAAA T ATGT ACCT CAT ATT ATCGGTCTG AATT ATTTTG AAAGTTT CATTGTAGTT ATTGTCAAT AATCCAAAT ATGTTG ACG AT A ACAACT G ACG ATGGT AAAATTTT CT TT AACATT CATG AT ATT ACTTTTT ACAAAAG ATTTT AT AATGG AATCGTTT AT CTT G AT AAT G GTTCGTT ATTTT AT CT CACAG AT AGTG AAATTT C AG AT C AAAAT GTAT GG AAACT AACTGG AT GTCAATTGTGTG AGCT AGCTG ATT CT ACCAT ATTT AT AGT G ATG AAATTT ACT CTTCGTCTG AATTT ATT GTT CT AAAATT AATT GG AAACAAAT ATTTTT ATT ATCCG GTTG AAAATTTT GATA CTGCT CAAG ATTTT AAGACAAGATGTGGGG AAATTT CATTG AAAAAT AATT CAGT T ATT ACT ATT ACT ATT ACT ATT ACT ATT ACT ATT ACT AT ACGG ATT ACT CAT AATT CTTTCG AG AT TGTTT AT CCTG ACACA AT AT CCGCAG AAT AT AT CTG AATT GT AGACGACAAAAATATTGGTTGGTATAATT AGATTT AGTAT ATT AT GAT ATT AT AAACCCACCG AAATT AAAT ATTTGGG AT C AATGTTT CAT ATG GTT AT CTGTC AAAAT AATT CTT CGTT ACCG ATT AG ATT ACCT CT AGCTTTTGCTGTAG AAATTT ATGG AAAAG AACCCACAATTG AT G AATT GG AAT ATTTTGCTT GT AATG AAG AT GAACT ATTT ATCCAGCCAAAT A CAAT CCCG AATT AGTT AAAG AATTCG GTT ATG AAT CTT ATG AACATT GTCT AAAA ACTTTGT GT AAAT AT AATT ATG AAG AT G AT ACTG AT AAAAAT AT CTTG ACG AAAA AAT ATTGCG AGCAATT AGCTGCCGGTTTT AAAAG AT ACGAT CA ACGAT ACT ATT AC ATTT CT G GCCCAT ACAAAATT AAT AG AACCAT ATT AATT AAT AAT CTTGTTTT AT CGGG AGGT AAGG AT AAAAAT A AT AATT ATTTGG AAATGTT CAAAG AATTT ATT AACT CTTTGT CTG AAAATG AATT AAAAAT CTTGCT AAAAAATT AGT ATT AGA ATTTCCAAAT CT AAAAACGCT AAAG CAG GT ATTCG ATTT G GT ACTT GT AATTT AG AAATT CAT ATCG ACG AAAAAATGTTGG ATG AACAT AAT AT TG AT ACAGT CAAAG AGGTTTT AATT ACACCTGCT CAAGG ATT CAAAG ATT AAG AT CCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAG CAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGA T AACAATT CCCCACT AG STW AATTTTGTTT AACTTT AAG AAGG AG AT AT AGCAA TGAACCGTCGTAACCGTAGCAACGACCTGAACCCGGAGCCGAGCATCGAAAACC CGAACAACC AGATTGCGGAGGAATTCCCGGGTAACAACAGCGTGTATAAAAGCG ACGGCTACGTTGATCTGAAGAACAACGGTCGTCTGTTCCCGATCTGGATTCTGA AGAACTTTAAACAATACAAGCTGCCGGAGATCATTCGTAAAGAGAACGAAGACCC GTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCAGGAATTCGTGGGCCA ATACCTGAACCCGCAGGGTCCGTATACCAGCATCCTGCTGTACCATGGTCTGGG TAGCGGCAAGACCGCGAGCGCGATCAACCTGATGAACATTCTGTACAACTATGA CAACGGCACCAACTTTATCGTTCTGATTAAAGCGAGCCTGCACAACGACCCGTGG ATGCAGG ACCTG AAGG AGTGGCTGGGTCGTG ATCCG AGCG AACAG AACGTGG AC AACGTTACCAAGCTGGATCGTTACAAAAACATCCACTTCGTGCACTATGACAGCC CGTTCGCGGATAGCAGCTTTATGAGCGTTATTAAGACCCTGGACCTGAGCAAAC CGACCATGTACATCATTGATGAGGCGCACAACTTTATCCGTAACGTGTATAGCAA CATT AACAGCAAACTGGGCAAGCGTGCG AAAGTT AT CT CAG AGT ACAT CAT G AA GGACAAGCGTGAAAACAAGAACACCCGTATCGTGCTGATTAGCGCGACCCCGGC GATCAACACCCCGTTCGAACTGGCGCTGATGTTTAACCTGCTGCGTCCGGGTAT TTTCCCG AGCAGCG AGCTGG ATTTCAACCGTACCTTTGTG CAGC AAAGCAG CTA CCCG ATCCTGAACCCG ATG AAG AAAAACATGTTTGAGCGTCGTATCCTGGGCCT GGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCGCGTCAAGAACTGAA GT ACAT CAACCTGCCG ATG AGCGCGTACCAGT ATG A T AT CT ACCGTATTTTCG AG AAACTGG AGGCGGAAATTCAAGAACGTGCGCGTCGTCGTGGCAAGCAGAGCCAA CTGTACCGTACCTATACCCGTCAGGCGTGCAACTTCGTGTTTCCGTACGTTAACA TGAACGTGAACGGTGAACTGCGTCCGCGTCCGGGCAAGTTCCGTCTGAGCGAAA AACTGGCGGACGATTTTAGCAAGGGCAAAAACCTGGACGTTCCGGATACCGAGA AAGAAATCCTGAACAAGTATACCAAAGCGATTGAGAACTACCTGAACGAGACCGA ACGTTATTTTCAGAACATCAACAAGAAAGACGCGGAGAACGGTCGTACCATCATT AACGACCTGGATGAATTCAAGAAAGGCTTTGGTACCAAGTTCAACAGCTTTCTGC AGTACTATCAAAGCGAGGGTCCGCGTAGCAGCCTGCTGACCGAAATGTACAACT GCAGCCCGAAAATGCTGGCGATCGCGTTCATGACCTATATTAGCCCGGGCAAGG TGATGATCTACAGCAACTATGTGGTTATGGAAGGCATCGACGTTATGAAAATTTA CTTTCGTCTGATCGGTTTCAACGATTTTACGATCGCGCGTGAGTACATGGGCTAT TGCGAATACCACGGTCGTATCGACCCGAAGGATCGTGTGCGTATCAAGAACATG TTCAACGACAAGAACAACGTGTACGGCAACAAGTGCAAAGTTATCATGCTGAGCC CGAGCGCGACCGAGGGTATTCAACTGCTGGATATCCGTCAGGAGCACATTATGG AACCGTATTGGACCGAAGTTCGTATCCAGCAAGTGATTGGCCGTGGTGTTCGTC AATGCAGCCACCGTGACCTGCCGATGAGCGAGCGTATCGTGGATATTTACCGTT ATAAGGTTATCAAACCGGAAAACCTGGACCCGGACGATACCGTGCGTCAAAGCA CCGACGAGTACGTTGAAGATCAGGCGAAGAGCAAAGCGAACCTGATTGAGAGCT TCCTGGGCGCTATGAAAGAAGCGGCGGTTGATTGCGAGCTGTTTAAGGAACACA ACATGATGAGCCAGAGCTACTATTGCTTCAAATTTCCGGAGAGCGCGGTGACCA AGAC CAACGTTGGCCCGGCGTACCGTGAAGACATCAAGGACGATGTGAAATATG ATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGCGTATTCGTGTGGTTAAGG TGAACGCGGTTTACCAAATCAACACCGACAACAACAACCCGGTGTATAGCAGCCC GACCAAGTACTGGTATAACAAGAAAACCGGCATGGTTTATGACTTCGAGACCCAC TACCCGGTGGGTCAGGTTGAATTTATCGATAACCTGCCGAACAAGCTGGACAAA GATACCTACATCATGCGTATTGATGTGATCATTCCGAGCATTACCGGTAGCGTTA ACACCT AACACT AG STW AATTTTGTTT AACTTT AAG AAGG AG AT AT AGCG ATG CCG ACATT AGCT ACT AT AACAACG AG ATCG AT AAAATT CT GTGG AACATCCTGGG TGACGATTATTTCACCCAAGACGAATTTGACGATCTGGTGAACAGCGTTGCGAAC ACCATTT ACCAGT ATG ACAACG GTAA G AGCATCG AT AAGCT G AAAGTG AT CAT CG AATTCGTTATCCTGAACAAGTTCAAGCTGTGCTACATCTACGATAACGACAGCAT CCTGAACCAAGTGAAATACGAGAAGAAAAGCGTTGGTAGCAAAACCATCGGCAA GAACAGCACCAACGACGATGAGGACGATGACGAAGATATCGCGGTGATTAAGCT GAGCGATATTGAGGCGGGCGAAAACTGGTTCAAGAAAAGCCCGAAAATCAGCAG CAAGCAGTTTCAAAGCGTTGACAAAGTTGAGGTGGCGACCTACGAAGACCTGAT CAGCCACAAGCACG ATT ACCCGAAAG AGATTT AAGGAAAGCCACT AT ACAT CCGT CGTAACACCCGTCTGGATGTGATCAAGAAAATTCCGCAATTCGAGCAGAAGAGC AAAGAATGGCTGAAA CAACGTACCGAGAGCCTGACCGCGACCGCGATTAGCGTG GTTTTTGATGAAGACCCGTATAAACACCCGATCGTTATTCTGCTGGACAAGTGCG GTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTTCACCACGGTAACAAGTATG AACAAATCGGCACCATGTTCTACAGCTTTCGTAACAACGTTGAGGTGGGTGAGT ACGGCCTGCTGCAGCACAGCGGTCACAAGTTTATCGCGGCGAGCCCGGATGGCA TCTGCAGCAAGAAAGCGAACACCGGTGGCCTGAGCAAACTGGTGGGTCGTCTGC TGGAGATTAAGTTCCCGTTTAGCCGTGAAATCAACAACAGCGGTGATCTGGACG GCG AT AT CTGCCCGCACT ACT ATTTT CTGCAGGTGCAAACCCAGCT GT ATGTT AC CGAGATGGACGAATGCGACTTCCTGCAGTGCAAAATTGACGAGTACGATAGCTG GGAAGACTTTGTGAAGGATAGCAACCCGATCGTTCCGGGTCTGAGCAAAACCAC CAACCTGGAGAAGGGCTGCCTGATTCAGCTGAGCGACAAAAACCTGATCGG CAG GTC ACG ACAAGG AAAAATGCCT GT AT AACAGCAAAT ACAT CT ATCCGCCG AAGCT G cacat G ACCAACG AGG STW CG AG AAGTGG ATT AGCAGCG STW CATG AACT AC CACAACAACGACCTGAGCGAGAACTATATGATTGATCGTGTGATCTACTGGCGT CTGAGCCAAGTTACCTGCAACCTGATTAAGCTGAACAAAGAAGCGTTCGAG GAA AAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTTCTGTTTTATCGTCAGCACA ACAAGCTGG GCG ATT AT AAACTG AAGTTTGTGG AG AAG AT AAGGTT AAAG AACA GCGCGGAGATTTTCAGCTACATCAACGAAGACTTTCTGAGCCTGAACAAAGATA GCAAGTACGAGCCGCTGTATCAGGAAGAGACCGAATGGCGTAAGAAATATAACC AAATCAAGGCGAAGAAAGCGCAGATGTACAAGAACAAGAGCTACAACAAGTACA CCAAGTTCAGCAACCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGC ATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG AACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCG CCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGC ACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGC CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGG ACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGAT TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC AAAAATTT AACGCG AATTTT AACAAAAT ATT AACGTTT ACAATTT CAGGTGGCAC TTTTCGGGG AAATGTGCGCGG AACCCCT ATTTGTTT ATTTTT CT STW ACATT CA AAT ATGTAT CGC CT C ATG AG AC AAT AACCCT G AT AAATGCTT C AAT AAT ATTG AA AAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGAT GCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGC GGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGC AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGA CCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC ACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAC TTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAAT CTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGG AT G AACG STW AG ACAG ATCGCT G AG AT AGGTGCCT CACTG ATT AAGCATTGGT AACT GTCAG ACCAAGTTT ACT CAT AT AT CADTC AG ATTG ATTT AAAACTT CATTT TT AATTT AAAAGG AT CT AGGTG AAG ATCCTTTTTG AT AAT CT CATG ACCAAAAT C CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAG G AT CTT CTTG AG AT CCTTTTTTT CTGCGCGTAAT CTGCTGCTTGCAAACAAAAAA ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG T AGCCGTAGTT AGGCCACCACTT CAAG AACT CTGTAGCACCGCCT ACAT ACCT CG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTT

