KR20230127265A - Regulation of the host microbiome through the delivery of DNA payloads with minimal spread - Google Patents

Abstract

The present invention relates to nucleic acids of interest for regulating the host microbiome, vectors encoding the nucleic acids, and methods for regulating the microbiome of a subject in vivo by delivering the nucleic acids of interest.

Description

Regulation of the host microbiome through the delivery of DNA payloads with minimal spread

The present invention relates to a nucleic acid of interest for regulating the microbiome of a host, a vector encoding the nucleic acid, and a method for regulating the microbiome of a host by delivering the nucleic acid of interest.

Delivery of DNA payloads to express genes of interest in bacterial populations outside the laboratory has many applications, including medicine, agriculture, biofuels, and cosmetics.

This strategy relies on the transfer of DNA by viral capsid, by bacterial conjugation or by other methods into target bacterial cells in a pure or mixed bacterial population such that one or several genes of interest are expressed at levels sufficient to produce the desired effect. . The effect is to kill the bacteria and thus reduce their colonization level or modify their proportion compared to other bacteria in the population when multiple species or multiple strains are present; It can be a direct therapeutic effect on the bacterium itself in or on the host by modifying its genome, or by altering its metabolism or its composition (proteins, lipids, sugars, metabolites, RNA, etc.). Effects can also affect target bacteria to produce, display or secrete one or multiple molecule(s) such as prophylactic or therapeutic molecule(s) that will have a direct or indirect effect on the host or other members of the host microbiome. It may be an indirect effect of

One of the major concerns of this strategy is that if the exogenous DNA remains stable in the cell to which it is transferred, or if it is transferred to other bacteria via other gene transfer mechanisms and then remains stable in these other populations, then progeny cells that it is transmitted to More generally, containment of exogenous DNA payloads is a concern when delivered to bacterial populations.

In order to solve this problem, the present inventors propose that the DNA payload, once delivered to the target bacteria, cannot be replicated in the target bacteria, but without the need for an antibiotic resistance selection marker on the DNA payload and without the need for a selection step using antibiotics. , developed new strategies to ensure that the gene(s) of interest are still expressed at levels sufficient to produce the expected results in the bacterium or host.

Plasmids carrying conditional origins of replication have a long history of use by microbiologists as tools to genetically modify bacterial strains of interest, thus creating stable genetically modified organisms. They are typically used to select for recombination events between a plasmid carrying this origin and the genome of the bacterium of interest.

Such plasmids carry antibiotic resistance selection markers and can be introduced into bacteria through transformation, conjugation or any other method. Because they lack an autonomously replicating origin of replication, only bacteria that have recombined the plasmid into their genome will stably retain the selection marker and survive the selection step. The plasmid is stably integrated and maintained into the progeny cells, which will also be able to survive in the presence of the selectable marker.

The most commonly used origin of conditional replication is based on the wild-type plasmid R6K and derivatives belonging to the IncX replication group, a group commonly found in various bacterial isolates. Replication of these plasmids is dependent on the binding of the pir- encoded П initiator protein to the origin of replication. These proteins can be expressed from a different replicon ( in trans ) than the plasmid carrying the R6K origin of replication. In this situation, replication of the R6K ori plasmid is conditional on the expression of the pir gene in trans. When delivered to the bacterium of interest, the plasmid will not replicate unless the pir gene is present and expressed.

A similar conditional origin is also the ColE1 origin (Panayotatos (1984) Nucleic Acids Res . 12 :2641-2648) or IncPalpha oriV (Matsumoto-Mashimo et al. (2004) Res. Microbiol. 155 :455-461). There are several drawbacks associated with these systems when attempting to construct systems in which the risk of spread of genetically modified material in an in vivo situation (eg, human, environment or animal) is minimized. In particular, this system is inspired by origins that are largely ubiquitous in nature, such as the ColE1 and R6K-types, which can be found, for example, in many Enterobacteria. Thus, having this origin on a recombinant plasmid that is transferred into the microbiome results in not only recombination between the recombinant plasmid and the wild-type component within the microbiome, but also the wild-type component resulting in the missing factor required for replication of the plasmid and thus the microbiome. significantly increases the chance of replication of these plasmids within Additionally, since inducible systems are generally leaky, conditional origins of replication that depend on these systems can be reduced to basal levels—sufficient for diffusion in the population—or even to full replication levels if the inducer is present in the target population ( For example, a LacI-based origin has a high chance of replicating (becoming active in the presence of lactose, which is very common in vivo given the modern diet).

It is not an object of the present invention to create and select recombination events between a DNA payload and a target bacterial genome in order to generate a stably genetically modified bacterium capable of transferring the modification to progeny cells, but rather the gene of interest carried on the DNA payload. Contrary to the purpose of limiting and/or preventing the production of genetically modified progeny cells while still enabling direct or indirect effects on the bacteria transferred to its host through efficient expression of DNA pages that can be delivered to the target bacterial population. To specifically engineer and efficiently produce a vehicle containing rod.

The desired effect to be obtained in the targeted bacterium or host includes, by way of non-limiting example, a therapeutic effect, a cosmetic effect, a bioremediation effect, an effect on plant growth or physiology, an effect on animal growth or physiology.

Obtaining a therapeutic or other tangible effect on the target bacterium or its environment using a non-replicating vector is provided that the DNA payload is efficiently delivered to the target bacterium and, despite its non-replicating nature, for a sufficient time and It is not an obvious development for the simple reason that it should be obtainable if it can be expressed to a sufficiently high level. The replicating plasmid itself will produce copies, resulting in gene doses and transfer to daughter cells allowing significant retention time in the bacterial population, but none of these effects occur with the non-replicating plasmid.

Here we demonstrate for the first time that it is possible to obtain an in vivo effect, such as a therapeutic effect, by delivery of a non-replicating vector to bacteria.

For this purpose, the present inventors based on a sparsely present two-system component, the phage-like inducible component, namely the primase of the phage-inducible chromosome island (PICI) and the origin of replication, to limit recombination events in the target population. have developed a novel origin of conditional replication that is particularly efficient for this application, which is a conditional origin of this type, even in the presence of a primase in trans, as a delivery vehicle, here a phage-derived particle or a packaged phage. It is demonstrated for the first time that midro enables efficient packaging of DNA payloads.

[Fillol-Salom et al. (2018) The ISME Journal 12 :2114-2128] or [Fillol-Salom et al. (2019) Mol. Cell 75 :1020-1030] are systems similar to P4-like components that hijack myoviridae, the main difference being that, according to the current study, they do not modify the size of the capsid to accommodate their genome. Since lambda PICIs are usually 10-13 kb in length, and the phages they hijack have genomes close to 50 kb, this means that they can insert several copies of their small genome into the large capsid.

Studies have shown that PICI can completely abolish phage production and only lead to the packaging of their genomes. PICI senses when hijacking lambda-like phages are induced, and they clone their genome by excising from the resident genome as prophage-like islands. Replication is based on a single protein, primase, containing primase and helicase activity, and a short DNA fragment immediately following the primase gene, which is usually recognized by the primase as the origin of replication. Additionally, many different PICIs have been described, each containing a different primase-ori phase.

[Fillol-Salom et al. (2018) The ISME Journal 12 :2114-2128]. PICI originating from E. coli strain CFT073 is specifically disclosed. In this document, the authors show that a genetic module containing primase and ori can function as an independent replication module when inserted cis into a thermosensitive-origin-containing plasmid; At permissive temperatures, plasmids replicate through the plasmid origin, but when shifted to non-permissive temperatures, the primase and oir modules act as the plasmid's primary sources of replication. However, it is clear from this observation whether even at non-permissive temperatures, to some extent replication can occur, whether it is due to a thermosensitive origin; whether the primase and ori can be physically separated (i.e. either away from each other on the same plasmid or have the system in trans) and still be able to replicate the plasmid; and finally, whether the ori located immediately downstream of the primase is the only component required for replication or whether a necessary second component is present and if the two components, ori and crr sequences, are also required for replication, in a particular orientation. As in the case of phosphorus, P4, it is not clear to those skilled in the art whether the particular orientation of the different components is important (Flensberg et al. (1987) J. Mol. Biol. 195 :439-445).

Other primase-based systems in which primase proteins are expressed in trans have been developed (Ziegelin et al. (2005) J. Bacteriol. 187 :3445-3454), but whether this type of replication is compatible with phagemid packaging remains to be seen. It is not known, and even if it could be, predicting whether packaging is efficient is much less clear.

It is indeed also very important that DNA payloads and their vehicles be produced very efficiently in order to be economically viable, and this is not an obvious development either. Indeed, some studies have shown that production titers of phage-derived particles packaging DNA payloads containing conditional ori were reduced by at least 3 log compared to DNA payloads containing non-conditional ori, even in multiple engineering trials. However, it was shown that this titer could not be increased.

Summary of the Invention

The present invention not only enables DNA payloads without antibiotic resistance markers and autonomous replication origins to be packaged into phage-derived particles at a high-titer price, but also allows these DNA payloads to be efficiently delivered to target bacteria and their DNA payloads. The payload is non-replicable, but comes from an unexpected discovery that it can exert its intended effect. In particular, the inventors also demonstrate for the first time that non-replicating DNA payloads expressing nucleases or engineered nucleases, such as base-editing agents, can cause similar killing or base-editing efficiencies to their replicative counterparts. did

Accordingly, the present invention relates to a method for modulating in vivo the microbiome of a host organism by delivering a nucleic acid of interest to a targeted recipient bacterial cell of the microbiome, wherein the nucleic acid of interest is transferred to the targeted recipient bacterial cell. produce a certain effect on

The method comprises administering to the host organism a nucleic acid vector comprising the nucleic acid of interest,

wherein the vector further comprises a conditional origin of replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell, wherein the vector is free of an antibiotic resistance marker;

thereby delivering said nucleic acid of interest to a targeted recipient bacterial cell,

Upon delivery to the targeted recipient bacterial cell, the nucleic acid of interest produces the desired effect on the targeted recipient bacterial cell even though the vector does not replicate in the targeted recipient bacterial cell.

The present invention also relates to a method for modulating in vivo the microbiome of a host organism by delivering a nucleic acid of interest to a targeted recipient bacterial cell of the microbiome, wherein the nucleic acid of interest is expressed in the targeted recipient bacterial cell. to produce a desired effect on the targeted recipient bacterial cell;

The method comprises administering to the host organism a nucleic acid vector comprising the nucleic acid of interest,

wherein the vector further comprises an origin of conditional replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell, wherein the vector is free of an antibiotic resistance marker;

thereby delivering said nucleic acid of interest to a targeted recipient bacterial cell;

Upon delivery to the targeted recipient bacterial cell, the nucleic acid of interest produces the desired effect on the targeted recipient bacterial cell, although the vector is non-replicated in the targeted recipient bacterial cell.

In certain embodiments, the predetermined effect on the targeted recipient bacterial cell directly or indirectly produces a response in the organism hosting the targeted recipient bacterial cell.

DETAILED DESCRIPTION OF THE INVENTION

Justice

As used herein, the term “ nucleic acid ” refers to a sequence of at least two covalently linked nucleotides, which may be single-stranded or double-stranded, or contain portions of both single-stranded and double-stranded. Nucleic acids of the invention may be naturally occurring, recombinant or synthetic. A nucleic acid may be in the form of a circular sequence or a linear sequence or a combination of both. A nucleic acid can be either genomic or cDNA, DNA, or RNA, or a combination of both. A nucleic acid can be any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine and isoguanine. may contain. Other examples of modified bases that can be used in the present invention are detailed in [Chemical Reviews 2016, 116 (20) 12655-12687]. The term “nucleic acid” also refers to other backbones including, without limitation, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidite linkages and/or deoxyribonucleotides and ribonucleotide nucleic acids. It encompasses any nucleic acid analog that may contain. Any combination of these properties of nucleic acids is also encompassed by the present invention.

As used herein, the term “ peptide ” refers to both short chains of at least two amino acids linked to each other and parts, subsets, or fragments of proteins that are not expressed independently from the rest of the protein. do. In some instances, a peptide is a protein. In some other examples, a peptide is not a protein, and a peptide refers only to a part, subset or fragment of a protein. Preferably, the peptide is from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 20, 25, 30, 40, 50, 100, 200 amino acid sizes.

in vivo Adjustment method

The present invention relates to methods for regulating the microbiome of a host organism in vivo.

" Microbiome ", as used herein, in the case of a mammalian organism, is skin, mammary gland, lichen, semen, vagina, uterus, ovarian follicle, lung, saliva, oral mucosa, conjunctiva, biliary tract, and gastrointestinal tract, blood, tumor, brain It refers to the collection of all microbiota that reside on or in organism tissues and biological fluids, together with the corresponding anatomical site in which they reside, including. In a specific embodiment, microbiome refers more particularly to the population of bacteria that form said microbiota.

By " modulation of the microbiome " is meant herein the exertion of a modifying or controlling influence on the microbiome. In the context of the present invention, modulation of the microbiome encompasses modulation of microbiome function and/or modulation of microbiome composition.

" Modulation of microbiome composition " means elimination of a particular species or strain of the microbiome, change in the ratio between different species or strains of the microbiome, or change of a particular species or strain of the microbiome to another species or strain. It means a change in the composition of the microbiome, including the replacement of. Such modulation of microbiome composition may be obtained directly or indirectly, typically by modifying the targeted bacterial cell, followed by effects of the microbiome on other bacteria not initially targeted by the vector; For example, it may have a killing effect.

" Modulation of microbiome function " is used herein, for example, to cause a particular species or strain to express a particular molecule, or to cause a particular species or strain to stop expressing a particular molecule, so that a particular species of the microbiome or to change the function of the strain.

By " host organism " is meant herein any multicellular organism, including any animal or any plant. In certain embodiments, the host organism is a host subject.

By "host subject" is meant herein any animal (eg, primate, eg, human) that hosts the microbiome. A subject according to the present invention is preferably a mammal, even more preferably a human. However, the term "subject" also refers to a non-human animal, particularly a mammal, such as a dog, cat, horse, cow, pig, sheep, donkey, rabbit, ferret, gerbil, hamster, chinchilla, rat, mouse, in need of treatment. , guinea pigs, and non-human primates, or non-mammals such as poultry.