SEQ. I D. N ° 26: sequence of the first spacer of the modified R349 gene (NB: do not take into account the first nu cleotide A added only to make a total of 10 nt as required by the sequence listing software)

 ATAATGAATC

SEQID ° 27: sequence of the second spacer of the modified R349 gene

CAATGAATCTGATCAATCATGTAGTGATTTTGACTGTGGACCTCGATC

SEQ. ID. N ° 28: sequence of the third spacer of the modified R349 gene

TG AAG AAGTTTTTGTTG AT CGTAATG AT AAT AATT CAG AT AAT ATTGGT AATT CA A AT AG T AT

SEQ. ID. N ° 29: sense sequence of RNA specific for the GFP gene

GCUGGACGGCGACGA

SEQ. ID. N ° 30: anti-sense sequence of RNA specific for the GFP gene ACGUCGCCGUCCAGC

SEQ.ID.N ° 31: sequence of Promoter T7

 T AAT ACG ACT CACT AT AG SEQ.ID.N ° 32: sequence of the Lac operon

 GAATTGTGAGCGGATAACAATTCC.

 SEQ.ID.N ° 33: T7 terminator sequence

 CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG SEQ.ID.N ° 34: sequence of the polyhistidine tag

 CACCACCACCACCACCAC

SEQ.ID. No. 35: Sequence of the plasmid pACYC184 carrying the tetracycline resistance gene

GAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATA AAACTTGTGCTT ATTTTT CTTT CTTT CAG GT AAAAAGG CCGT ATCCAG AAT CTG ACG GTCT A G GT GTTATAG ACATTG AG C AACTG ACTG AAATGCCT CAAAATGTT CTT CAG T ATGCCATTGGG AT AT AT AT CAACGGTGGTAT CCAGTG ATTTTTTT CT CCAT TTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGT GATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTC ATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGAT TTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGC GTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTG CTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGT GGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTG GCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGA GAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCG CTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAA TGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACA GGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGA CAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACT ATAAAGATACCAGGCGTTTCCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCT GCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCA CGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCAC GAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGT CCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGAT TTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACA AGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTA GCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAG CAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGA T AAAAT ATTT CT AG ATTT C AGTG CAATTT AT CT CTT CAAATGTAG C ACCTG GTAA CAGCCCCAT ACG AT AT AT AAGTTGTAATT CT CATGTTTG ACAGCTT CAT CG AT AA GCTTTAATGCGGTAGTTTATCACAGTTAAATT GCTAACGCAGTCAGGCACCGTGT ATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTGGATGCTG TAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCC ATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGA TGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCC GCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGG CGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCA CCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGG AAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGG TGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCAT TCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAA TGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACC CAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGA CTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCA TTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTG CGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCAC CAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCT GGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTAT GATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTT GCAGGCCATGCTGTC CAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCT TACCAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCGCC TCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTT GTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGA ATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCA ATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATC CATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGG GTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGG CTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAAC GTG AAGCGACTGCTGCTGCAAAACGTCTGCG ACCTG AGCAACAACATGAATGGT CTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCCCCTACGTGCTG CTGAAGTTGCCCGCAACAGAGAGTGGAACCAACCGGTGATACCACGATACTATG ACTGAGAGTCAACGCCATGAGCGGCCTCATTTCTTATTCTGAGTTACAACAGTCC GCACCGCTGTCCGGTAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCC GCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCG CCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCG CCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGT GG AACACCT ACAT CT GT ATT AACG AAGCGCT AACCGTTTTT AT CAGGCT CTGGG A GGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGC AAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAA TAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAA CCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATC ACTTATTCAGGCGTAGCACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAA ATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCT GCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCAT CAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAA GAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGA TTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGT TTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATC GT CGTGGTATT CACT GCC ATG AGCG AAAACGTTT CAGTTTGCT CATGG AAAAC GGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCC ATACG

SEQ.ID. # 36: tetracycline resistance gene sequence

AT G STW CT AACAAT GCGCT CAT CGT CAT CCT CGGCACCGT CACCCT GG ATGCTGT AGGCAT A GGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGC CAGTCACTATGGCGTGCTGCTAGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCT CGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGCCA CT AT GTC ACT ACGCG AT CATGGCG ACCACACCCGT CTC GTGG AT CTC CT ACGCCGG ACGCAT CG TGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGAT GGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGG CAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCG GCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGG AGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGG G CAT G ACT AT CGT CGCCGCACTT AT G ACT CTT GT CNT AT GCAACT CAT CGT AGG GC ACAGGT CGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGC CTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCC ACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTA CGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTC CG GCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATC AGGG AT ACAGCTTCAAGG CGCT CGCGGCT ACCAGCCT AACTT CTT GG CG AT CACT ACCGCTG ATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGG CGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCT CGACCTGA SEQ.ID. N ° 37: sequences organized in operon including the modified R349 gene with specific sequence of 15 nucleotides of the tetracycline resistance gene (SEQ ID N ° 39) and the R350 and 354 genes.

TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAAT AATTTTGTTT AACTTT AAG AAGG AG AT AT AG AT ATGG AATT AATT AGTCGTGT CT TT ACT CATGG AG AAAAT ATTTT ACTTGTT AGTT CT ACAAAT AAGTT AT ATATATTG AGT ATTGAAAGTCCAGTATATATTGACATTAAATTAGATGATGATGATTCTGTTAAAG CGTTTT ATT CTTGT AATTT ATT CACG ATG ATT CAT ACATCCAAAGG AAAAATTT A TCTATCAAGATCATTTATTTGTGGTGGAGGTGAAATCGATGCATATGATAGTGAA AGTGATGTTGGAAGTGATGCTGAGTCTGATGCTGAGTCTGATGCTGAATCAGAT T CAG AAAAT C AT ACT C AAAAT AAT AC STW ACTCCC AT STW AAT AT C ACACT AAT T AATTTGG ATT CAT CAAAT AATTCC ACT C AATCCG G CT CTT ACC AG CTC AAT G AA TCCGGCTCTTACCAGCCCAATGAATCTGATCAATCATGTAGTGATTTTGACTGTG GACCTCGATCCGGCTCTTACCAGCCTGAAGAAGTTTTTGTTGATCGTAATGATAA T AATT CAG AT AAT ATT G GT AATT CAAAT AGT ATCG GCT CTT ACCAG CCAT CT AT G ACTG AAAAAGCTGG AAT AATTTTGTT AAACG AAATT AAAACCATGGTAG ATG AAA TTTTGTCT CAAAAAAAT ATT AATT CTGTTGG AATT AACG AT ATTGTT ATT CGTCC AAGCGGACAGTATGTGAGTAATATGGGATATATTACAGAATCAGGTAGTGTATC ATTT CAT ACCAAT AAAAAT G CTWG CTT ATT ATT CAAGTCCAATGTT C ATG AAAT C CTTTT CGTTG AAG AAATGTTT ATGTACT CT AAAAAT AATTTT ATTT ATTTGGCCA T ACCATTT AAAAAAT ACCAGGCAT CT CT AAG AG AT ATTGCTCCTTTT CAT ACAAT T AAT AAAAC AAAAAAT G GT ATT AATT C AAAAT AGTGTTTCCATTT G ACACTG AAAAAAT AG AATTTTGT G AT AATTT CTTTT AT ACTT ATG AAT CCAAT A CTT GTT AT CAT CATGTT ATTT C ATT CT AC AAAAATGTAAATT ATT ATCCTT CTT GG AT AT ATTT CAAAT CTG AG ATTTT AT ATT ATAT AGTTT AAT AGTGT GT ATGTT AAAG AT AAT AACAACGTGT AT STW CGTA AATTT CAAT AATT CT CTTG AAAAAT ACATCG AT AACAAACTCG ACTTGG ATGTCGT AATT GTGCCTG AT AGTT AT AG ACCAATGG AAATG AG ACT ACT ATT AAAGTT AGGT AT AA CATTGTATAGCGATTACAATTTTAATGGATGTGAGGATGATGAAGAACATATTTT CG AAATT ATG AAAAAT CAAT ATGTACCT CAT ATT ATCGGTCTG AATT ATTTTG AA AGTTT CATTGT AGTT ATTGTC AAT AATCC STW ATGTTG ACG AT AAC AACTG ACG ATGGT AAAATTTT CTTT AACATT CATG AT ATT ACTTTTT ACAAAAG ATTTT AT AAT GG AATCGTTT AT CTTG AT AATGGTTCGTT ATTTT AT CT CACAG AT AGTG AAATTT CAGATCAAAATGTATGGAAACTAACTGGATGTCAATTGTGTGAGCTAGCTGATTC T CGTA AT AT AGTT ACCT ATTT AATTT ACCGGACAAAATTG ATG AAATTT ACT CT TCGTCTG AATTT ATTGTT CT AAAATT AATTGG AAACAAAT ATTTTT ATT AT CCGG TTG AAAATTTTG AT ACTGCT CAAG ATTTT AAG ACAAG AT GTGGGG AAATTT CATT G AAAAAT AATT CAGTT CTTG AACT CGTT AAT ACT AGTATT ATT AACAG ACAAT CT AAAAGTT AT CAT ACT ACAGT AT CT ATT AAT ATTG AT ACGG ATTGT ACT ACT CAT A ATT CTTT CG AG AG ATT GTTT AT CCTG ACACAAT CT A TTCTATTCGAATTGTAGACGACAAAAATATTGGTTTTGGTGATGGGCCTAAAATT G AATTTTGTG AATCGGCT ATT ATGCAATTTT ATT AT GTAA ATTT AATTGCT CAT A ATTTTCACACAGAGTTTAATTTACAAGAATTTGCCAAACTGAAACCCACCGAAAT T STW ATTTGGG AT CAATGTTT CAT ATGGTT AT CTGTCAAAAT AATT CTT CGTT A GCC ATT AG ATT ACCT CT AGCTTTTGCTGTAG AAATTT ATGG AAAAG AACCCACAA TTG ATG AATTGG AAT ATTTTGCTTGTAATG AAG AT G AAACTGG ATT CAAACAT AT TT AT CCAGCCAAAT ACAAT CCCT ATT AGT AAT CGGTT ATG AAT CTT AT G AACATTGTCT AAAA ATTT ATG AAG ATG AT ACT G AT AAAA ATATCTTGACGAAAAAATATTGCGAGCAATT AGATATATGAT AATGAT AAT CTTGTTTT ATCGGG AG GT AAGG AT AAAAAT AAT AATT ATTTGG AAATGTT CAAAG AATTT ATT AACT CTTT GT CTG AAAATG AATT AAAAAT CTTGCT AAAAAATTGG ACAGCAT CAACTTGTGT A AG ACC AG AT AAC AAAT AT AG AATT AT CATT ATTT CC AAAT CT AAAAACG CT AAAG CAGGT ATTCG ATTTGGT ACTTGTAATTT AG AAATT CAT ATCG ACG AAATAT ATTAG AATT ACACCTGCT CAAGG A TTCAAAGATTAAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCT GCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTC TTG AGGGGTTTTTTG AG AT CT CG AT CCCGCG AAATT AAT ACG ACT CACT AT AGG GG AATTGTG AGCGG AT AACAATTCCCCACT AG STW AATTTTGTTT AACTTT AAG AAGGAGATATAGCAATGAACCGTCGTAACCGTAGCAACGACCTGAACCCGGAGC CGAGCATCGAAAACCCGAACAACCAGATTGCGGAGGAATTCCCGGGTAACAACA GCGTGTATAAAAGCGACGGCTACGTTGATCTGAAGAACAACGGTCGTCTGTTCC CGATCTGGATTCTGAAGAACTTTAAACAATACAAGCTGCCGGAGATCATTCGTAA AGAGAACGAAGACCCGTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCA GGAATTCGTGGGCCAATACCTGAACCCGCAGGGTCCGTATACCAGCATCCTGCT GTACCATGGTCTGGGTAGCGGCAAGACCGCGAGCGCGATCAACCTGATGAACAT T CT GT ACAACT AT G ACAACGGCACCAACTTT ATCGTT CTG ATT AAAGCG AGCCT G CACAACGACCCGTGGATGCAGGACCTGAAGGAGTGGCTGGGTCGTGATCCGAGC GAACAGAACGTGGACAACGTTACCAAGCTGGATCGTTACAAAAACATCCACTTCG TGCACT ATG ACAGCCCGTTCGCGG AT AGCAGCTTT ATG AGCGTT ATT AAG ACCCT GGACCTGAGCAAACCGACCATGTACATCATTGATGAGGCGCACAACTTTATCCGT AACGTGTATAGCAACATTAACAGCAAACTGGGCAAGCGTGCGAAAGTTATCTATAGAG GACAAGCGTGAAAACAAGAACACCCGTATCGTGCTGATT AGCGCGACCCCGGCGATCAACACCCCGTTCGAACTGGCGCTGATGTTTAACCTG CTGCGTCCGGGTATTTTCCCGAGCAGCGAGCTGGATTTCAACCGTACCTTTGTG ACCGAAAGCAGCTACCCGATCCTGAACCCGATGAAGAAAAACATGTTTGAGCGT CGTATCCTGGGCCTGGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCG CGTCAAGAACTGAAGTACATCAACCTGCCGATGAGCGCGTACCAGTATGATATCT ACCGTATTTTCGAGAAACTGGAGGCGGAAATTCAAGAACGTGCGCGTCGTCGTG GCAAGCAGAGCCAACTGTACCGTACCTATACCCGTCAGGCGTGCAACTTCGTGT TTCCGTACGTTAACATGAACGTGAACGGTGAACTGCGTCCGCGTCCGGGCAAGT TCCGTCTGAGCGAAAAACTGGCGGACGATTTTAGCAAGGGCAAAAACCTGGACG TTCCGGATACCGAGAAAGAAATCCTGAACAAGTATACCAAAGCGATTGAGAACTA CCTGAACGAGACCGAACGTTATTTTCAGAACATCAACAAGAAAGACGCGGAGAA CGGT CGT TIIAC CATT AACG ACCTGG ATG AATT CAAG AAAGGCTTTGGTACCAA GTTCAACAGCTTTCTGCAGTACTATCAAAGCGAGGGTCCGCGTAGCAGCCTGCT GACCGAAATGTACAACTGCAGCCCGAAAATGCTGGCGATCGCGTTCATGACCTA TATTAGCCCGGGCAAGGTGATGATCTACAGCAACTATGTGGTTATGGAAGGCAT CGACGTTATGAAAATTTACTTTCGTCTGATCGGTTTCAACGATTTTACGATCGCG CGTGAGTACATGGGCTATTGCGAATACCACGGTCGTATCGACCCGAAGGATCGT GTGCGTATCAAGA ACATGTTCAACGACAAGAACAACGTGTACGGCAACAAGTGC AAAGTTATCATGCTGAGCCCGAGCGCGACCGAGGGTATTCAACTGCTGGATATC CGTCAGGAGCACATTATGGAACCGTATTGGACCGAAGTTCGTATCCAGCAAGTG ATTGGCCGTGGTGTTCGTCAATGCAGCCACCGTGACCTGCCGATGAGCGAGCGT ATCGTGGATATTTACCGTTATAAGGTTATCAAACCGGAAAACCTGGACCCGGAC GATACCGTGCGTCAAAGCACCGACGAGTACGTTGAAGATCAGGCGAAGAGCAAA GCGAACCTGATTGAGAGCTTCCTGGGCGCTATGAAAGAAGCGGCGGTTGATTGC GAGCTGTTTAAGGAACACAACATGATGAGCCAGAGCTACTATTGCTTCAAATTTC CGGAGAGCGCGGTGACCAAGACCAACGTTGGCCCGGCGTACCGTGAAGACATCA AGGACGATGTGAAATATGATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGC GTATTCGTGTGGTTAAGGTGAACGCGGTTTACCAAATCAACACCGACAACAACAA CCCGGTGTATAGCAGCCCGACCAAGTACTGGTATAACAAGAAAACCGGCATGGT TTATGACTTCGAGACCCACTACCCGGTGGGTCAGGTTGAATTTATCGATAACCTG CCGAACAAGCTGGACAAAGATACCTACATCATGCGTATTGATGTGATCATTCCGA GCATT ACCGGTAGCGTT AACACCT AACACT STW AG AATTTTGTTT AACTTT AAG AAGG AG AT AT ACCG AGCG ATG ACT AT AACAACG ACATT AGCT AG AT AT CG AAAAT TCTGTGGAACATCCTGGGTGACGATTATTTCACCCAAGACGAATTTGACGATCTG GTGAACAGCGTTGCGAACACCATTTACCAGTATGACAACGAAGTGAGCATCGAT AAGCTGAAAGTGATCATCGAATTCGTTATCCTGAACAAGTTCAAGCTGTGCTACA TCTACGATAACGACAGCATCCTGAACCAAGTGAAATACGAGAAGAAAAGCGTTG GTAGCAAAACCATCGGCAAGAACAGCACCAACGACGATGAGGACGATGACGAAG ATATCGCGGTGATTAAGCTGAGCGATATTGAGGCGGGCGAAAACTGGTTCAAGA AAAGCCCGAAAATCAGCAGCAAGCAGTTTCAAAGCGTTGACAAAGTTGAGGTGG CGACCTACGAAGACCTGATCAGCCACAAGCACGATTACCCGAAAGAGATTTATAA GGAAAGCCACTACATCCGTCGTAACACCCGTCTGGATGTGATCAAGAAAATTCCG CAATTCGAGCAGAAGAGCAAAGAATGGCTGAAACAACGTACCGAGAGCCTGACC GCGACCGCGATTAGCGTGGTTTTTGATGAAGACCCGTATAAACACCCGATCGTT ATTCTGCTGGACAAGTGCGGTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTT CACCACGGTAACAAGTATG AACAAATCGGCACCAT GTT CT ACAGCTTT CGTAACA ACGTTGAGGTGGGTGAGTACGGCCTGCTGCAGCACAGCGGTCACAAGTTTATCG CGGCGAGCCCGGATGGCATCTGCAGCAAGAAAGCGAACACCGGTGGCCTGAGCA AACTGGTGGGTCGTCTGCTGGAGATTAAGTTCCCGTTTAGCCGTGAAATCAACA ACAGCGGTGATCTGGACGGCGATATCTGCCCGCACTAC TATTTTCTGCAGGTGC AAACCCAGCTGTATGTTACCGAGATGGACGAATGCGACTTCCTGCAGTGCAAAA TTGACGAGTACGATAGCTGGGAAGACTTTGTGAAGGATAGCAACCCGATCGTTC CGGGTCTGAGCAAAACCACCAACCTGGAGAAGGGCTGCCTGATTCAGCTGAGCG ACAAAAACCTGATCGGCAGCGACGACAAGGAAAAATGCCTGTATAACAGCAAATA CATCTATCCGCCGAAGCTGCACATGACCAACGAGGAAATCGAGAAGTGGATTAG CAGCGAAATCATGAACTACCACAACAACGACCTGAGCGAGAACTATATGATTGAT CGTGTGATCTACTGGCGTCTGAGCCAAGTTACCTGCAACCTGATTAAGCTGAAC AAAGAAGCGTTCGAGGAAAAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTT CTGTTTTATCGTCAGCACAGCGACAAGCTGGATAAACTGATTAAGTTTGTGGAG AAGGTTAAAGAAGATAACAGCGCGGAGATTTTCAGCTACATCAACGAAGACTTTC TGAGCCTGAACAAAGATAGCAAGTACGAGCCGCTGTATCAGGAAGAGACCGAAT GGCGTAAGAAATATAACCAAATCAAGGCGAAGAAAGCGCAGATGTACAAGAACA AGAGCTACAACAAGTACACCAAGTTCAGCAACCTCGAGCACCACCACCACCACCA CTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCAC CGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGG TTTTTTG SEQ. I D. N ° 38: Gene R349 modified with repeated sequences of the tetra cycl ine resistance gene (SEQ IDN ° 39)