A human subject according to the present invention may be a prenatal human, a newborn, a child, an infant, an adolescent or an adult of any age.

In the methods of the invention, a nucleic acid of interest is delivered to a targeted recipient bacterial cell of the microbiome or a group of targeted recipient bacterial cells of the microbiome, wherein the nucleic acid of interest is incorporated into a vector provided by the donor bacterial cell. do.

A " donor bacterial cell " herein refers to a bacterium capable of hosting a vector comprising the nucleic acid of interest, producing a vector comprising the nucleic acid of interest, and/or capable of transferring the vector comprising the nucleic acid to other bacteria. means In certain embodiments, the vector may be a phagemid and the donor bacterial cell may be a bacterial cell capable of producing the phagemid, more particularly in the form of a packaged phagemid. In an alternative embodiment, the vector may be a plasmid, more particularly a conjugation plasmid, and the donor bacterial cell may be a bacterium capable of transferring the conjugation plasmid to other bacteria, in particular through conjugation.

By “ recipient bacterial cell ” herein is meant any bacterium from the host microbiome specifically targeted to be delivered with the nucleic acid of interest.

The targeted recipient bacterium may be any bacterium, particularly present in an organism, more particularly a mammalian organism. It may be any commensal, commensal, or pathogenic bacteria of the microbiota or microbiome.

The microbiome may include a variety of endogenous bacterial species, any of which may be targeted according to the present disclosure. In some embodiments, the genus and/or species of the targeted recipient bacterial cell may depend on the type of bacteriophage used to make the vector and/or bacterial delivery vehicle. For example, some bacteriophages exhibit tropism for, or preferentially target, certain host species of bacteria. Other bacteriophages do not exhibit this tropism and can be used to target a number of different genera and/or species of endogenous bacterial cells.

Examples of recipient bacterial cells include, but are not limited to, genera Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella ) spp., Neisseria spp., Aeromonas spp., Francisella spp., Corynebacterium spp., Citrobacter spp., Chlamydia (Chlamydia) spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas ( Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp ., Streptococcus spp., Staphylococcus spp., Bacteroides spp., Prevotella spp., Clostridium spp., Bifidovac Bifidobacterium spp., Clostridium spp., Brevibacterium spp., Lactococcus spp., Leuconostoc spp., Actinobacillus ) spp., Selnomonas spp., Shigella spp., Zymonas spp., Mycoplasma spp., Treponema spp., Leuconostoc spp ., Corynebacterium spp., Enterococcus spp., Enterobacter spp., Pyrococcus spp., Serratia spp., Morganella ( Morganella) spp., Parvimonas spp., Fusobacterium spp., Actinomyces spp., Porphyromonas spp., Propinibacterium spp., Cutibacterium spp. Micrococcus spp., Bartonella spp., Borrelia spp., Brucelia spp., Campylobacter spp., Chlamydophilia spp ., Cutibacterium (formerly Propionibacterium) spp., Ehrlichia spp., Haemophilus spp., Leptospira spp., Listeria spp., Mycoplasma spp., Nocardia spp., Rickettsia spp., Ureaplasma spp., and Lactobacillus spp., and mixtures thereof. do.

Thus, the targeted recipient bacterial cell may be from any one or more of the aforementioned genera of bacteria.

In one embodiment, the targeted recipient bacterium is Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp. ., Helicobacter spp., Vibrio spp, Salmonella spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp., Clostridium spp., Shigella spp., Enterococcus spp., Enterobacter spp., Propionibacterium spp., Cutibacterium spp. and Listeria spp.

In some embodiments, targeted recipient bacterial cells of the present disclosure are anaerobic bacterial cells (eg, cells that do not require oxygen for growth). Anaerobic bacterial cells include conditionally anaerobic cells such as, without limitation, Escherichia coli, Shewanella oneidensis and Listeria. Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species. In humans, anaerobic bacteria are most commonly present in the gastrointestinal tract. In some specific embodiments, the targeted recipient bacteria are thus the bacteria most commonly present in the gastrointestinal tract.

In some embodiments, the targeted recipient bacterial cells include, but are not limited to, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacillus Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Claw Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis), Actinobacillus actinomycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selenomonas ruminatium , Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, star Staphylococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosus , Lactobacillus casei, Lactobacillus acidophilus, Enterococcus faecalis, Bacillus coagulans, Bacillus cereus, Bacillus popilla Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas ruminantium, Lactobacillus hilgar D (Lactobacillus hilgardii), Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Streptomyces Streptomyces phaechromogenes, Streptomyces ghanaenis, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes , Serratia marcescens, Morganella morganii, Citrobacter freundii, Pseudomonas aeruginosa, Parvimonas micra, Freundi Botella Intermedia (Prevotella intermedia), Fusobacterium nucleatum, Prevotella nigrescens, Actinomyces israelii, Porphyromonas Endodonthalis ( Porphyromonas endodontalis), Porphyromonas gingivalis, Micrococcus luteus, Bacillus megaterium, Aeromonas hydrophila, Aeromonas caviae caviae), Bacillus anthracis, Bartonella henselae, Bartonella Quintana, Bordetella pertussis, Borrelia burgdorferi ), Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitten Brucella melitensis, Brucella suis, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Chlamydia pneumoniae), Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens ), Clostridium tetani, Corynebacterium diphtheria, Cutibacterium acnes (formerly Propionibacterium acnes), Elicia canis ( Ehrlichia canis), Ehrlichia chaffeensis, Enterococcus faecium, Francisella tularensis, Haemophilus influenza, Legionella pneumophila , Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium lepra Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae , Neisseria meningitides, Nocardia asteroids, Rickettsia rickettsia, Salmonella enteritidis, Salmonella typhi, Salmonella paratyphi ( Salmonella paratyphi), Salmonella typhimurium, Shigella flexneri, Shigella dysenteriae, Staphylococcus saprophyticus, Streptococcus new Moniae (Streptococcus pneumoniae), Streptococcus pyogenes, Streptococcus viridans, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholera, Vibrio parahaemolyticus, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Actino Actinobacter baumanii, Pseudomonas aeruginosa, and mixtures thereof. In one embodiment, the bacterium of interest targeted is Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae ), Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacter aerogenes, and mixtures thereof do.

In some embodiments, the targeted bacterial cell is, without limitation, Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerocco Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium ( Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium , Fusobacterium, Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus , Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter, Papillibacter, Propioni Spira (Proprionispira), Pseudobutyrivibrio (Pseudobutyrivibrio), Pseudoramibacter (Pseudoramibacter), Roseburia (Roseburia), Ruminococcus (Ruminococcus), Sarcina (Sarcina), Seinonella (Seinonella), Suttlewortia (Shuttleworthia), Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum, Syntrophococcus, Thermobacillus, Turibacter (Turibacter), Weisella, Clostridium, Bacteroides, Ruminococcus, Faecalibacterium, Treponema, Fascolactobacter Phascolarctobacterium, Megasphaera, Faecalibacterium, Bifidobacterium, Lactobacillus, Sutterella, and/or Prevotella. .

In another embodiment, the targeted bacterial cell is, without limitation, Achromobacter xylosoxidans, Acidaminococcus fermentans, Acidaminococcus intestini, Acida Acidaminococcus sp., Acinetobacter baumannii, Acinetobacter junii, Acinetobacter lwoffii, Actinobacillus capsulatus, evil Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces radingae, Adlerk Adlercreutzia equolifaciens, Aeromicrobium massiliense, Aggregatibacter actinomycetemcomitans, Akkermansia muciniphila, Aliagarivorans marinus, Alistipes finegoldii, Alistipes indistinctus, Alistipes inops, Alistipes ondedonchii onderdonkii), Alistipes putredinis, Alistipes senegalensis, Alistipes shahii, Alistipes timonensis, Alloscardo Alloscardovia omnicolens, Anaerobacter polyendosporus, Anaerobaculum hydrogeniformans, Anaerococcus hydrogenalis, Anaerococcus hydrogenalis Anaerococcus prevotii, Anaerococcus senegalensis, Anaerofustis stercorihominis, Anaerostipes caccae, Anaer Anaerostipes hadrus, Anaerotruncus colihominis, Aneurinibacillus aneurinilyticus, Bacillus licheniformis, Bacillus massillio Bacillus massilioanorexius, Bacillus massiliosenegalensis, Bacillus simplex, Bacillus smithii, Bacillus subtilis, Bacillus thuringiensis thuringiensis), Bacillus timonensis, Bacteroides xylanisolvens, Bacteroides acidifaciens, Bacteroides caccae, Bacteroides ca Bacteroides capillosus, Bacteroides cellulosilyticus, Bacteroides clarus, Bacteroides coprocola, Bacteroides coprophilus ), Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides finegoldii, Bacteroides fluxus ), Bacteroides fragilis, Bacteroides gallinarum, Bacteroides intestinalis, Bacteroides nordii, Bacteroides oleicipple Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides plebeius, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroides sp., Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis (Bacteroides uniformis), Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides pectinophilus ATCC, Barnesiella intestinihominis), Bavariicoccus seileri, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum gallicum), Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium stercoris, Bilophila wadsworthia, Blau Blautia faecis, Blautia hansenii, Blautia Hydrogenotrophica, Blautia luti, Blautia obeum, Blautia producta, Blautia wexlerae, Brachymonas chironomi, Brevibacterium senegalense, Briantella formatexigens ( Bryantella formatexigens), Butyrate-producing bacteria, Butyricicoccus pullicaecorum, Butyricimonas virosa, Butyrivibrio crossotus, Butyricicoccus pullicaecorum (Butyricimonas virosa), Butyrivibrio fibrisolvens ( Butyrivibrio fibrisolvens), Caldicoprobacter faecalis, Campylobacter concisus, Campylobacter jejuni, Campylobacter upsaliensis, Catenybacterium Michuokai (Catenibacterium mitsuokai), Cedecea davisae, Cellulomonas massiliensis, Cetobacterium somerae, Citrobacter braakii, Sheet Citrobacter freundii, Citrobacter pasteurii, Citrobacter sp., Citrobacter youngae, Cloacibacillus evryensis, Clostridiales bacterium, Clostridioides difficile, Clostridium asparagiforme, Clostridium bartlettii, Clostridium boliviensis ( Clostridium boliviensis), Clostridium bolteae, Clostridium hathewayi, Clostridium hiranonis, Clostridium hylemonae, Clostridium Clostridium leptum, Clostridium methylpentosum, Clostridium nexile, Clostridium orbiscindens, Clostridium ramosum, Clostridium Clostridium scindens, Clostridium sp, Clostridium sp., Clostridium spiroforme, Clostridium sporogenes, Clostridium Clostridium symbiosum, Collinsella aerofaciens, Collinsella intestinalis, Collinsella stercoris, Collinsella tanakaei, Corp. Coprobacillus cateniformis, Coprobacter fastidiosus, Coprococcus catus, Coprococcus comes, Coprococcus eutactus), Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium pseudodiphtheriticum, Cutibacterium acnes, Dermabacter hominis, Desulfitobacterium hafniense, Desulfovibrio fairfieldensis, Desulfovibrio piger, Dialister succinatyphilus succinatiphilus), Dielma fastidiosa, Dorea formicigenerans, Dorea longicatena, Dysgonomonas capnocytophagoides, Dysgonomonas capnocytophagoides Dysgonomonas gadei, Dysgonomonas mossii, Edwardsiella tarda, Eggerthella lenta, Eisenbergiella tayi, Enorma Massili Enorma massiliensis, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter Enterobacter massiliensis, Enterococcus casseliflavus, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus sp., Enterovibrio nigricans, Erysipella toclostridium lamosum ( Erysipelatoclostridium ramosum), Escherichia coli, Escherichia sp., Eubacterium biforme, Eubacterium dolichum, Eubacterium hallii ), Eubacterium limosum, Eubacterium ramulus, Eubacterium rectale, Eubacterium siraeum, Eubacterium ventriosum ( Eubacterium ventriosum), Exiguobacterium marinum, Exiguobacterium undae, Faecalibacterium cf, Faecalibacterium prausnitzii, Paecali Faecalitalea cylindroides, Ferrimonas balearica, Finegoldia magna, Flavobacterium daejeonense, Flavonifractor plautii, Fusicatenibacter saccharivorans, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium necrophorum, Fusobacterium necrophorum Fusobacterium nucleatum, Fusobacterium periodonticum, Fusobacterium sp., Fusobacterium ulcerans, Fusobacterium barium ( Fusobacterium varium), Gallibacterium anatis, Gemmiger formicilis, Gordonibacter pamelaeae, Hafnia alvei, Helicobacter bilis), Helicobacter bills, Helicobacter canadensis, Helicobacter canis, Helicobacter cinaedi, Helicobacter macacae, Helicobacter pametensis pamethensis), Helicobacter pullorum, Helicobacter pylori, Helicobacter rodentium, Helicobacter winghamensis, Herbaspirillum massiliense, Holdermanella Holdemanella biformis, Holdemania fdiformis, Holdemania filiformis, Holdemania massiliensis, Holdemania filiformis, Hungatella Hungatella hathewayi, Intestinibacter bartlettii, Intestinimonas butyriciproducens, Klebsiella oxytoca, Klebsiella pneumoniae (Klebsiella pneumoniae), Kurthia massiliensis, Lachnospira pectinoschiza, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus Lactobacillus animalis, Lactobacillus antri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus curvatus , Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus iners, Lactobacillus intestinalis, Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus paracasei, Lactobacillus pla Lantarum (Lactobacillus plantarum), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus ultunensis , Lactobacillus vaginalis, Lactobacillus plantarum subsp., Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Listeria green grayi), Listeria innocua, Mannheimia granulomatis, Marvinbryantia formatexigens, Megamonas funiformis, Megamonas hypermegal (Megamonas hypermegale), Methanobrevibacter smithii, Methanobrevibacter smithii F1, Micrococcus luteus, Microvirgula Aerodenitrificans (Microvirgula aerodenitrificans), Mitsuokella jalaludinii, Mitsuokella multacida, Mollicutes bacterium, Murimonas intestini, Neisseria macacae ), Nitriliruptor alkaliphilus, Oceanobacillus massiliensis, Odoribacter laneus, Odoribacter splanchnicus, Ornitobacterium linotra Ornithobacterium rhinotracheale, Oxalobacter formigenes, Paenibacillus barengoltzii, Paenibacillus chitinolyticus, Paenibacillus lautus , Paenibacillus motobuensis, Paenibacillus senegalensis, Paenisporosarcina quisquiliarum, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides johnsonii, Parabacteroides merdae, Paraprevotella Paraprevotella xylaniphila, Parasutterella excrementihominis, Parvimonas micra, Pediococcus acidilactici, Peptoclostridium difficile ( Peptoclostridium difficile), Peptoniphilus harei, Peptoniphilus obesi, Peptoniphilus senegalensis, Peptoniphilus timonensis, Phascolarctobacterium succinatutens, Porphyromonas asaccharolytica, Porphyromonas uenonis, Prevotella baroniae, Prevotella baboon Prevotella bivia, Prevotella copri, Prevotella dentalis, Prevotella micans, Prevotella multisaccharivorax, Prevotella Prevotella oralis, Prevotella salivae, Prevotella stercorea, Prevotella veroralis, Propionibacterium acnes , Propionibacterium avidum, Propionibacterium freudenreichii, Propionimicrobium lymphophilum, Proteus mirabilis, Proteuspenneri ATCC, Providencia alcalifaciens, Providencia rettgeri, Providencia rustigianii, Providencia stuartii, Pseudoflavonifractor Pseudoflavonifractor capillosus, Pseudomonas aeruginosa, Pseudomonas luteola, Ralstonia pickettii, Rheinheimera perlucida, Reinheimera texassen Rheinheimera texasensis, Riemerella columbina, Romboutsia lituseburensis, Roseburia faecis, Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus bicirculans, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus campanellensis (Ruminococcus champanellensis), Ruminococcus faecis, Ruminococcus gnavus, Ruminococcus lactaris, Ruminococcus obeum, Ruminococcus ) sp, Ruminococcus sp., Ruminococcus torques, Sarcina ventriculi, Sellimonas intestinalis, Senegalima Cilia anaerobia ( Senegalimassilia anaerobia), Shigella sonnei, Slackia piriformis, Staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus Staphylococcus nepalensis, Staphylococcus pseudintermedius, Staphylococcus xylosus, Stenotrophomonas maltophilia, Streptococcus agalac Streptococcus agalactiae, Streptococcus anginosus, Streptococcus australis, Streptococcus caballi, Streptococcus castoreus, Streptococcus Streptococcus didelphis, Streptococcus equinus, Streptococcus gordonii, Streptococcus henryi, Streptococcus hyovaginalis, Streptococcus infantarius, Streptococcus infantis, Streptococcus lutetiensis, Streptococcus merionis, Streptococcus mytis mitis), Streptococcus mutans, Streptococcus oralis, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus flurextorum ( Streptococcus plurextorum), Streptococcus porci, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sobrinus, Streptococcus sobrinus Streptococcus thermophilus, Streptococcus thoraltensis, Streptomyces albus, Subdoligranulum variabile, Succinatimonas hippei, Sute Sutterella parvirubra, Sutterella wadsworthensis, Terrisporobacter glycolicus, Terrisporobacter mayombei, Thalassobacillus devorans ( Thalassobacillus devorans), Timonella senegalensis, Turicibacter sanguinis, unknown sp, unknown sp., Varibaculum cambriense, Veillonella atypica , Veillonella dispar, Veillonella parvula, Vibrio cincinnatiensis, Virgibacillus salexigens, and Weissella paramesenteroides).

In another embodiment, the targeted bacterial cells are those commonly found in skin microbiota, including but not limited to Acetobacter farinalis, Acetobacter malorum, Acetobacter oleanensis ( Acetobacter orleanensis), Acetobacter sicerae, Achromobacter anxifer, Achromobacter denitrificans, Achromobacter marplatensis, Achromobacter Achromobacter spanius, Achromobacter xylosoxidans subsp. xylosoxidans, Acidovorax konjaci, Acidovorax radicis, Acinetobacter johnsonii, Actinomadura citrea, Acti Actinomadura coerulea, Actinomadura fibrosa, Actinomadura fulvescens, Actinomadura jiaoheensis, Actinomadura luteofluorea Sense (Actinomadura luteofluorescens), Actinomadura mexicana, Actinomadura nitritigenes, Actinomadura verrucosispora, Actinomadura yumaensis, Actinomyces odontolyticus, Actinomycetospora atypica, Actinomycetospora corticicola, Actinomycetospora rhizophila rhizophila), Actinomycetospora rishiriensis, Aeromonas australiensis, Aeromonas bestiarum, Aeromonas bivalvium ), Aeromonas encheleia, Aeromonas eucrenophila, Aeromonas Hydrophila subsp. Hydrophila, Aeromonas piscicola, Aeromonas popoffii, Aeromonas rivuli, Aeromonas salmonicida (Aeromonas salmonicida) subsp. Pectinolytica, Aeromonas salmonicida subsp. smithia, Amaricoccus kaplicensis, Amaricoccus veronensis, Aminobacter aganoensis, Aminobacter ciceronei, amino Aminobacter lissarensis, Aminobacter niigataensis, Ancylobacter polymorphus, Anoxybacillus flavithermus subsp. Yunnanensis, Aquamicrobium aerolatum, Archangium gephyra, Archangium gephyra, Archangium minus, Ar Archangium violaceum, Arthrobacter viscosus, Bacillus anthracis, Bacillus australimaris, Bacillus drentensis, Bacillus mycoides (Bacillus mycoides), Bacillus pseudomycoides, Bacillus pumilus, Bacillus safensis, Bacillus vallismortis, Bosea thiooxidans, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum, Brevundimonas aurantiaca, Brevundimonas intermedia, Burkholde Burkholderia aspalathi, Burkholderia choica, Burkholderia cordobensis, Burkholderia diffusa, Burkholderia insulsa, Burkholderia rhynchosiae, Burkholderia terrestris, Burkholderia udeis, Buttiauxella gaviniae, Caenimonas terrae), Capnocytophaga gingivalis, Chitinophaga dinghuensis, Chryseobacterium gleum, Chryseobacterium greenlandense, Chryseobacter Chryseobacterium jejuense, Chryseobacterium piscium, Chryseobacterium sediminis, Chryseobacterium tructae, Chryseobacterium ureil Chryseobacterium ureilyticum, Chryseobacterium vietnamense, Corynebacterium accolens, Corynebacterium afermentans subsp. Lipophilum, Corynebacterium minutissimum, Corynebacterium sundsvallense, Cupriavidus metallidurans, Cupriavidus nantongensis (Cupriavidus nantongensis), Cupriavidus necator, Cupriavidus pampae, Cupriavidus yeoncheonensis, Cutobacterium flaccumfaciens , Devosia epidermidihirudinis, Devosia riboflavina, Devosia riboflavina, Diaphorobacter oryzae, Dietzia picralcali Dietzia psychralcaliphila, Ensifer adhaerens, Ensifer americanus, Enterococcus malodoratus, Enterococcus pseudoavium, Enterococcus pseudoavium Enterococcus viikkiensis, Enterococcus xiangfangensis, Erwinia rhapontici, Falsirhodobacter halotolerans, Flavobacterium araucananum ), Flavobacterium frigidimaris, Gluconobacter frateurii, Gluconobacter thailandicus, Gordonia alkanivorans, Halomonas aqua Halomonas aquamarina, Halomonas axialensis, Halomonas meridiana, Halomonas olivaria, Halomonas songnenensis, Halomonas bariabilis (Halomonas variabilis), Herbaspirillum chlorophenolicum, Herbaspirillum frisingense, Herbaspirillum hiltneri, Herbaspirillum huttiense subsp. Putei, Herbaspirillum lusitanum, Herminiimonas fonticola, Hydrogenophaga intermedia, Hydrogenophaga pseudoflava, Kleb Klebsiella oxytoca, Kosakonia sacchari, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus modestisalitolerans, Lactobacillus plantarum subsp. Argentoratensis, Lactobacillus xiangfangensis, Lechevalieria roselyniae, Lentzea albida, Lentzea californiensis, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc gelidum subsp. Gasicomitatum, Leuconostoc mesenteroides subsp. Suionicum, Luteimonas aestuarii, Lysobacter antibioticus, Lysobacter koreensis, Lysobacter oryzae, Magneto Magnetospirillum moscoviense, Marinomonas alcarazii, Marinomonas primoryensis, Massilia aurea, Massilia jejuensis, Massilia key Massilia kyonggiensis, Massilia timonae, Mesorhizobium acaciae, Mesorhizobium qingshengii, Mesorhizobium shonense, Methylobacter Methylobacterium haplocladii, Methylobacterium platani, Methylobacterium pseudosasicola, Methylobacterium zatmanii, Microbacterium oxydan (Microbacterium oxydan), Micromonospora chaiyaphumensis, Micromonospora chalcea, Micromonospora citrea, Micromonospora coxensis , Micromonospora echinofusca, Micromonospora halophytica, Micromonospora kangleipakensis, Micromonospora maritima, Micromonospora maritima Micromonospora nigra, Micromonospora purpureochromogene, Micromonospora rhizosphaerae, Micromonospora saelicesensis , Microvirga subterranea, Microvirga zambiensis, Mycobacterium alvei, Mycobacterium avium subsp. Silvaticum, Mycobacterium colombiense, Mycobacterium conceptionense, Mycobacterium conceptionense, Mycobacterium fascinogenes ( Mycobacterium farcinogenes), Mycobacterium fortuitum subsp. Fortuitum, Mycobacterium goodii, Mycobacterium insubricum, Mycobacterium llatzerense, Mycobacterium neoaurum neoaurum), Mycobacterium neworleansense, Mycobacterium obuense, Mycobacterium peregrinum, Mycobacterium saopaulense , Mycobacterium septicum, Mycobacterium setense, Mycobacterium smegmatis, Neisseria subflava, Nocardia regiogen Nocardia lijiangensis, Nocardia thailandica, Novosphingobium barchaimii, Novosphingobium lindaniclasticum, Novosphingobium lindaniclasticum , Novosphingobium mathurense, Ochrobactrum pseudogrignonense, Oxalicibacterium solurbis, Paraburkholderia glathei, Paraburkholderia Paraburkholderia humi, Paraburkholderia phenazinium, Paraburkholderia phytofirmans, Paraburkholderia sordidicola, Paraburkholderia tericola (Paraburkholderia terricola), Paraburkholderia xenovorans, Paracoccus laeviglucosivorans, Patulibacter ginsengiterrae, Polymorphospora rubra ( Polymorphospora rubra), Porphyrobacter colymbi, Prevotella jejuni, Prevotella melaninogenica, Propionibacterium acnes subsp. elongatum), Proteus vulgaris, Providencia rustigianii, Pseudoalteromonas agarivorans, Pseudoalteromonas atlantica, Pseudoalteromonas paragorgicola (Pseudoalteromonas paragorgicola), Pseudomonas asplenii, Pseudomonas asuensis, Pseudomonas benzenivorans, Pseudomonas cannabina, Pseudomonas cissicola , Pseudomonas congenlance (Pseudomonas congelans), Pseudomonas costantinii, Pseudomonas ficuserectae, Pseudomonas frederiksbergensis, Pseudomonas graminis, Pseudomonas jesenii senii) , Pseudomonas koreensis, Pseudomonas koreensis, Pseudomonas kunmingensis, Pseudomonas marginalis, Pseudomonas mucidolens, Pseudomonas panasis acis ), Pseudomonas plecoglossicida, Pseudomonas poae, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas Reinekei, Pseudomonas rhizospaera Pseudomonas rhizosphaerae, Pseudomonas seleniipraecipitans, Pseudomonas umsongensis, Pseudomonas zhaodongensis, Pseudonocardia alaniniphila, Pseudo Pseudonocardia ammonioxydans, Pseudonocardia autotrophica, Pseudonocardia kongjuensis, Pseudonocardia yunnanensis, Pseudoferax Pseudoxanthomonas soli, Pseudoxanthomonas daejeonensis, Pseudoxanthomonas indica, Pseudoxanthomonas kaohsiungensis, Psychrobacter aquaticus, Sike Psychrobacter arcticus, Psychrobacter celer, Psychrobacter marincola, Psychrobacter nivimaris, Psychrobacter okhotskensis, Psychrobacter okhotskensis, Psychrobacter piscatorii, Psychrobacter pulmonis, Ramlibacter ginsenosidimutans, Rheinheimera japonica , Reinheimera muenzenbergensis, Rheinheimera soli, Rheinheimera tangshanensis, Rheinheimera texasensis, Reinheimera tilapiae ( Rheinheimera tilapiae), Rhizobium alamii, Rhizobium azibense, Rhizobium binae, Rhizobium daejeonense, Rhizobium endophyticum, Rhizobium etli, Rhizobium fabae, Rhizobium freirei, Rhizobium gallicum, Rhizobium loessense, Rhizobium sophoriradisis (Rhizobium sophoriradicis), Rhizobium taibaishanense, Rhizobium vallis, Rhizobium vignae, Rhizobium vignae, Rhizobium yanglingens ( Rhizobium yanglingense), Rhodococcus baikonurensis, Rhodococcus enclensis, Rhodoferax saidenbachensis, Rickettsia canadensis, Rickett Rickettsia heilongjiangensis, Rickettsia honei, Rickettsia raoultii, Roseateles aquatilis, Roseateles aquatilis ), Salmonella enterica subsp. Salamae, Serratia ficaria, Serratia myotis, Serratia vespertilionis, Shewanella aestuarii, Shuwa Shewanella decolorationis, Sphingobium amiense, Sphingobium baderi, Sphingobium barthaii, Sphingobium chlorophenolicum , Sphingobium cupriresistens, Sphingobium czechense, Sphingobium fuliginis, Sphingobium indicum, Sphingobium indicum indicum), Sphingobium japonicum, Sphingobium lactosutens, Sphingomonas dokdonensis, Sphingomonas pseudosanguinis, Sphingomonas pseudosanguinis Sphingopyxis chilensis, Sphingopyxis fribergensis, Sphingopyxis granuli, Sphingopyxis indica, Sphingopyxis witflariensis, Staphylococcus agnetis, Staphylococcus aureus subsp. aureus, Staphylococcus epidermidis, Staphylococcus hominis subsp. Novobiosepticus, Staphylococcus nepalensis, Staphylococcus saprophyticus subsp. bovis, Staphylococcus sciuri subsp. Carnaticus, Streptomyces caeruleatus, Streptomyces canarius, Streptomyces capoamus, Streptomyces siskaucasicus ciscaucasicus), Streptomyces griseorubiginosus, Streptomyces olivaceoviridis, Streptomyces panaciradicis, Streptomyces paeopurpureus (Streptomyces phaeopurpureus), Streptomyces pseudovenezuelae, Streptomyces resistomycificus, Tianweitania sediminis, Tsukamurella paurometabola, Variovorax guangxiensis, Vogesella alkaliphila, Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas cassavae, Xantho Xanthomonas cucurbitae, Xanthomonas cynarae, Xanthomonas euvesicatoria, Xanthomonas fragariae, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas pisi, Xanthomonas populi, Xanthomonas vasicola, Xenophilus aerolatus, Yersinia numii (Yersinia nurmii), Abiotrophia defectiva, Acidocella aminolytica, Acinetobacter guangdongensis, Acinetobacter parvus, Acineto Acinetobacter radioresistens, Acinetobacter soli, Acinetobacter variabilis, Actinomyces cardiffensis, Actinomyces dental dentalis), Actinomyces europaeus, Actinomyces gerencseriae, Actinomyces graevenitzii, Actinomyces haliotis , Actinomyces johnsonii, Actinomyces massiliensis, Actinomyces meyeri, Actinomyces meyeri, Actinomyces Actinomyces naeslundii, Actinomyces neuii subsp. Anitratus, Actinomyces odontolyticus, Actinomyces oris, Actinomyces turicensis, Actinomycetospora cortisi Actinomycetospora corticicola, Actinotignum schaalii, Aerococcus christensenii, Aerococcus urinae, Aeromicrobium flavum flavum), Aeromicrobium massiliense, Aeromicrobium tamlense, Aeromonas sharmana, Aggregatibacter aphrophilus , Aggregatibacter segnis, Agrococcus baldri, Albibacter Methylovorans, Alcaligenes faecalis subsp. faecalis, Algoriphagus ratkowskyi, Alkalibacterium olivapovliticus, Alkalibacterium pelagium, Alkalibacterium pelagium, allo Alloprevotella rava, Alsobacter metallidurans, Amaricoccus kaplicensis, Amaricoccus veronensis, Anaerococcus hydrogenalis ( Anaerococcus Hydrogenalis), Anaerococcus lactolyticus, Anaerococcus murdochii, Anaerococcus octavius, Anaerococcus prevotii, Anaerococcus prevotii Anaerococcus vaginalis, Aquabacterium citratiphilum, Aquabacterium olei, Aquabacterium olei, Aquabacterium parvum, Aquin Aquincola tertiaricarbonis, Arcobacter venerupis, Arsenicicoccus bolidensis, Arthrobacter russicus, Astica caulis excelricus (Atopobium deltae), Atopobium parvulum, Atopobium rimae, Atopobium vaginae, Aureimonas alta Aureimonas altamirensis, Aureimonas rubiginis, Azospira oryzae, Azospirillum oryzae, Bacillus circulans, Bacillus Drenten Bacillus drentensis, Bacillus fastidiosus, Bacillus lehensis, Bacillus oceanisediminis, Bacillus rhizosphaerae, Bacteriovorax stolpie (Bacteriovorax stolpii), Bacteroides coagulans, Bacteroides dorei, Bacteroides fragilis, Bacteroides ovatus, Bacteroides ster Bacteroides stercoris, Bacteroides uniformis, Bacteroides vulgatus, Bdellovibrio bacteriovorus, Bdellovibrio exovorus, Bellna Belnapia moabensis, Belnapia soli, Blautia hansenii, Blautia obeum, Blautia wexlerae, bonded Bosea lathyri, Brachybacterium fresconis, Brachybacterium muris, Brevibacterium ammoniilyticum, Brevibacterium casei , Brevibacterium epidermidis, Brevibacterium iodinum, Brevibacterium luteolum, Brevibacterium paucivorans, Brevibacterium paucivorans Brevibacterium pityocampae, Brevibacterium sanguinis, Brevundimonas albigilva, Brevundimonas diminuta, Brevundimonas vancanneytii), Caenimonas terrae, Calidifontibacter indicus, Campylobacter concisus, Campylobacter gracilis, Campylobacter hominis (Campylobacter hominis), Campylobacter rectus, Campylobacter showae, Campylobacter ureolyticus, Capnocytophaga gingivalis, Capnocytophaga leadbetteri, Capnocytophaga ochracea, Capnocytophaga