ATGG AATT AGTCGTCTTT ACT CATGG AG AAAAT ATTTT ACTTGTT AGTT CTATAT ATG ATAT ATAT ATT ATT ATT ATT ATT ATT ATT ATT ATT AGT CATTT ATTTGTG GTG G AG GT GA AATCGATGCATATGATAGTGAAAGTGATGTTGGAAGTGATGCTGAGTCTGATGC TG AGTCTG ATGCTG AAT AGC ATT CAG AAAAT CAT ACT CAAAAT AAT ACAAAT ACT CCCAT STW AAT AT CAC ACT AATT AATTT GG ATT CAT CAAAT AATTCC ACT CAAT C CGGCTCTTACCAGCCTAATGAATCCGGCTCTTACCAGCCCAATGAATCTGATCAA TCATGTAGTGATTTTGACTGTGGACCTCGATCCGGCTCTTACCAGCCTGAAGAA GTTTTTGTTG ATCGT AATG AT AAT AATT AGC AT AAT ATT G GT AATT CAAAT AGTA TCGGCTCTTACCAGCCATCTATGACTGAAAAAGCTGGAATAATTTTGTTAAACGA AATT AAAACCATGGTAG ATG AAATTTTGTCT CAAAAAAAT ATT AATT CTGTTGG A ATTAACGATATTGTTATTCGTCCAAGCGGACAGTATGTGAGTAATATGGGATATA TT ACAG AAT CAGGTAGTGT AT CATTT CAT ACCAAT AAAAATGGTTT CTT ATT ATT CAAGTCCAATGTT C ATG AAATCCTTTTCGTTG AAG AAATGTTT ATGT ACT CT AAA AAT AATTTT ATTT ATTTGGCCAT ACCATTT AAAAAAT ACCAGGCATCT CT AAGAG AT ATTGCTCCTTTT C AT AC AATT AAT AAAACAAAAAAT G GT ATT AAAT GG AAGT A TTT CAAAAT AGTGTTT CCATTTGACACTG AAAAAAT AG AATTTTGT G AT AATTT C TTTT AT ACTT ATG AAT CCAAT ACTTGTT AT CAT CATGTT ATTT CATT CT ACAAAAA TGT AAATT ATT ATCCTT CTTGG AT AT ATTT CAAAT ATT AT AGT ATAT ATAT ATTAT AGTAT CAAGTG AT ACT AAT AGTGTGT ATGTT AAAG AT AAT AACA ACGT GT AT STW CGTA AATTT CAAT AATT CT CTTG AAAAAT ACATCG AT AACAA ACTCGACTTGGATGTCGTAATTGTGCCTGATAGTTATAGACCAATGGAAATGAG ACT ACT ATT AAAGTT AGGT AT AACATT GT AT AGCG ATT ACAATTTT AATGG ATGT GAGGATGATGAAGAACATATTTTCGAAATTATGAAAAATCAATATGTACCTCATA TT ATCGGTCTG AATT ATTTTG AAAGTTT CATTGT AGTT ATTGTCAAT AATCCAAA T ATGTTG ACG AT AACAACTG A CG ATGGTAAAATTTT CTTT AACATT CATG AT ATT ACTTTTTACAAAAGATTTTATAATGGAATCGTTTATCTTGATAATGGTTCGTTAT TTTATCTCACAGATAGTGAAATTTCAGATCAAAATGTATGGAAACTAACTGGATG T CAATTGTGTG AG CT AG CT GATT CT CGTA AT AT AGTT ACCT ATTT AATTT CAGC G ACAAAATTG ATG AAATTT ACT CTTCGTCTG AATTT ATTGTT CT AAAATT AATT GG AAAC STW ATTTTT ATT AT CGC GTTG AAAATTTTG AT ACT G CT C AAG ATTTT AA G ACAAGATGTGGGG AAATTT CATTG AAAAAT AATT CAGTT CTTG AACT CGTT AAT ACT AGTATT ATT AACAG ACAAT CT AAAAGTT AT CAT ACT ACAGT AT CT ATT AAT AT TGATACGGATTGTACTACTCATAATTCTTTCGAGAGATTGTTTATCCTGACACAA T CT CTT AGTT ATTCCG CAG AAT ATT CT ATTCGAATTGTAG ACG AC AAAAAT ATT G GTTTTGGTGATGGGCCTAAAATTGAATTTTGTGAATCGGCTATTATGCAATTTTA TT AT AAGTATTT AATTGCT CAT AATTTT CACACAG AGTTT AATTT ACAAG AATTT GCCAAACTGAAACCCACCG AAATT AAAT ATTTGGG AT CAATGTTT CAT ATGGTT A T CTGTCAAAAT AATT CTTCGTT ACCG ATT AG ATT ACCT CT AGCTTTTGCTGTAG A AATTT ATGG AAGT AATT AGT AATAT AG CCAAAT AC AAT CCCG AATT AAG AATT CGGTT ATG AAT ATG AACATTGT AAA ATAT TTGACGAAT ATAT ATAT ATAT ACAT ATAT ATCG GG AG GT AAG G AT AAAAAT AAT AATT ATTT GG AAAT G TT CAAAG AATTT ATT AACT CTTTGTCTG AAAATG AATT AAAAAT CTTGCT AAAAA ATTGG ACAGCAT CAACTTGTGT AAG ACCAG AT AACAAAT AT AG AATT AT CATT AT TT CCAAAT CT AAAA GTACTTGTAATTT AG AA ATTCATATCGACGAAAAAATGTTGGATGAACATAATATTGATACAGTCAAAGAGG TTTT AATT AC ACCTGCT C AAG G ATT CAAAG ATT AA