sputigena, Cardiobacterium hominis, Cardiobacterium Cardiobacterium valvarum, Carnobacterium divergens, Catonella morbi, Caulobacter henricii, Cavicella subterranea, Cellulomonas Cellulomonas xylanilytica, Cellvibrio vulgaris, Chitinimonas taiwanensis, Chryseobacterium arachidis, Chryseobacterium cheongense, Chryseobacterium formosense, Chryseobacterium formosense, Chryseobacterium greenlandense, Chryseobacterium indologenes, Chryseo Chryseobacterium piscium, Chryseobacterium rigui, Chryseobacterium solani, Chryseobacterium taklimakanense, Chryseobacterium ureil Chryseobacterium ureilyticum, Chryseobacterium ureilyticum, Chryseobacterium zeae, Chryseomicrobium aureum, Cloacibacterium haliotis), Cloacibacterium normanense, Cloacibacterium normanense, Collinsella aerofaciens, Comamonas denitrificans, Comamonas Comamonas terrigena, Corynebacterium accolens, Corynebacterium afermentans subsp. Lipophilum, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium aurimuco Corynebacterium aurimucosum, Corynebacterium coylae, Corynebacterium durum, Corynebacterium freiburgense, Corynebacterium glaucum ), Corynebacterium glyciniphilum, Corynebacterium imitans, Corynebacterium jeikeium, Corynebacterium jeikeium, Cory Corynebacterium kroppenstedtii, Corynebacterium lipophiloflavum, Corynebacterium massiliense, Corynebacterium mastitidis, Corynebacterium mastitidis Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium mucifaciens, Corynebacterium mustelae, Corynebacterium mustelae Bacterium mycetoides, Corynebacterium pyruviciproducens, Corynebacterium simulans, Corynebacterium singulare, Corynebacterium Corynebacterium sputi, Corynebacterium suicordis, Corynebacterium tuberculostearicum, Corynebacterium tuberculostearicum, Corynebacterium ureicelerivorans, Corynebacterium variabile, Couchioplanes caeruleus subsp. Caeruleus, Cupriavidus metallidurans, Curtobacterium herbarum, Dechloromonas agitata, Deinococcus actinosclerus), Deinococcus antarcticus, Deinococcus caeni, Deinococcus ficus, Deinococcus geothermalis, Deinococcus radio Deinococcus radiodurans, Deinococcus wulumuqiensis, Deinococcus xinjiangensis, Dermabacter hominis, Dermabacter vaginalis, Dermaco Dermacoccus nishinomiyaensis, Desemzia incerta, Desertibacter roseus, Dialister invisus, Dialister micraerophilus , Dialister propionicifaciens, Dietzia aurantiaca, Dietzia cercidiphylli, Dietzia timorensis, Dietzia timo Dietzia timorensis, Dokdonella koreensis, Dokdonella koreensis, Dolosigranulum pigrum, Eikenella corrodens, Elisabeth kinggia Myricola (Elizabethkingia miricola), Elstera litoralis, Empedobacter brevis, Enhydrobacter aerosaccus, Enterobacter xiangfangensis, Enterococcus Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus olivae, Erwinia rhapontici, Eubacterium eligens, oil flakes Eubacterium infirmum, Eubacterium rectale, Eubacterium saphenum, Eubacterium sulci, Exiguobacterium mexicanum, Pak Facklamia tabacinasalis, Falsirhodobacter halotolerans, Finegoldia magna, Flavobacterium cutihirudinis, Flavobacterium lindani Flavobacterium lindanitolerans, Flavobacterium resistens, Friedmanniella capsulata, Fusobacterium nucleatum subsp. Polymorphum, Gemella haemolysans, Gemella morbillorum, Gemella palaticanis, Gemella sanguinis, Gemmobacter aqua Gemmobacter aquaticus, Gemmobacter caeni, Gordonia jinhuaensis, Gordonia kroppenstedtii, Gordonia polyisoprenivorans, Gor Gordonia polyisoprenivorans, Granulicatella adiacens, Granulicatella elegans, Haemophilus parainfluenzae, Haemophilus sputorum (Haemophilus sputorum), Halomonas sulfidaeris, Herpetosiphon aurantiacus, Hydrocarboniphaga effusa, Idiomarina maris, Johnnybacter anno Janibacter anophelis, Janibacter hoylei, Janibacter indicus, Janibacter limosus, Janibacter melonis, Zeot Gallicocus halophilus (Jeotgalicoccus halophilus), Jonquetella anthropi, Kaistia geumhonensis, Kingella denitrificans, Kingella oralis, Klebsiella oxytoca (Klebsiella oxytoca), Knoellia aerolata, Knoellia locipacati, Kocuria atrinae, Kocuria carniphila, Kocuria cristinae (Kocuria kristinae), Kocuria palustris, Kocuria turfanensis, Lachnoanaerobaculum saburreum, Lachnoanaerobaculum saburreum ), Lactobacillus crispatus, Lactobacillus iners, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. Lactis, Lactococcus piscium, Lapillicoccus jejuensis, Lautropia mirabilis, Legionella beliardensis, Leptotrichia Leptotrichia buccalis, Leptotrichia goodfellowii, Leptotrichia hofstadii, Leptotrichia hongkongensis, Leptotrichia shahii, Lepto Leptotrichia trevisanii, Leptotrichia wadei, Luteimonas terricola, Lysinibacillus fusiformis, Lysobacter spongiicola, Lyso Lysobacter xinjiangensis, Macrococcus caseolyticus, Marmoricola pocheonensis, Marmoricola scoriae, Massilia alkalitolerans ), Massilia alkalitolerans, Massilia aurea, Massilia plicata, Massilia timonae, Megamonas rupellensis, Meiothermus silvanus, Methylobacterium dankookense, Methylobacterium goingingense, Methylobacterium goingingense, Methylobacterium goingingense Methylobacterium isbiliense, Methylobacterium jeotgali, Methylobacterium oxalidis, Methylobacterium platani, Methylobacterium pseudosasi Methylobacterium pseudosasicola, Methyloversatilis universalis, Microbacterium foliorum, Microbacterium Hydrothermale, Microbacterium Hydrothermale ), Microbacterium lacticum, Microbacterium lacticum, Microbacterium laevaniformans, Microbacterium paludicola, Microbacterium Microbacterium petrolearium, Microbacterium phyllosphaerae, Microbacterium resistens, Micrococcus antarcticus, Micrococcus cohnii , Micrococcus flavus, Micrococcus lylae, Micrococcus terreus, Microlunatus aurantiacus, Microflu or glycogenica ( Micropruina glycogenica), Microvirga aerilata, Microvirga aerilata, Microvirga subterranea, Microvirga vignae, Microvirga zambiensis, Microvirgula aerodenitrificans, Mogibacterium timidum, Moraxella atlantae, Moraxella catarrhalis ( Moraxella catarrhalis), Morganella morganii subsp. morganii, Morganella psychrotolerans, Murdochiella asaccharolytica, Mycobacterium asiaticum, Mycobacterium chubuense , Mycobacterium crocinum, Mycobacterium gadium, Mycobacterium holsaticum, Mycobacterium iranicum, Mycobacterium Mycobacterium longobardum, Mycobacterium neoaurum, Mycobacterium neoaurum, Mycobacterium obuense, Negatycoccus succini Negativicoccus succinicivorans, Neisseria bacilliformis, Neisseria oralis, Neisseria sicca, Neisseria subflava, Nesterenkonia lacucecoensis ( Nesterenkonia lacusekhoensis), Nesterenkonia rhizosphaerae, Nevskia persephonica, Nevskia ramosa, Niabella yanshanensis, Niveibacterium Niveibacterium umoris, Nocardia niwae, Nocardia thailandica, Nocardioides agariphilus, Nocardioides dilutus, Nocardioides ganghwensis, Nocardioides hwasunensis, Nocardioides nanhaiensis, Nocardioides sediminis, Nosocomyco Nosocomimiicoccus ampullae, Noviherbaspirillum malthae, Novosphingobium lindaniclasticum, Novosphingobium rosa, Ocrobactrum rhizospaerae (Ochrobactrum rhizosphaerae), Olsenella uli, Ornithinimicrobium murale, Ornithinimicrobium tianjinense, Oryzobacter terrae, Autowia Beijing Ottowia beijingensis, Paenalcaligenes suwonensis, Paenibacillus agaridevorans, Paenibacillus phoenicis, Paenibacillus xylanexedens ), Paludibacterium yongneupense, Pantoea cypripedii, Parabacteroides distasonis, Paraburkholderia andropogonis, Paracoccus alcaliphilus, Paracoccus angustae, Paracoccus kocurii, Paracoccus laeviglucosivorans, Paracoccus sedi Paracoccus sediminis, Paracoccus sphaerophysae, Paracoccus yeei, Parvimonas micra, Parviterribacter multiflagellatus ), Patulibacter ginsengiterrae, Pedobacter aquatilis, Pedobacter ginsengisoli, Pedobacter xixiisoli, Peptococcus niger ), Peptoniphilus coxii, Peptoniphilus gorbachii, Peptoniphilus harei, Peptoniphilus koenoeneniae, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, Phascolarctobacterium faecium, Phenyl Phenylobacterium haematophilum, Phenylobacterium kunshanense, Pluralibacter gergoviae, Polymorphobacter multimanifer, Porphyromonas Benno Nice (Porphyromonas bennonis), Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas pasteri, Porphyromonas gingivicanis Porphyromonas pogonae, Porphyromonas somerae, Povalibacter uvarum, Prevotella aurantiaca, Prevotella baroniae ( Prevotella baroniae), Prevotella bivia, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella corporis ( Prevotella corporis), Prevotella denticola, Prevotella enoeca, Prevotella histicola, Prevotella intermedia, Prevotella Jeju Prevotella jejuni, Prevotella jejuni, Prevotella maculosa, Prevotella melaninogenica, Prevotella melaninogenica, Prevotella Prevotella micans, Prevotella multiformis, Prevotella nanceiensis, Prevotella nigrescens, Prevotella oris, Prevotella oris Prevotella oulorum, Prevotella pallens, Prevotella pleuritidis, Prevotella saccharolytica, Prevotella saccharolytica salivae), Prevotella shahii, Prevotella timonensis, Prevotella veroralis, Propionibacterium acidifaciens, Propioni Propionibacterium acnes subsp. acnes, Propionibacterium acnes subsp. acnes, Propionibacterium acnes subsp. elongatum, Propionibacterium granulosum, Propionimicrobium lymphophilum, Propionispira arcuata, Pseudokineococcus Lusitanus ( Pseudokineococcus lusitanus), Pseudomonas aeruginosa, Pseudomonas chengduensis, Pseudonocardia benzenivorans, Pseudorhodoplanes sinuspersici, Cycrobacter sangui Nice (Psychrobacter sanguinis), Ramlibacter ginsenosidimutans, Rheinheimera aquimaris, Rhizobium alvei, Rhizobium daejeonense, Rhizobium larimo Rhizobium larrymoorei, Rhizobium rhizoryzae, Rhizobium soli, Rhizobium taibaishanense, Rhizobium vignae, Rhodanobacter glycinis (Rhodanobacter glycinis), Rhodobacter veldkampii, Rhodococcus enclensis, Rhodococcus fascians, Rhodococcus fascians, Rhodococcus reposi Rhodovarius lipocyclicus, Rivicola pingtungensis, Roseburia inulinivorans, Rosenbergiella nectarea, Roseomonas aerilata ), Roseomonas aquatica, Roseomonas mucosa, Roseomonas rosea, Roseomonas vinacea, Rothia aeria , Rothia amarae, Rothia dentocariosa, Rothia endophytica, Rothia mucilaginosa, Rothia nasimurium , Rubellimicrobium mesophilum, Rubellimicrobium roseum, Rubrobacter bracarensis, Rudaea cellulosilytica, Ruminococcus gna Ruminococcus gnavus, Runella zeae, Saccharopolyspora rectivirgula, Salinicoccus qingdaonensis, Scardovia wiggsiae ), Sediminibacterium ginsengisoli, Selenomonas artemidis, Selenomonas infelix, Selenomonas noxia, Selenomonas spooty Gena (Selenomonas sputigena), Shewanella aestuarii, Shuttleworthia satelles, Simonsiella muelleri, Skermanella aerolata , Skermanella stibiiresistens, Slackia exigua, Smaragdicoccus niigatensis, Sneathia sanguinegens, Solilubrobacter solai (Solirubrobacter soli), Sphingobacterium caeni, Sphingobacterium daejeonense, Sphingobacterium hotanense, Sphingobacterium kyonggiense, Sphingobacterium multivorum, Sphingobacterium nematocida, Sphingobacterium spiritivorum, Sphingobium amiense, Sphingobium ind Sphingobium indicum, Sphingobium lactosutens, Sphingobium subterraneum, Sphingomonas abaci, Sphingomonas aestuarii, Sphingomonas canadensis, Sphingomonas daechungensis, Sphingomonas dokdonensis, Sphingomonas echinoides, Sphingomonas ponticola ( Sphingomonas fonticola), Sphingomonas fonticola, Sphingomonas formosensis, Sphingomonas gei, Sphingomonas hankokensis, Sphingomonas hankokensis ( Sphingomonas hankokensis), Sphingomonas koreensis, Sphingomonas kyeonggiensis, Sphingomonas laterariae, Sphingomonas mucosissima, Sphingomonas Sphingomonas oligophenolica, Sphingomonas pseudosanguinis, Sphingomonas sediminicola, Sphingomonas yantingensis, Sphingomonas yunnanensis yunnanensis), Sphingopyxis indica, Spirosoma rigui, Sporacetigenium mesophilum, Sporocytophaga myxococcoides, Staphylococcus suba Staphylococcus auricularis, Staphylococcus epidermidis, Staphylococcus epidermidis, Staphylococcus hominis subsp. Novobiosepticus, Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Stenotrophomonas koreensis, Stenotrophomonas rhizo Stenotrophomonas rhizophila, Stenotrophomonas rhizophila, Streptococcus agalactiae, Streptococcus canis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus intermedius, Streptococcus mutans, Streptococcus oligofermentans (Streptococcus oligofermentans), Streptococcus oralis, Streptococcus sanguinis, Streptomyces iconiensis, Streptomyces yanglinensis, Tabrijicola aquatica (Tabrizicola aquatica), Tahibacter caeni, Tannerella forsythia, Tepidicella xavieri, Tepidimonas fonticaldi, Terracoccus luteus ( Terracoccus luteus), Tessaracoccus flavescens, Thermus thermophilus, Tianweitania sediminis, Tianweitania sediminis, Treponema Treponema amylovorum, Treponema denticola, Treponema lecithinolyticum, Treponema medium, Turicella otitidis, Turi Turicibacter sanguinis, Undibacterium oligocarboniphilum, Undibacterium squillarum, Vagococcus salmoninarum, Varibaculum cambriense), Vibrio metschnikovii, Xanthobacter tagetidis, Xenophilus aerolatus, Xenophilus arseniciresistens, Imella ru Yimella lutea, Zimmermannella alba, Zimmermannella bifida and/or Zoogloea caeni.

In another embodiment, the targeted bacterial cells are those commonly found in the vaginal microbiota, including but not limited to Acinetobacter antiviralis, Acinetobacter baumannii, Acinetobacter baumannii, Acinetobacter cal Acinetobacter calcoaceticus, Acinetobacter johnsonii, Actinobaculum massiliense, Actinobaculum schaalii, Actinomyces europaeus , Actinomyces graevenitzii, Actinomyces israelii, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces naeslundii, evil Actinomyces neuii, Actinomyces odontolyticus, Actinomyces turicensis, Actinomyces urogenitalis, Actinomyces Actinomyces viscosus, Aerococcus christensenii, Aerococcus urinae, Aerococcus viridans, Aeromonas enkeleia ( Aeromonas encheleia), Aeromonas salmonicida, Afipia massiliensis, Agrobacterium tumefaciens, Algoriphagus aquatilis, Alibibrio Aliivibrio wodanis, Alistipes finegoldii, Alloiococcus otitis, Alloprevotella tannerae, Alloscardovia omnicolens ), Altererythrobacter epoxidivorans, Ammoniphilus oxalaticus, Amnibacterium kyonggiense, Anaerococcus hydrogenalis, Anae Anaerococcus lactolyticus, Anaerococcus murdochii, Anaerococcus obesiensis, Anaerococcus prevotii, Anaerococcus tetradius (Anaerococcus tetradius), Anaerococcus vaginalis, Anaeroglobus geminatus, Anoxybacillus pushchinoensis, Aquabacterium parvum ), Arcanobacterium phocae, Arthrobacter aurescens, Asticcacaulis excentricus, Atopobium minutum, Atopobium par Atopobium parvulum, Atopobium rimae, Atopobium vaginae, Avibacterium gallinarum, Bacillus acidicola, Bacillus atropae Bacillus atrophaeus, Bacillus cereus, Bacillus cibi, Bacillus coahuilensis, Bacillus gaemokensis, Bacillus methanolicus, Bacillus oleronius, Bacillus pumilus, Bacillus shackletonii, Bacillus sporothermodurans, Bacillus subtilis, Bacillus wakoensis wakoensis), Bacillus weihenstephanensis, Bacteroides barnesiae, Bacteroides coagulans, Bacteroides dorei, Bacteroides faesis (Bacteroides faecis), Bacteroides forsythus, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salier Bacteroides salyersiae, Bacteroides stercoris, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides xylanisolvens , Bacteroides zoogleoformans, Barnesiella viscericola, Bhargavaea cecembensis, Bifidobacterium adolescentis, Bifidobacteria Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium logum subsp. Infantis, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium scardovii, Bilophila wadsworthia , Blautia Hydrogenotrophica, Blautia obeum, Blautia producta, Brachybacterium faecium, Bradyrhizobium japoni Bradyrhizobium japonicum, Brevibacterium mcbrellneri, Brevibacterium otitidis, Brevibacterium paucivorans, Bulleidia extructa), Burkholderia fungorum, Burkholderia phenoliruptix, Caldicellulosiruptor saccharolyticus, Caldimonas taiwanensis, Cam Campylobacter gracilis, Campylobacter hominis, Campylobacter sputorum, Campylobacter ureolyticus, Capnocytophaga ochracea), Cardiobacterium hominis, Catonella morbi, Chlamydia trachomatis, Chlamydophila abortus, Chondromyces robustus ( Chondromyces robustus), Chryseobacterium aquaticum, Citrobacter youngae, Cloacibacterium normanense, Clostridium cavendishii, Clostridium coli Canis (Clostridium colicanis), Clostridium jejuense, Clostridium perfringens, Clostridium ramosum, Clostridium sordellii, Clostridium Clostridium viride, Comamonas terrigena, Corynebacterium accolens, Corynebacterium appendicis, Corynebacterium coyleae ), Corynebacterium glucuronolyticum, Corynebacterium glutamicum, Corynebacterium jeikeium, Corynebacterium croppenstedtii (Corynebacterium kroppenstedtii), Corynebacterium lipophiloflavum, Corynebacterium minutissimum, Corynebacterium mucifaciens, Corynebacterium nuruki ), Corynebacterium pseudogenitalium, Corynebacterium pyruviciproducens, Corynebacterium singulare, Corynebacterium striatum ), Corynebacterium tuberculostearicum, Corynebacterium xerosis, Cryobacterium psychrophilum, Curtobacterium flaccumfaciens ), Cutibacterium acnes, Cutibacterium avidum, Cytophaga xylanolytica, Deinococcus radiophilus, Delftia churuhaten Delftia tsuruhatensis, Desulfovibrio desulfuricans, Dialister invisus, Dialister micraerophilus, Dialister pneumosintes, Dial Lister propionicifaciens, Dickeya chrysanthemi, Dorea longicatena, Eggerthella lenta, Eggerthia catenaformis , Eikenella corrodens, Enhydrobacter aerosaccus, Enterobacter asburiae, Enterobacter cloacae, Enterococcus avium (Enterococcus avium), Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Erwinia persina ( Erwinia persicina), Erwinia rhapontici, Erwinia toletana, Escherichia coli, Escherichia fergusonii, Eubacterium brachy brachy), Eubacterium eligens, Eubacterium nodatum, Eubacterium rectale, Eubacterium saphenum, Eubacterium siraeum ( Eubacterium siraeum), Eubacterium sulci, Eubacterium yurii, Exiguobacterium acetylicum, Facklamia ignava, Paecalibacterium prausnit Faecalibacterium prausnitzii, Filifactor alocis, Finegoldia magna, Fusobacterium gonidiaformans, Fusobacterium nucleatum , Fusobacterium periodonticum, Gardnerella vaginalis, Gemella asaccharolytica, Gemella bergeri, Gemella haemollisans haemolysans), Gemella sanguinis, Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus thermoglucosidasius, Geobacter glvisiae (Geobacter grbiciae), Granulicatella elegans, Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus parahaemolyticus , Haemophilus parainfluenzae, Hafnia alvei, Halomonas meridiana, Halomonas phoceae, Halomonas venusta, her Herbaspirillum seropedicae, Janthinobacterium lividum, Jonquetella anthropi, Klebsiella granulomatis, Klebsiella oxytoca ( Klebsiella oxytoca), Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus cholehominis (Lactobacillus coleohominis), Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri ( Lactobacillus gasseri), Lactobacillus helveticus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis , Lactobacillus kefiranofaciens, Lactobacillus kimchicus, Lactobacillus kitasatonis, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus ultunensis, Lactobacillus vaginalis, Lactococcus lactis, Leptotrichia buccalis, Leuconostoc carnosum (Leuconostoc carnosum), Leuconostoc citreum, Leuconostoc garlicum, Leuconostoc lactis, Leuconostoc mesenteroides, Rishinimonas creep Lysinimonas kribbensis, Mageebacillus indolicus, Maribacter orientalis, Marinomonas protea, Marinospirillum insulare, Masilia timona Massilia timonae, Megasphaera elsdenii, Megasphaera micronuciformis, Mesorhizobium amorphae, Methylobacterium radiotolerans , Methylotenera versatilis, Microbacterium halophilum, Micrococcus luteus, Microterricola viridarii, Mobiluncus curtisii), Mobiluncus mulieris, Mogibacterium timidum, Moorella glycerini, Moraxella osloensis, Morganella morganii ), Moryella indoligenes, Murdochiella asaccharolytica, Mycoplasma alvi, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma muris, Mycoplasma salivarium, Negativicoccus succinicivorans, Neisseria flava, Neisseria gonorrhoeae ), Neisseria mucosa, Neisseria subflava, Nevskia ramosa, Nevskia soli, Nitriliruptor alkaliphilus , Odoribacter splanchnicus, Oligella urethralis, Olsenella uli, Paenibacillus amylolyticus, Paenibacillus humicus ), Paenibacillus pabuli, Paenibacillus pasadenensis, Paenibacillus pini, Paenibacillus validus, Pantoea Agglomerans agglomerans), Parabacteroides merdae, Paraburkholderia caryophylli, Paracoccus yeei, Parastreptomyces abscessus, Parvimonas Micra (Parvimonas micra), Pectobacterium Betabasculorum (Pectobacterium betavasculorum), Pectobacterium carotovorum, Pediococcus acidilactici, Pediococcus ethanolidurans, Pedobacter alluvionis, Pedobacter wanjuense, Pelomonas aquatica, Peptococcus niger, Peptoniphilus asaccharolyticus, Peptoniphilus gorbachii , Peptoniphilus harei, Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilus massiliensis, Peptostreptococcus Peptostreptococcus anaerobius, Peptostreptococcus massiliae, Peptostreptococcus stomatis, Photobacterium angustum, Photobacterium frigidiphyllum ( Photobacterium frigidiphilum), Photobacterium phosphoreum, Porphyromonas asaccharolytica, Porphyromonas bennonis, Porphyromonas catoniae, Porphyromonas Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas somerae, Porphyromonas uenonis, Prevotella amnii, Prevotella amnii Prevotella baroniae, Prevotella bergensis, Prevotella bivia, Prevotella buccae, Prevotella buccalis, Prevotella colorans, Prevotella copri, Prevotella corporis, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella intermedia, Prevotella loescheii, Prevotella marshii, Prevotella melaninogenica , Prevotella micans, Prevotella nigrescens, Prevotella oris, Prevotella pleuritidis, Prevotella rumicola ruminicola), Prevotella shahii, Prevotella stercorea, Prevotella timonensis, Prevotella veroralis, Propionimicrobium Propionimicrobium lymphophilum, Proteus mirabilis, Pseudomonas abietaniphila, Pseudomonas aeruginosa, Pseudomonas amygdali, Pseudomonas azotoformans (Pseudomonas azotoformans), Pseudomonas chlororaphis, Pseudomonas cuatrocienegasensis, Pseudomonas fluorescens, Pseudomonas fulva, Pseudomonas lutea , Pseudomonas mucidolens, Pseudomonas oleovorans, Pseudomonas orientalis, Pseudomonas pseudoalcaligenes, Pseudomonas psychrophila, Pseudomonas putida udomonas putida), Pseudomonas synxantha, Pseudomonas syringae, Pseudomonas tolaasii, Pseudopropionibacterium propionicum, Rahnella aquatilis, Ralstonia pickettii, Ralstonia solanacearum, Raoultella planticola, Rhizobacter dauci, Rhizobium etli , Rhodococcus fascians, Rhodopseudomonas palustris, Roseburia intestinalis, Roseburia inulinivorans, Rotia Musillaginosa ( Rothia mucilaginosa), Ruminococcus bromii, Ruminococcus gnavus, Ruminococcus torques, Sanguibacter keddieii, Sedimibacterium Salmoneum (Sediminibacterium salmoneum), Selenomonas bovis, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Shewanella algae algae), Shewanella amazonensis, Shigella boydii, Shigella sonnei, Slackia exigua, Sneathia amnii, stea Sneathia sanguinegens, Solobacterium moorei, Sorangium cellulosum, Sphingobium amiense, Sphingobium japonicum, Sphingobium yanoikuyae, Sphingomonas wittichii, Sporosarcina aquimarina, Staphylococcus aureus, Staphylococcus auri Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis ( Staphylococcus hominis), Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus simiae ), Staphylococcus simulans, Staphylococcus warneri, Stenotrophomonas maltophilia, Stenoxybacter acetivorans, strepto Streptococcus agalactiae, Streptococcus anginosus, Streptococcus australis, Streptococcus equinus, Streptococcus gallolyticus ( Streptococcus gallolyticus), Streptococcus infantis, Streptococcus intermedius, Streptococcus lutetiensis, Streptococcus marimammalium, Streptococcus Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus phocae, Streptococcus Streptococcus pseudopneumoniae, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus thermophilus, Sutterella wadsworthensis), Tannerella forsythia, Terrahaemophilus aromaticivorans, Treponema denticola, Treponema maltophilum, Treponema Treponema parvum, Treponema vincentii, Trueperella bernardiae, Turicella otitidis, Ureaplasma parvum, Ureaplasmau Ureaplasma urealyticum, Varibaculum cambriense, Variovorax paradoxus, Veillonella atypica, Veillonella dispar, veil Veillonella montpellierensis, Veillonella parvula, Virgibacillus proomii, Viridibacillus arenosi, Viridibacillus arvi , Weissella cibaria, Weissella soli, Xanthomonas campestris, Xanthomonas vesicatoria, Zobellia laminariae and / or Zoogloea ramigera.