SEQ. I D. N ° 39: specific sequence of 15 n clotides of the tetracycline resistance gene

CGGCTCTTACCAGCC SEQ. ID. N ° 40: vector PP37 CCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGT CGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACC GAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTAC AATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTG ACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGAC GGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGA GCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGC GGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCAT CCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAA GCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTA AGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCT CACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGG TAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCA ATGCCAG CGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCA TCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTC CAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCA GACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTC TGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGC ACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCG CCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAA GATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCG GTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCAT GATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCG GAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGC CTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAG TCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA GGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGG CAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGG GATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCA CCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGA TCGTTGGCAACCAGC ATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATG GTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCG AGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGA CCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTT GATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGC AGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCG CTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATT TAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAA CGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAA TGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATA CTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTC TCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATG GTG TCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAG TAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGA GATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAA ACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTC GGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGAT GCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACT AT AGGGG AATTGT G AGCGG AT AACAATT CCCCT CT AG STW AATTTTGTTT AACT TTAAGAAGGAGATATAGATATGGAATTAATTAGTCGTGTCTTTACTCATGGAGAA AAT ATTTT ACTTGTT AGTT CT AC STW AAGTT AT AT ATT ATG G GT AAT AATG AAT ATG GTT C ATGTG CTWG C AAAAT AG GT AC AG AT AAAACTT AT ATTG AAAGTCC AGT AT AT ATTG ACATT AAATT AG ATG ATG AT GATT CTGTT AAAGCGTTTT ATT CTTGT AATTT ATT CACG AT GATT CAT ACATCCAAAGG AAAAATTT AT CT AT CAAG AT CAT TTATTTGTGGTGGAGGTGAAATCGATGCATATGATAGTGAAAGTGATGTTGGAA GTGATGCTGAGTCTGATGCTGAGTCTGATGCTGAATCAGATTCAGAAAATCATA CT CAAAAT AAT ACAAAT ACTCCCAT STW AAT AT CACACT AATT AATTTGG ATT CA T CAAAT AATTCCACT C AAT CCG G CT CTT ACC AGCCT AATG AAT GCC G CT CTT ACC AGCCCAATGAATCTGATCAATCATGTAGTGATTTTGACTGTGGACCTCGATCCGG CT CTT ACC AG CCTG AAG AAGTTTTTGTTG ATCGT AAT G AT AAT AATT CAG AT AAT ATTGGT AATT CAAAT AGTATCGGCT CTT ACCAGCCAT CT ATG ACTG AAAAAGCT GG AAT AATTTTGTT AAACG AAATT AAAACCATGGT AG ATG AAATTTTGTCT CAAAA AAATATTAATTCTGTTGGAATTAACGATATTGTTATTCGTCCAAGCGGACAGTAT GTG AGT AAT ATGGG AT AT ATT ACAG AAT CAGGTAGTGTAT CATTT CAT ACCAAT A AAAATGGTTT CTT ATT ATT CAAGTCCAATGTT CATG AAATCCTTTTCGTTG AAG A AATGTTT ATGT ACT CT AAAAAT AATTTT ATTT ATTTGG CCAT ACC ATTT AAAAAAT ACCAG G CAT CT CT AAG AG AT ATTGCTCCTTTT CAT AC AATT AAT AAAAC AAAAAA TGGTATTAAATGGAAGTATTTCAAAATAGTGTTTCCATTTGACACTGAAAAAATA AATTTTGTG G AT AT AATTT CTTTT ACTT AATCCAAT ACTTGTT ATG CAT CAT AT G C ATT CT TT ATTT AC AAAAATGTAAATT ATCCTT ATT CTT G GATAT ATTT CAAAT CT GAGATTGATATTAATAGTAAAAATATGTTCTTTTCAAGTGATACTAATAGTGTGT ATGTT AAAG AT AT AAT AACAACGTGT STW CGTA AATTT CAAT AATT CT CTTG A AAAATACATCGATAACAAACTCGACTTGGATGTCGTAATTGTGCCTGATAG TTAT AG ACCAATGG AAATG AG ACT ATT AAAGTT AGGT AT AACATTGT AT AGCG ATT ACAATTTT AATGG ATGTG AGG ATG ATG AAG AACAT ATTTTCG AAATT ATG AAAAA T CAAT ATGT ACAT ATTGG ATCTG ATTG ATTG ATTG ATGT AACATT CATG AT ATT ACTTTTT ACAAAAG ATTTT AT AATGG AATCGTTT AT CTT G AT AATGGTTCGTT ATTTT AT CT CACAG AT AGTG AAATTT CAG AT CAAAATGTAT GGAAACTAACTGGATGTCAATTGTGTGAGCTAGCTGATTCTACCATATATAGTTA CTC ATTT AATTT ACCGG ACAAAATT G ATG AAATTT ACT CTTCGTCTG AATTT ATT GTT CT AAAATT AATTGG AAACAAAT ATTTTT ATT ATCCGGTTG AAAATTTTG AT A CT G CT CAAG ATTTT AAG AC AAG ATGTG GGG AAATTT C ATTG AAAAAT AATT CAGT T CTTG AACT CGTT AAT ACT AGTATT ATT AACAG ACAAT CT AAAAGTT AT CAT ACT AC AGT AT CT ATT AAT ATTG AT ACGG ATT GT ACT ACT CAT AATT CTTTCG AG AT CCTG ACACAAT CT CTT AGTT ATT CCGCAG AAT ATT CT ATTCG AATTGT AG ACG ACAAAAATATTGGTTTTGGTG ATGGGCCTAAAATTG AATTTTGTG AATCG GCT ATT ATGCAATTTT ATT AT AAGT ATTT AATTGCT CAT AATTTT C ACACAG AGT TT AATTT ACAAG AATTTGCCAAACT G AAACCCACCG AAATT AAAT ATTTGGG AT C AATGTTT CAT ATGGTT AT CTGTC AAAAT AATT CTT CGTT ACCG ATT AG ATT ACCT CT AGCTTTTGCTGTAG AAATTT ATGG AAAAG AACCCACAATTG AT GATT ATTTTGCTTGTAATG AAG AT G AAACTGG ATT CAAACAT ATTT ATCCAGCCAAAT A CAAT CCCG AATT AGTT AAAG AATTCG GTT ATG AAT CTT ATG AAC ATT GTCT AAAA ACTTTGT GT AAAT AT AATT ATG AAG AT G AT ACTG AT AAAAAT AT CTTG ACGAA AGC CAAG AACAT AAAACAAATG AATTT ACCC ACATT GG ACT ATT AC ATTT CT G GCCCAT AC AAAATT AAT AG AACCAT ATT AATT AAT AAT CTTGTTTT AT CGGG AGGT AAGG AT AAAAAT A AT AATT ATTTGG AAATGTT CAAAG AATTT ATT AAT CTTTGT ACT AAA CTTTGT CAA AACAAA T AT AG AATT AT C ATT ATTTCCAAAT CT AAAAACGCT AAAG CAG GT ATTCG ATTT G GT ACTT GT AATTT AG AAATT CAT ATCG ACG AAAAAATGTTGG ATG AACAT AAT AT TG AT ACAGTCAAAG AGGTTTT AATT ACACCTGCT CAAGG ATT CAAAG ATT AAG AT CCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAG CAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGA T AACAATT CCCCACT AG AAAT AATTTTGTTT AACTTT AAG AAGG AG AT AT AGCAA TGAACCGTCGTAACCGTAG CAACGACCTGAACCCGGAGCCGAGCATCGAAAACC CGAACAACCAGATTGCGGAGGAATTCCCGGGTAACAACAGCGTGTATAAAAGCG ACGGCTACGTTGATCTGAAGAACAACGGTCGTCTGTTCCCGATCTGGATTCTGA AGAACTTTAAACAATACAAGCTGCCGGAGATCATTCGTAAAGAGAACGAAGACCC GTGCAACGTGCAGGTTAAGCTGGAGCTGCGTAAATATCAGGAATTCGTGGGCCA ATACCTGAACCCGCAGGGTCCGTATACCAGCATCCTGCTGTACCATGGTCTGGG TAGCGGCAAGACCGCGAGCGCGATCAACCTGATGAACATTCTGTACAACTATGA CAACGGCACCAACTTTATCGTTCTGATTAAAGCGAGCCTGCACAACGACCCGTGG ATGCAGGACCTGAAGGAGTGGCTGGGTCGTGATCCGAGCGAACAGAACGTGGAC AACGTTACCAAGCTGGATCGTTACAAAAACATCCACTTCGTGCACTATGACAGCC CGTTCGCGGATAGCAGCTTTATGAGCGTTATTAAGACCCTGGACCTGAGCAAAC CGACCATGTACATCATTGATGAGGCGCACAACTTTATCCGTAACGTGTATAGCAA CATT AACAGCAAACTGGGCAAGCGTGCG AAAGTT AT CT CAG AGT CAT G AA ACAT GGACAAGCGTGAAAACAAGAACACCCGTATCGTGCTGATTAGCGCGACCCCGGC GATCAACACCCCGTTCGAACTGGCGCTGATGTTTAACCTGCTGCGTCCGGGTAT TTTCCCGAGCAGCGAGCTGGATTTCAACCGTACCTTTGTGACCGAAAGCAG CTA ATG AAG CCCG ATCCTGAACCCG AAAAACATGTTTGAGCGTCGTATCCTGGGCCT GGTTAGCTACTATATTGGTGCGACCCCGGACCTGTATGCGCGTCAAGAACTGAA GT ACAT CA ACCTGCCG AGCGCGTACCAGT ATG ATG AG AT AT CT ACCGTATTTTCG AAACTGG AGGCGGAAATTCAAGAACGTGCGCGTCGTCGTGGCAAGCAGAGCCAA CTGTACCGTACCTATACCCGTCAGGCGTGCAACTTCGTGTTTCCGTACGTTAACA TGAACGTGAACGGTGAACTGCGTCCGCGTCCGGGCAAGTTCCGTCTGAGCGAAA AACTGGCGGACGATTTTAGCAAGGGCAAAAACCTGGACGTTCCGGATACCGAGA AAGAAATCCTGAACAAGTATACCAAAGCGATTGAGAACTACCTGAACGAGACCGA ACGTTATTTTCAGAACATCAACAAGAAAGACGCGGAGAACGGTCGTACCATCATT AACG ACCTGG AATT ATG CAAG AAAGGCTTTGGTACCAAGTTCAACAGCTTTCTGC AGTACTATCAAAGCGAGGGTCCGCGTAGCAGCCTGCTGACCGAAATGTACAACT GCAGCCCGAAAATGCTGGCGATCGCGTTCATGACCTATATTAGCCCGGGCAAGG ATG TG AT CT ACAGCAACT ATGTGGTT ATGG AAGGCATCG ACGTT ATG AAAATTT A CTTTCGTCTGATCGGTTTCAACGATTTTACGATCGCGCGTGAGTACATGGGCTAT TGCGAATACCACGGTCGTATCGACCCGAAGGATCGTGTGCGTATCAAGAACATG TTCAACGACAAGAACAACGTGTACGGCAACAAGTGCAAAGTTATCATGCTGAGCC CGAGCGCGACCGAGGGTATTCAACTGCTGGATATCCGTCAGGAGCACATTATGG AACCGTATTGGACCGAAGTTCGTATCCAGCAAGTGATTGGCCGTGGTGTTCGTC AATGCAGCCACCGTGACCTGCCGATGAGCGAGCGTATCGTGGATATTTACCGTT ATAAGGTTATCAAACCGGAAAACCTGGACCCGGACGATACCGTGCGTCAAAGCA CCGACGAGTACGTTGAAGATCAGGCGAAGAGCAAAGCGAACCTGATTGAGAGCT TCCTGGGCGCTATGAAAGAAGCGGCGGTTGATTGCGAGCTGTTTAAGGAACACA ACATGATGAGCCAGAGCTACTATTGCTTCAAATTTCCGGAGAGCGCGGTGACCA AGACCAACGTTGGCCCGGCGTACCGTGAAGACATCAAGGACGATGTGAAATATG ATAGCGGTCTGAACAGCAAAAACAGCATCGTTGAGCGTATTCGTGTGGTTAAGG TGAACGCGGTTTACCAAATCAACACCGACAACAACAACCCGGTGTATAGCAGCCC GACCAAGTACTGGTATAACAAGAAAACCGGCATGGTTTATGACTTCGAGACCCAC TACCCGGTGGGTCAGGTTGAATTTATCGATAACCTGCCGAACAAGCTGGACAAA GATACCTACATCATGCGTATTGATGTGATCATTCCGAGCATTACCGGTAGCGTTA ACACCT AACACT AG STW AATTTTGTTT AACTTT AAG AAGG AG AT AT AGCG ATG CCG ACATT AGCT ACT AT AACAACG AG ATCG AT AAAATT CT GTGG AACATCCTGGG TGACGATTATTTCACCCAAGACGAATTTGACGATCTGGTGAACAGCGTTGCGAAC ACCATTT ACCAGT ATG ACAACG GTAA G AGCATCG AT AAGCT G AAAGTG AT CAT CG AATTCGTTATCCTGAACAAGTTCAAGCTGTGCTACATCTACGATAACGACAGCAT CCTGAACCAAGTGAAATACGAGAAGAAAAGCGTTGGTAGCAAAACCATCGGCAA GAACAGCACCAACGACGATGAGGACGATGACGAAGATATCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTG GAAAACTGGTTCAAGAAAAGCCCGAAAATCAGCAG CAAGCAGTTTCAAAGCGTTGACAAAGTTGAGGTGGCGACCTACGAAGACCTGAT CAGCCACAAGCACG ATT ACCCG AAAG AG ATTT AT AAGG AAAGCCACT ACAT CCGT CGTAACACCCGTCTGGATGTGATCAAGAAAATTCCGCAATTCGAGCAGAAGAGC AAAGAATGGCTGAAACAACGTACCGAGAGCCTGACCGCGACCGCGATTAGCGTG GTTTTTGATGAAGACCCGTATAAACACCCGATCGTTATTCTGCTGGACAAGTGCG GTCGTGGCCTGCCGTTCGTGGAGAACAAATTTGTTCACCACGGTAACAAGTATG AACAAATCGGCACCATGTTCTACAGCTTTCGTAACAACGTTGAGGTGGGTGAGT ACGGCCTGCTGCAGCACAGCGGTCACAAGTTTATCGCGGCGAGCCCGGATGGCA TCTGCAGCAAGAAAGCGAACACCGGTGGCCTGAGCAAACTGGTGGGTCGTCTGC TGGAGATTAAGTTCCCGTTTAGCCGTGAAATCAACAACAGCGGTGATCTGGACG GCG AT AT CTGCCCGCACT ACT ATTTT CTGCAGGTGCAAACCCAGCT GT ATGTT AC CGAGATGGACGAATGCGACTTCCTGCAGTGCAAAATTGACGAGTACGATAGCTG GGAAGACTTTGTGAAGGATAGCAACCCGATCGTTCCGGGTCTGAGCAAAACCAC CAACCTGGAGAAGGGCTGCCTGATTCAGCTGAGCGACAAAAACCTGATCGGCAG CG ACG ACAAGG AAAAATGCCT GT AT AACAGCAAAT ACAT CT ATCCGCCG AAGCT G cacat G ACCAACG AGG AAAT CG AG AAGTGG ATT AGCAGCG AAAT CATG AACT AC CACAACAACGACCTGAGCGAGAACTATATGATTGATC GTGTGATCTACTGGCGT CTGAGCCAAGTTACCTGCAACCTGATTAAGCTGAACAAAGAAGCGTTCGAGGAA AAAATCCCGCTGCTGCAGCAATTCTGGGACTACGTTCTGTTTTATCGTCAGCACA ACAAGCTGG GCG ATT AT AAACTG AAGTTTGTGG AG AAG AT AAGGTT AAAG AACA GCGCGGAGATTTTCAGCTACATCAACGAAGACTTTCTGAGCCTGAACAAAGATA GCAAGTACGAGCCGCTGTATCAGGAAGAGACCGAATGGCGTAAGAAATATAACC AAATCAAGGCGAAGAAAGCGCAGATGTACAAGAACAAGAGCTACAACAAGTACA CCAAGTTCAGCAACCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGC ATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGG AACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCG CCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGC ACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGC CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGG ACT CTTGTTCCAAACT GG AACAACACT CAACCCT ATCTCG GTCT ATT CTTTT G AT TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC AAAAATTT AACGCG AATTTT AACAAAAT ATT AACGTTT ACAATTT CAGGTGGCAC TTTTCGGGG AAATGTGCGCGG AACCCCT ATTTGTTT ATTTTT CT STW ACATT CA AAT ATGTAT CGC CT C ATG AG AC AAT AACCCT G AT AAATGCTT C AAT AAT ATTG AA AAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGAT GCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGC GGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGC AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGT CACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGA CCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC ACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTAC TTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG CAGGACCACTTC TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAAT CTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGG ATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGT AACTGTCAG ACCAAGTTT ACT CAT AT AT CADTC AG ATTG ATTT AAAACTT CATTT TT AATTT AAAAGG AT CT AGGTG AAG ATCCTTTTTG AT AAT CT CATG ACCAAAAT C CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAG G AT CTT CTTG AG AT CCTTTTTTT CTGCGCGTAAT CTGCTGCTTGCAAACAAAAAA ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG T AGCCGTAGTT AGGCCACCACTTCAAG AACT CTGTAGCACCGCCT ACAT ACCTCG CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTA CCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAA CGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTT

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Claims

REVEN DICATIONS  1. Method for modification by cutting and / or total or partial degradation of a target nucleic acid sequence of a host cell comprising bringing said host cell into contact in vitro or ex vivo with a biological system comprising: a first component comprising at least one protein chosen from the proteins R350 and R354 of giant virus Mimivirus of line A or at least one expression vector of at least one nucleic acid sequence of the R350 and / or R354 genes coding for respectively at least one said protein R350 and / or R354, and a second component comprising at least one nucleic acid sequence, called affinity sequence, said affinity sequence comprising at least one sequence complementary to a specific sequence of said target sequence, said specific sequence of said target sequence comprising at least 10 nucleotides, preferably 15 nucleotides. 2. Method according to claim 1, characterized in that said second component comprises said affinity sequence in the form of DNA or preferably in the form of RNA. 3. Method according to claim 1 or 2, characterized in that said affinity sequence comprises at least 2 same said complementary sequences of the same specific sequence, preferably from 2 to 4, preferably still 4 same d ites complementary sequences of the same specific sequence, repeated spaced apart. 4. Method according to one of claims 1 to 3, characterized in that said or each said complementary sequence of the same said specific sequence of the affinity sequence is flanked in 3 'and 5' by adjacent sequences framing a sequence of 15 nucleotides of Zamilon virophage contained and repeated 4 times in the R349 gene of giant Mimivirus virus. 5. Method according to one of claims 1 to 4, characterized in that the said same sequences complementary to the same specific sequence of the target sequence are flanked in 3 'and 5' of flanking sequences different from each other between the different same complementary sequences and said flanking sequences respectively corresponding to the flanking sequences of the same same repeated sequences of the zamilon virophage contained in said gene R349 of Mimivirus. 6. Method according to one of claims 1 to 5, characterized in that said affinity sequence comprises the modified R349 gene in which only the 4 repeated sequences of 15 nucleotides of the zamilon virophage are replaced at the same locations by said 4 same sequences complementary to a said specific sequence of the said target sequence. 7. Method according to one of claims 1 to 6, characterized in that said affinity sequence is integrated into a cloning vector of said affinity sequence capable of transforming or transfecting a said host cell and replicating the so-called affinity sequence. 8. Method according to claim 7, characterized in that the said affinity sequence is integrated in the form of DNA placed under the control of a transcription promoter capable of transcribing the said affinity sequence from DNA to RNA in said host cell. 9. Method according to one of claims 1 to 8, characterized in that said host cell is a bacterium or a eukaryotic cell. 10. Method according to one of claims 1 to 9, characterized in that said first component comprises an expression vector of at least one nucleic acid sequence of gene R350 and / or R354 encoding for and capable of respectively expressing at least one said protein R350 and / or R354 in a said host cell. 11 Method according to one of claims 1 to 10, characterized in that said first and second components are integrated in the same said vector, preferably a plasmid. 12. Method according to one of claims 1 to 11, for at least cutting said target sequence at a specific site, characterized in that said first component comprises at least the R350 protein or at least one expression vector for at least one nucleic acid sequence of the R350 gene coding for respectively at least one said R350 protein. 13. Method according to one of claims 1 to 12, for at least cutting into a specific site and at least partially degrading said target sequence, characterized in that said first component comprises at least the protein R350 and the protein R354 or at least one expression vector for at least nucleic acid sequences of the R350 and R354 genes coding for at least the said proteins R350 and R354 respectively. 14. Method according to one of claims 10 to 12, characterized in that the said first and second components are integrated into the same said vector comprising the sequences of Mimivirus of line A including: at least one modified part of the R349 gene, preferably the complete modified R349 gene, said modified part of the R349 gene including a part of the 349 gene including at least one of the 4 repeated sequences of 15 nucleotides of the zamilon virophage of the R349 gene, preferably the 4 repeated sequences, the said modification of the said modified part of the R349 gene consisting in that the said repeating sequence (s) of 15 nucleotides of the zamilon virophage of the R349 gene is (or are ) replaced by the same so-called complementary sequence of a specific sequence of said target sequence, and a sequence allowing the transcription into RNA of said modified part of the R349 gene, preferably of the complete modified R349 gene in a said host cell, and - the R350 and 354 genes, as well as sequences allowing the expression of the R350 and R354 genes in a said host cell. 15. Biological system useful for implementing a process for modifying by cut and / or total or partial degradation of a target nucleic acid sequence of a host cell comprising bringing said host cell into contact in in vitro or ex vivo, as defined in one of claims 1 to 14 comprising: a first component comprising at least one protein chosen from the proteins R350 and R354 of giant virus Mimivirus of line A or at least one expression vector of at least one nucleic acid sequence of the R350 and / or R354 genes coding for respectively at least one said protein R350 and / or R354, and a second component comprising a nucleic acid sequence, called an affinity sequence, said affinity sequence comprising at least one sequence complementary to a specific sequence of said target sequence, said specific sequence of said target sequence comprising at least minus 10 nucleotides, preferably 15 nucleotides, preferably the or each said complementary sequence of a specific sequence of said target sequence being flanked in 3 'and 5' by adjacent sequences flanking a sequence of 15 nucleotides of the zamilon virophage contained and repeated 4 times in the R349 gene of giant virus Mimivirus of line A.