In one embodiment, the targeted recipient bacterium is Escherichia coli.

In one embodiment, the targeted recipient bacterium is Klebsiella pneumoniae.

In one embodiment, the targeted recipient bacteria are Bacteroides thetaiotaomicron and/or Bacteroides faecis.

In one embodiment, the targeted recipient bacterium is Roseburia intestinalis.

In one embodiment, the targeted bacterium is Cutibacterium acnes, more particularly clade IA1 or RT4, RT5, RT8, RT9, RT10 or clone complex (CC) CC1, CC3, CC4, more particularly ST1, Acne-associated Cutibacterium acnes from ST3, ST4.

In one embodiment, the targeted recipient bacterium is a pathogenic bacterium. The targeted recipient bacteria may be virulent bacteria.

In certain embodiments, the targeted recipient bacteria are involved in infection in the host. In certain embodiments, the targeted recipient bacteria are associated with the triggering, progression, or exacerbation of an auto-immune disease in the host. In certain embodiments, the targeted recipient bacteria are associated with initiation, progression, or exacerbation of tumors or metastases in the host. In certain embodiments, the targeted recipient bacteria are associated with the initiation, progression, or exacerbation of a neurodegenerative disease in the host. In certain embodiments, the targeted recipient bacteria are associated with the initiation, progression, or exacerbation of a CNS-related disease in the host. In certain embodiments, the targeted recipient bacteria are associated with resistance of the host to treatment for infections, tumors, neurodegenerative diseases, CNS-related diseases, autoimmune diseases, and/or cancer.

Targeted recipient bacteria include extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiella pneumoniae, vancomycin-resistant Enterococcus (VRE) ), methicillin-resistant Staphylococcus aureus (MRSA), multiple drug-resistant (MDR) Acinetobacter baumannii, MDR Enterobacter spp., and combinations thereof It may be antibacterial agent-resistant bacteria including those selected from the group consisting of. The targeted recipient bacteria may be selected from the group consisting of extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli strains. In certain embodiments, the targeted recipient bacteria are ESBL Escherichia coli and/or ESBL Klebsiella pneumoniae.

Alternatively, the targeted recipient bacteria may be bacteria of a given species of microbiome, particularly bacteria of the human microbiota.

Certain effects and corresponding nucleic acids of interest

In the control method of the present invention, the nucleic acid of interest, as defined above, produces a desired effect on the targeted recipient bacterial cell.

" A nucleic acid that produces a desired effect on the targeted recipient bacterial cell " is used herein to indicate that delivery of the nucleic acid to the targeted recipient bacterial cell is a response to the targeted recipient bacterial cell (e.g. expression of RNA, expression of protein). or activation or inhibition of an activity), wherein said response in said targeted recipient bacterial cell preferably directly or indirectly induces a response in said organism hosting said targeted recipient bacterial cell. create more

In certain embodiments, the nucleic acid of interest is expressed in the targeted recipient bacterial cell to produce the desired effect. Expression of the nucleic acid of interest includes expression as coding or non-coding RNA, or expression as a protein. Alternatively, in certain embodiments, the nucleic acid of interest is not expressed in the targeted recipient bacterial cell, and the presence of the nucleic acid of interest in the targeted recipient bacterial cell (e.g., already present in the targeted recipient bacterial cell) by providing a binding region to a molecule that produces the desired effect.

In the context of the present invention, the predetermined effect comprises killing the recipient bacterial cell, causing the recipient bacterial cell to stop producing the desired molecule, causing the recipient bacterial cell to reduce its level of production of the predetermined molecule, and causing the recipient bacterial cell to stop producing the desired molecule. can be selected from the group consisting of steps that result in the production of the molecule of interest.

Making the recipient bacterial cell produce the molecule of interest

In certain embodiments, the predetermined effect is to cause the recipient bacterial cell to produce a molecule of interest, particularly a host regulatory molecule.

In another particular embodiment, the predetermined effect is to cause the recipient bacterial cell to produce, as the molecule of interest, a transcription factor and/or a modified nuclease, particularly to activate a particular pathway or gene in bacteria that are naturally turned off. .

In another specific embodiment, the predetermined effect preferably temporarily increases or decreases the fitness of the recipient bacterial cell to its environment, compared to other members of the microbiome that are not particularly recipient bacterial cells. to cause the production of the molecule of interest.

In another specific embodiment, the predetermined effect is to cause the recipient bacterial cell to produce, as a molecule of interest, a molecule of interest that functions in the microbiome environment, particularly without producing an effect at the level of the host organism's cells.

" Host Regulatory Molecule " or " HMM " means herein any molecule, produced by the recipient bacterial cell, that acts, directly or indirectly, at the level of the host organism.

The HMM may be of any nature. In particular, the HMM may be selected from the group consisting of non-coding nucleic acids, coding nucleic acids, proteins, lipids, sugars, LPS, metabolites, and small molecules.

Examples of non-coding nucleic acids typically include non-coding DNA or non-coding RNA, such as siRNA.

Examples of coding nucleic acids typically include coding DNA or coding RNA.

Examples of proteins typically include cytokines such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors and anti-inflammatory cytokines; surface layer proteins such as SlpB, especially from Propionibacterium freudenreichii; microbial anti-inflammatory molecules (MAMs), such as MAMs from Faecalibacterium prausnitz; antibodies such as monoclonal antibodies, multispecific antibodies, chimeric antibodies, antibody fragments and derivatives thereof; enzymes, particularly enzymes that cause the production of other HMMs; peptides such as Immune Selective Anti-Inflammatory Derivatives (FEG, salivary gland derived peptides), and mimetic proteins or peptides derived from the microbiome that mimic a subject's cell-derived antigens.

Mimetic peptides of particular interest include mimetic peptides associated with auto-immune diseases, eg, those incorporated herein by reference [Negi et al. (2017) Plos One 12 :e0180518]. Of particular interest are gene sequences encoding any of the mimetic peptides in the S1 table of Negi et al.

Examples of lipids typically include SCFAs such as butyrate.

Examples of small molecules typically include cyclosporine, non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs) and ROS.

The HMM may further have an arbitrary effect. In certain embodiments, the HMM may be a molecule that affects the host's immune system, host CNS, and/or host metabolism.

In particular, the HMM is an anticancer molecule, an antibiotic molecule, an anti-viral molecule, an anti-parasitic molecule, an anti-protozoal molecule, an anesthetic molecule, an anticoagulant molecule, an enzyme inhibitor, a steroidal molecule, an anti-inflammatory molecule, an antihistamine molecule, an immune molecule Inhibitory molecule, anti-neoplastic molecule, antigen, vaccine, antibody, hemorrhagic molecule, sedative molecule, analgesic molecule, antipyretic molecule, hormone, anti-hormonal molecule, anticholinergic, antidepressant molecule, antipsychotic molecule, neurotoxin molecule , sleep molecule, tranquilizer molecule, anticonvulsant molecule, muscle relaxant molecule, anti-aging molecule, anti-neurodegenerative molecule, neuromodulator, antispasmodic molecule, muscle contraction molecule, channel blocker molecule, miotic molecule, anti-secretion anti-thrombotic agent molecules, diuretic molecules, cardiovascular active molecules, vasoactive molecules, vasodilator molecules, anti-hypertensive agent molecules, angiogenic molecules, modulators of cell-extracellular matrix interactions (e.g., cell growth inhibitors and anti-adhesion molecules), growth factors, differentiation factors, antioxidant molecules, inhibitors of DNA, RNA, or protein synthesis, apoptotic factors, anti-apoptotic molecules, or anti-UV molecules.

The HMM may also be of any origin. In particular, the HMM may be selected from the group consisting of host endogenous molecules, host exogenous molecules naturally expressed by other organisms, and synthetic compounds.

By " host endogenous molecule " is meant herein any molecule naturally produced by a host subject, particularly a healthy host subject.

A "host exogenous molecule naturally expressed by another organism " herein is not produced by the host subject (or subject of the same species as the host species), but is produced by another organism, particularly another species, another sex, another family, means any molecule naturally produced by an organism from another class or from another kingdom. Typically, the host exogenous molecules naturally expressed by other organisms may be molecules produced by bacteria, particularly microbiota.

In certain embodiments, the nucleic acid of interest encodes a bacteriocin or lysin, which can be a proteinaceous toxin produced by a recipient bacterium to kill or inhibit the growth of other bacteria. Bacteriocins are classified in several ways, including production strains, common mechanisms of resistance, and mechanisms of death. Such bacteriocins have been described from gram-negative bacteria (eg microcins, cholicin-like bacteriocins and teilosins) and gram-positive bacteria (eg class I, class II, class III or class IV bacteriocins).

In one embodiment, the nucleic acid of interest encodes a toxin selected from the group consisting of microcins, cholicin-like bacteriocins, teilosins, class I, class II, class III, and class IV bacteriocins.

In certain embodiments, corresponding immune polypeptides (ie, anti-toxins) can be used to protect recipient bacterial cells (see review in Cotter et al., Nature Reviews Microbiology 11: 95, 2013).

A " synthetic compound " as used herein is a compound that is not naturally produced by a host subject (or subject of the same species as the host species) or another organism, particularly an organism of another species, another sex, another family, another class, or another kingdom. means any molecule.

The molecule of interest may be further produced by the targeted recipient bacterial cell in any form. In particular, the HMM may be selected from the group consisting of secreted molecules, intracellular molecules and membrane-expressed molecules.

Production of the molecule of interest by the targeted recipient bacterial cell may require delivery of a nucleic acid of interest comprising one or more type(s) of gene(s) or group(s) of genes. In particular, the nucleic acid of interest comprises a gene encoding the molecule of interest, in particular the HMM, several genes encoding a protein complex that is a molecule of interest, in particular HMM, an enzyme(s) of a metabolic pathway responsible for the production of the molecule of interest, in particular HMM. It may be selected from the group consisting of a gene or group of genes that encodes, a nucleic acid encoding a molecule of interest, particularly a HMM, and a non-coding nucleic acid that is a molecule of interest, particularly a HMM.

Making recipient bacterial cells stop producing certain molecules

In certain embodiments, the desired effect is to cause the recipient bacterial cell to stop producing the desired molecule.

By “causing a recipient bacterial cell to cease production of a given molecule” herein is meant to reduce or abolish the production of said molecule by said bacterial cell and/or cause a recipient bacterial cell to produce a variant of said molecule. do.

Typically, the given molecule whose production is to be stopped has a negative effect on the host organism.

In certain embodiments, the given molecule whose production is to be stopped affects the fitness of the recipient bacterial cell for its environment. In certain embodiments, causing a recipient bacterial cell to stop producing the given molecule preferably temporarily reduces the recipient bacterial cell's fitness for its environment, particularly compared to other members of the microbiome that are not the recipient bacterial cell. , increase or decrease.

In certain embodiments, the given molecule may be selected from the group consisting of toxins, virulence factors, virulence proteins, virulence factors, proteins encoded by antibiotic resistance genes, proteins encoded by remodeling genes or regulatory genes. In certain embodiments, the predetermined effect is to selectively eliminate antibiotic resistance from antibiotic resistant bacterial strains.

In certain embodiments, the nucleic acid of interest is a gene or group of genes that encode one or more exogenous enzyme(s) to effect genetic modification.

In certain embodiments, the nucleic acid of interest is a gene encoding a base-editor or prime-editor.

In some embodiments, genetic modifications are made by one or more of the following enzymes and systems.

Incorporated herein by reference, [Rees et al. (2018) Nat Rev Genet 19 :770-788, cytosine base editor (CBE) and adenosine base editor (ABE).

There are seven types of DNA base editors described so far:

● Cytosine base editor (CBE) converting C:G to T:A (Komor et al . (2016) Nature 533 :420-424)

● Adenine base editor (ABE) converting A:T to G:C (Gaudelli et al. (2017) Nature 551 :464-471)

● Cytosine guanine base editor (CGBE) converting C:G to G:C (Chen et al. (2020) Biorxiv "Precise and programmable C:G to G:C base editing in genomic DNA"; Kurt et al. ( 2020) Nat. Biotechnol. "CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells")

● Cytosine adenine base editor (CABE) that converts C:G to A:T (Zhao et al. (2020) Nature Biotechnol. "New base editors change C to A in bacteria and C to G in mammalian cells")

● Adenine cytosine base editor (ACBE) converting A:T to C:G (WO2020181180)

● Adenine thymine base editor (ATBE) converting A:T to T:A (WO2020181202)

● Thymine adenine base editor (TABE) converting T:A to A:T (WO2020181193, WO2020181178, WO2020181195)

Base editors have different base modifying enzymes. CBE is particularly dependent on the following ssDNA cytidine deaminase: APOBEC1, rAPOBEC1, APOBEC1 mutant or evolved form (evoAPOBEC1), and APOBEC homologues (APOBEC3A (eA3A), Anc689), cytidine deaminase 1 ( CDA1), evoCDA1, FERNY, evoFERNY.

ABE can convert adenosine to inosine on ssDNA, E. coli. It relies on the deoxyadenosine deaminase activity of the E. coli tRNA adenosine deaminase enzyme, the tandem fusion TadA-TadA*, where TadA* is an evolved form of TadA. TadA* includes TadA-8a-e and TadA-7.10.

Aside from base modifying enzymes, there are modifications implemented to base editors to increase editing efficiency, precision and modularity:

• addition of one or two uracil DNA glycosylase inhibitor domains (UGI) to prevent base excision repair mechanisms that revert base editing;

• Addition of Mu-GAMs, which inhibit the non-homologous end joining mechanism (NHEJ) in cells, thereby reducing indel rates;

• the use of a nickase active Cas9 (nCas9 D10A) which creates a nick on the non-edited strand, favoring its repair and consequent fixation of the edited base;

• Use of various Cas proteins, eg from different organisms, different PAM motifs or mutants with different fidelities or different families (eg Cas12a).

Non-limiting examples of DNA-based editor proteins include BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, TARGET-AID, TARGET-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Cas12a-BE, Ea3A-BE3, A3A -BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRY-CBE , SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4, enAsBE1.1, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBE1.1, AsBE1.4, CRISPR -Includes Abest, CRISPR-Cbest, eA3A-BE3, AncBE4.

The cytosine guanine base editor (CGBE) consists of the nickase CRISPR fused to:

a. Cytosine deaminase (rAPOBEC) and base cleavage reversion proteins (e.g., rXRCC1) (Chen et al. (2020) Biorxiv “Precise and programmable C:G to G:C base editing in genomic DNA”).

b. Rat APOBEC1 variant (R33A) protein and E. E. coli-derived uracil DNA N-glycosylase (eUNG) (Kurt et al. (2020) Nat. Biotechnol. "CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells").

Cytosine adenine base editor (CABE) consists of Cas9 nickase, cytidine deaminase (e.g., AID), and uracil-DNA glycosylase (Ung) (Zhao et al. (2020) Nature Biotechnol. "New base editors change C to A in bacteria and C to G in mammalian cells").

ACBE includes nucleic acid programmable DNA-binding protein and adenine oxidase (WO2020181180).

ATBE consists of a Cas9 nickase and one or more adenosine deaminase or oxidase domains (WO2020181202).

TABE consists of the Cas9 nickase and adenosine methyltransferase, thymine alkyltransferase, or adenosine deaminase domains (WO2020181193, WO2020181178, WO2020181195).

The base editor molecule may also consist of one or more of the editor enzymes listed above (eg, a combination of ABE and CBE) fused to a Cas protein. These biomolecules are termed dual base editors and can edit two different bases (Grunewald et al. (2020) Nature Biotechnol. "A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing"; Li et al. (2020) Nature Biotechnol. "Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors").

Incorporated herein by reference, [Anzalone et al. (2019) Nature 576 :149-157, the prime editor (PE) is an nCas9 fused to a reverse transcriptase used in combination with a prime editing RNA (pegRNA, a guide RNA containing a template region for reverse transcription). made up of

Prime editing allows insertions, introduction of deletions (indels) and 12 base to base conversions. Prime editing relies on the ability of reverse transcriptase (RT), fused to a Cas nickase variant, to convert the RNA sequence taken by the prime editing guide RNA (pegRNA) into DNA at the nick site created by the Cas protein. DNA flips generated by this process may or may not be included in the targeted DNA sequence.

The prime editing system includes:

- Cas nickase variants such as reverse transcriptase domains such as M-MLV RT or mutant forms thereof (M-MLV RT (D200N), M-MLV RT (D200N / L603W), M-MLV RT (D200N / L603W / T330P / T306K /W313F) Cas9-H840A fused to;

● Prime editing guide RNA (pegRNA).

To facilitate editing, prime editing systems design sgRNAs to anneal with the edited strand rather than the original strand, ideally only after cleavage of the edited strand flaps, with additional sgRNAs targeting Cas nickase activity against non-edited DNA. can include the expression of

Non-limiting examples of prime editing systems include PE1, PE1-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b.

CRISPEY (Cas9 Retron precISe Parallel Editing via homologY) is a retron RNA fused to sgRNA and expressed with at least a retron protein containing reverse transcriptase and Cas9 (Sharon et al. (2018) Cell 175 :544-557. e16).

SCRIBE strategy: The retron system is expressed in combination with a recombinase that promotes recombination of single-stranded DNA, also known as single-stranded annealing protein (SSAP) (Farzadfard & Lu (2014) Science 346 :1256272). Such recombinases include phage recombinases such as lambda red, recET, Sak, Sak4, and Wannier et al. (2020) Proc Natl Acad Sci USA 117 (24): 13689-13698;

Group II intron-based targetron systems [Karberg et al. (2001) Nat Biotechnol 19 :1162-7, incorporated herein by reference and adapted for many bacterial species.

Other letron-based gene targeting approaches are described in Simon et al., incorporated herein by reference. (2019) Nucleic Acids Res 47 :11007-11019.

In certain embodiments, the CRISPR system is comprised of a nucleic acid of interest. The CRISPR system contains two separate components: i) an endonuclease, in this case a CRISPR-associated nuclease (Cas or "CRISPR-associated protein") and ii) a guide RNA. The guide RNA may be in the form of a chimeric RNA consisting of a combination of CRISPR (RNAcr) bacterial RNA and RNAtracr (trans-activating RNA CRISPR) (Jinek et al. (2012) Science 337 (6096):816-21). The guide RNA combines the targeting specificity of RNAcr, which corresponds to a "spacing sequence" that serves as a guide to the Cas protein, and the conformational nature of RNAtracr of a single transcript. When the guide RNA and Cas protein are co-expressed in cells, the target genomic sequence can be permanently modified or blocked. The deformation is advantageously guided through a recovery matrix. In general, CRISPR systems include two major classes that rely on pre-existing nuclease action. Class 1 is composed of multi-subunit effector complexes and includes types I, III and IV. Class 2 consists of single-unit effector modules such as the Cas9 nuclease, type II (II-A,II-B,II-C,II-C variants), type V (VA,VB,VC,VD,VE , V-U1, V-U2, V-U3, V-U4, V-U5) and VI types (VI-A, VI-B1, VI-B2, VI-C, VI-D).

A nucleic acid of interest according to the present disclosure may include a nucleic acid sequence encoding a Cas protein. A variety of CRISPR enzymes are available for use as sequences of interest on plasmids. In some embodiments, the CRISPR enzyme is a type II CRISPR enzyme. In some embodiments, a CRISPR enzyme catalyzes DNA cleavage. In some other embodiments, the CRISPR enzyme catalyzes RNA cleavage. Preferably, the CRISPR enzyme does not make double strand breaks. In some embodiments, the CRISPR enzyme makes single strand breaks or nicks. In some embodiments, the CRISPR enzyme does not make any breaks in DNA or RNA. In one embodiment, a Cas13-deaminase fusion is used to base edit RNA.

In one embodiment, the CRISPR enzyme can be coupled to the sgRNA. In some embodiments, the sgRNA targets a gene encoding a given molecule as defined above.

Non-limiting examples of Cas proteins as part of a multi-subunit effector or as a single-unit effector include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10 , Cas11 (SS), Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2), Cas13b (C2c6) , Cas13c (C2c7), Cas13d, Csa5, Csc1, Csc2, Cse1, Cse2, Csy1, Csy2, Csy3, Csf1, Csf2, Csf3, Csf4, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx13, Csx1, Csx15, SdCpf1, CmtCpf1, TsCpf1, CmaCpf1, PcCpf1, ErCpf1, FbCpf1, UbcC pf1, AsCpf1, LbCpf1, Mad4, Mad7, Cms1, a homolog thereof, an ortholog thereof, a variant thereof, or a modified form thereof. In some embodiments, the CRISPR enzyme cleaves both strands of a target nucleic acid at a Protospacer Adjacent Motif (PAM) site.

In various embodiments, the invention encompasses fusion proteins comprising a Cas9 (eg, Cas9 nickase) domain and a deaminase domain. In some embodiments, the fusion protein is Cas9 and a cytosine deaminase enzyme, such as the APOBEC enzyme, or adenosine deaminase, eg, as disclosed in US Patent Publication No. 2015/0166980, incorporated herein by reference. second enzyme, such as the ADAT enzyme. In one embodiment, the deaminase is an ACF1/ASE deaminase.

In various embodiments, APOBEC deaminase is APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In various embodiments, the fusion protein comprises a Cas9 domain, a cytosine deaminase domain, and a uracil glycosylase inhibitor (UGI) domain.

In one embodiment, the deaminase is an adenosine deaminase that deaminates adenosine in DNA, eg, as disclosed in US Pat. No. 10,113,163, incorporated herein by reference. In some embodiments, the fusion protein further comprises an inhibitor of base repair, such as a nuclease killing inosine specific nuclease (dISN), as disclosed, for example, in US Pat. No. 10,113,163. In various embodiments, the present invention is described in, for example, Anzalone et al. (2019) Nature 576 :149-157, a catalytically damaged Cas9 endo fused to an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) that specifies the target site and encodes a preferred editing region. It encompasses fusion proteins that include nucleases.

In certain embodiments, the CRISPR enzyme is any Cas protein, in particular any Cas9 protein, including any naturally occurring bacterial Cas9, as well as any variant, chimera, homologue or ortholog thereof.

"Cas9" refers to the protein Cas9 (also called Csn1 or Csx12) or a functional protein, peptide or polypeptide fragment thereof, i.e., capable of interacting with guide RNA(s) and exerting enzymatic activity (nuclease) to target It means allowing to perform double-strand breaks of genomic DNA. Thus, "Cas9" means truncated to remove domains of a modified protein, eg domains of the protein that are not essential for the protein's predefined function, in particular domains that are not necessary for interaction with the gRNA(s).

The sequence encoding Cas9 (whole protein or fragment thereof) used in the context of this disclosure can be obtained from any known Cas9 protein (Fonfara et al. (2014) Nucleic Acids Res 42 (4):2577-90; Shmakov et al. (2017) Nat Rev Microbiol 15 (3):169-182). Examples of Cas9 proteins useful in the present disclosure include Streptococcus pyogenes (SpCas9), Streptococcus thermophiles (St1Cas9, St3Cas9), Streptococcus mutans, Star Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida (FnCas9) and Neisseria meningitides ( NmCas9), but is not limited thereto.

The sequence encoding Cpf1 (Cas12a) (whole protein or fragment thereof) used in the context of this disclosure can be obtained from any known Cpf1 (Cas12a) protein (Koonin et al. (2017) Current Opinion in Microbiology 37 : 67-78). Examples of Cpf1 (Cas12a) proteins useful in the present disclosure include the Cpf1 (Cas12a) proteins of Acidaminococcus sp, Lachnospiraceae bacteriu and Francisella novicida Including, but not limited to.

The sequence encoding Cas13a (whole protein or fragment thereof) can be obtained from any known Cas13a (C2c2) protein (Abudayyeh et al. (2017) Nature 550 :280). An example of a Cas13a (C2c2) protein useful in the present disclosure includes, but is not limited to, the Cas13a (C2c2) protein (LwaCas13a) of Leptotrichia wadei.

Sequences encoding Cas13d (whole protein or fragments thereof) can be obtained from any known Cas13d protein (Yan et al. (2018) Mol Cell 70 (2):327-339). Examples of Cas13d proteins useful in the present disclosure include, but are not limited to, Cas13d proteins from Eubacterium siraeum and Ruminococcus sp.

The sequence encoding Mad4 (whole protein or fragment thereof) used in the context of the present invention is disclosed in International Patent Application Publication No. WO2018/236548.

The sequence encoding Mad7 (whole protein or fragment thereof) used in the context of the present invention is disclosed in International Patent Application Publication No. WO2018/236548.

The sequence encoding Cms1 (whole protein or fragment thereof) used in the context of the present invention is disclosed in International Patent Application Publication No. WO2017/141173.

In some embodiments, other programmable nucleases may be used. These include engineered TALENs (Transcription Activator-Like Effector Nucleases; Transcription Activator-Like Effector Nucleases) and variants, engineered zinc finger nuclease (ZFN) variants, natural, evolved or engineered meganucleases or recombi Nase variants, and any combination or hybrid of programmable nucleases. Thus, the programmable nucleases provided herein can be used to selectively modify DNA encoding a gene of interest such as, for example, a toxin gene, virulence factor gene, antibiotic resistance gene, remodeling gene or regulatory gene ( See: WO2014124226 and US2015/0064138).

In some embodiments, genetic modifications are made at the RNA level. RNA base editing is based on the same principle as DNA base editing: the enzyme that catalyzes the conversion of an RNA base to another must come close to the target base to effect its conversion locally. In one embodiment, the enzyme used for RNA editing is an adenosine deaminase of the ADAR family that converts adenosine to inosine in the dsRNA structure. Several seminal studies have used this specificity for dsRNA and fused the ADAR deaminase domain (ADAR _DD ) to antisense oligos to program local RNA base editing. More recently, the ability of some CRISPR-Cas systems to bind RNA molecules has been repurposed for RNA editing. Using a catalytically dead Cas13b enzyme (dPspCas13b) fused to hyperactive mutants of the ADAR2 deaminase domain (ADAR2DD-E488Q for REPAIRv1 and ADAR2 _DD -E488Q-T375G for REPAIRv2), Cox et al. Compared to improved specificity and efficiency. Non-limiting examples of RNA-based editor proteins include REPAIRv1, REPAIRv2.

In certain embodiments, the modification is an antibiotic resistance gene, a virulence factor or protein gene, a toxin factor or protein gene, a gene expressing a bacterial receptor, a membrane protein, a structural protein, a secreted protein, and a gene expressing resistance to common drugs. It is made in a gene selected from the group consisting of.

In one embodiment, modifications are made to target and inactivate virulence factors. A virulence factor can be any substance produced by a pathogen that alters host-pathogen interactions by increasing the degree of damage done to the host. Virulence factors can be used to colonize niches or cell adhesion in the host, for example, to evade the host's immune response, facilitate entry into host cells, obtain nutrients from the host, or inhibit other physiological processes in the host. It is used by pathogens in many ways, including Virulence factors may include enzymes, endotoxins, adhesion factors, motor factors, factors involved in complement evasion, and biofilm formation promoting factors. For example, such targeted virulence factor genes are E. coli. E. coli virulence factor genes, such as, but not limited to, EHEC-HlyA, Stx1 (VT1), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c (VT2c), Stx2d (VT2d), Stx2e (VT2e) and Stx2f ( VT2f), Stx2h (VT2h), fimA, fimF, fimH, neuC, kpsE, sfa, foc, iroN, aer, iha, papC, papGI, papGII, papGIII, hlyC, cnf1, hra, sat, ireA, usp ompT, ibeA , malX, fyuA, irp2, traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aaiC, aatA, TEM, CTX, SHV, csgA, csgB, csgC, csgD, csgE, csgF, csgG, csgH, T1SS , T2SS, T3SS, T4SS, T5SS, T6SS (secretion system). For example, such targeted virulence factor genes can be Shigella dysenteriae virulence factor factors such as, without limitation, stx1 and stx2. For example, such a targeted virulence factor gene can be a Yersinia pestis virulence factor gene such as, but not limited to, yscF (plasmid-derived (pCDL) T3SS external salivary subunit). For example, such a targeted virulence factor gene can be a Francisella tularensis virulence factor gene such as, without limitation, fslA. For example, such a targeted virulence factor gene can be a Bacillus anthracis virulence factor gene such as, without limitation, pag (anthrax toxin, cell-associated protective antigen). For example, such targeted virulence factor genes include Vibrio cholera virulence factor genes such as, without limitation, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulatory gland), and toxT (master virulence regulator) can be For example, such targeted virulence factor genes include Pseudomonas aeruginosa virulence factor genes such as, without limitation, pyoverdin (eg, sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA , pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT, opmQ), siderophore pyokelins (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG, and toxins (eg, exoU, exoS and exoT) For example, such targeted virulence factor genes may be Klebsiella pneumoniae virulence factor genes such as, without limitation, fimA (attachment, type I fimbriae major subunit), and cps (capsule polysaccharide) For example, such targeted virulence factor genes may be Acinetobacter baumannii virulence factor genes such as, without limitation, ptk (capsule polysaccharide) Polymerization) and epsA (assembly) For example, such targeted virulence factor genes may be Salmonella enterica Typhi virulence factor genes such as, without limitation, MIA (Invasion, SPI-1 Regulator), ssrB (SPI-2 regulator), and efflux pump genes acrA, acrB and tolC, which may be associated with bile tolerance. For example, these targeted virulence factor genes are nucleatum) virulence factor gene such as, without limitation, FadA and TIGIT For example, this targeted virulence factor gene may be a Bacteroides fragilis virulence factor gene such as, without limitation, bft.

In another embodiment, the modification is an antibiotic resistance gene such as, without limitation, GyrB, ParE, ParY, AAC(1), AAC(2'), AAC(3), AAC(6'), ANT(2"), ANT (3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"), APH(3'), APH(4), APH(6), APH (7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-M, KPC, SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA β-lactamase, mecA, Omp36, OmpF, PIB, bla (blaI, blaR1) and mec (mecI, mecR1) operons, chloramphenicol acetyltransferase (CAT), chloramphenicol phosphotransferase, ethambutol-resistant Arabinosyltransferase (EmbB), MupA, MupB, integral membrane protein MprF, Cfr 23S rRNA methyltransferase, rifampin ADP-ribosyltransferase (Arr), rifampin glycosyltransferase, rifampin monooxygenase, rifampin phospho Transferase, DnaA, RbpA, Rifampin-resistant-beta-subunit of RNA polymerase (RpoB), Erm 23S rRNA methyltransferase, Lsa, MsrA, Vga, VgaB, streptogramin Vgb lyase, Vat acetyltransferase, fluo Roquinolone acetyltransferase, fluoroquinolone-resistant DNA topoisomerase, fluoroquinolone-resistant GyrA, GyrB, ParC, quinolone resistant protein (Qnr), FomA, FomB, FosC, FosA, FosB, FosX, VanA, VanB , VanD, VanR, VanS, lincosamide nucleotidyltransferase (Lin), EreA, EreB, GimA, Mgt, Ole, macrolide phosphotransferase (MPH), MefA, MefE, Mel, streptothricin acetyl Transferase (sat), Sul1, Sul2, Sul3, sulfonamide-resistant FolP, tetracycline inactivating enzyme TetX, TetA, TetB, TetC, Tet30, Tet31, TetM, TetO, TetQ, Tet32, Tet36, MacAB-TolC, MsbA , MsrA, VgaB, EmrD, EmrAB-TolC, NorB, GepA, MepA, AdeABC, AcrD, MexAB-OprM, mtrCDE, EmrE, adeR, acrR, baeSR, mexR, phoPQ, mtrR, or Comprehensive Antibiotic Resistance Database Database) (CARD https://card.mcmaster.ca/).

In a preferred embodiment, the antibiotic is a penicillin such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, blood bampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; Cephalosporins, such as cefacetril, cefadroxil, cephalexin, cephaloglycin, cephalonium, cephaloridine, cephalothin, cefapyrin, cefatrizine, cefazaflur, cefazedone, cefazolin, Pradine, ceproxadine, ceftesol, cefaclor, seponicide, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef, cefbuferazone, cefminox , cefotetan, cefoxitin, sefotiam, cefcapen, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixim, cefmenoxime, cefodizim, cefotaxime, cefovexin, ceffimizole, cefpodoxime, cefteram, ceftamer, ceftibuten, cefthiopur, ceftiolene, ceftizoxim, ceftriaxone, cefoperazone, ceftagidim, ratamoxef, cefclidine, cefepime, Ceffluprenam, Sefocellis, Sefozofran, Cefpyrom, Cefquinom, Flomoxef, Ceftobiprole, Ceftarolin, Ceftolozan, Cephaloram, Cefpharole, Cefcanel, Cefedrolor , cefimpidone, cefetrizole, cefibitril, cefmatylene, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cefuracetim, and nitrocepin; polymyxins such as polysporin, neosporin, polymyxin B, and polymyxin E, rifampicins such as rifampicin, rifapentine, and rifaximin; fidaxomycin; Quinolones such as cinoxacin, nalidic acid, oxolinic acid, pyromidic acid, pipemidic acid, losoxacin, ciprofloxacin, enoxacin, fleoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin reaper, lufloxacin, balofloxacin, grepafloxacin, levofloxacin, pajufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, squarenoxacin, and zabofloxacin; Sulfonamides such as sulfafurazole, sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole, sulfisomidine, sulfadoxine, sulfamethoxazole, sulfamoxol, sulfanitran, sulfadimethoxine, sulfamethoxypyr minced, sulfamethoxydiazine, sulfadoxine, sulfamethopyrazine, and terepthyl; macrolides such as azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, medicamycin, oleandomycin, solithromycin, pyramycin, trolls leandomycin, tylosin, and roxithromycin; ketolides such as telithromycin, and setromycin; fluoroketolides such as solithromycin; lincosamides such as lincomycin, clindamycin, and pilimycin; tetracyclines such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline; aminoglycosides such as amikacin, dibecacin, gentamicin, kanamycin, neomycin, netylmicin, sisomicin, tobramycin, paromomycin, and streptomycin; ansamycins such as geldanamycin, herbimycin, and rifaximin; carbacephem such as laurakarbef; carbapenems such as eltapenem, doripenem, imipenem (or cilastatin), and meropenem; glycopeptides such as teicoplanin, vancomycin, telavancin, dalba reply, and orita reply; lincosamides such as clindamycin, and lincomycin; lipopeptides such as daptomycin; monobactams such as aztreonam; nitrofurans such as furazolidone, and nitrofurantoin; oxazolidinones such as linezolid, pocizolide, radezolide, and torezolide; Teixobactin, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin , platensimycin, quinupristine (or dalfopristine), thiamphenicol, tigecycline, tinidazole, trimethoprim, alatrofloxacin, fidaxomycin, nalidixic acid, rifampin, derivatives thereof, and combinations thereof. selected from the group.

When the antibiotic resistance gene is placed in a bacterium on a plasmid without an intoxication system, it is possible to eliminate antibiotic resistance through cutting either in the antibiotic resistance gene or elsewhere in the plasmid.

In another embodiment, modifications are made to the bacterial toxin gene. Bacterial toxins can be classified as either exotoxins or endotoxins. Exotoxins are produced and actively secreted, while endotoxins remain part of the bacteria. Responses to bacterial toxins can include severe inflammation, resulting in sepsis. Such toxins include, for example, botulinum neurotoxin, tetanus toxin, staphylococcal toxin, diphtheria toxin, anthrax toxin, alpha toxin, pertussis toxin, Shiga toxin, heat-stable enterotoxin (E. coli ST), colibactin, BFT (B. fragilis toxin) or any toxin described in [Henkel et al., (Toxins from Bacteria in EXS. 2010; 100: 1-29)]. In certain embodiments, the toxin is a Shiga toxin.

In some embodiments, modifications are made to the mimetic peptide gene sequence to reduce homology with human peptide sequences, and thus create a mimetic peptide that is no longer recognized by the host immune system. Mimetic peptides of particular interest include bacterial mimetic peptides associated with auto-immune diseases, eg, Negi et al. (2017) Plos One 12 :e0180518]. Of particular interest are gene sequences encoding any of the mimetic peptides in the S1 table of Negi et al.

In a preferred embodiment, the mimetic peptide is from Proteobacteria or Firmicutes. Of particular interest are Negi et al., which have homology to four human peptides from low molecular weight phosphotyrosine protein phosphatases, aldehyde dehydrogenase family 3 member B1, maleylacetoacetate isomerase and uracil-DNA glycosylase. These are the gene sequences encoding the 24 intestinal bacterial peptides identified by this. These gene sequences can be modified to reduce homology to human sequences and prevent cross-reactivity of human counterparts with those recognized by the host immune system.

In a preferred embodiment, the genetic modification is in the Bacteroides faecis or Bacteroides thetaiotaomicron beta-galactosidase gene. Preferably, the Bacteroides paesis or Bacteroides thetaiotaomicron beta-galactosidase protein with genetic modification is Bacteroides paesis or Bacteroides thetaiotaomicron beta-gal without genetic modification. It shows lower homology to the human MYH5 cardiac peptide compared to the lactosidase protein. Preferably genetic modifications are performed on peptide fragments recognized as epitopes by the human immune system, resulting in weaker or absent epitope recognition by the human immune system.

In a preferred embodiment, the genetic modification is present in a human commensal bacterium encoding a Ro60 ortholog gene. Preferably, the genetically modified Ro60 protein exhibits lower homology to the human Ro60 peptide compared to the original protein. Preferably genetic modifications are performed on DNA sequences corresponding to peptide fragments recognized as epitopes by the human immune system, resulting in weaker or absent epitope recognition by the human immune system. Human commensal bacteria that are preferably targeted for genetic modification are: Propionibacterium propionicum, Corynebacterium amycolatum, Actinomyces masiliensis. massiliensis), Bacteroides thetaiotaomicron. Even more preferably, the human commensal bacterium targeted for genetic modification is Propionibacterium propionicum.

In a preferred embodiment, the genetic modification is present in a human commensal bacterial DNA sequence encoding a peptide mimicking insulin B 9-25, a self-epitope implicated in type 1 diabetes. The gene mutation reduces homology to the insulin B9-25 epitope SHLVEALYLVCGERGFF (SEQ ID NO: 1). In a preferred embodiment, the target bacteria belong to the phylum Firmicutes. In a preferred embodiment, the target gene of the target bacterium is part of the transketolase N superfamily.

In a preferred embodiment, the genetic modification is an epitope of the autoantigen β2-glycoprotein I (β2GPI), which encodes a peptide mimicking a self-epitope involved in antiphospholipid syndrome (APS) Roseburia interioris (Roseburia intestinalis). Gene mutations reduce homology to the T cell (β2GPI) epitope KVSFFCKNKEKKCSY (SEQ ID NO: 2) and/or the B cell epitope VSRGGMRKFIC (SEQ ID NO: 3).

Genetic alterations may be present in translated or untranslated regions of genes. Genetic modifications may be present in the promoter region of a gene or in any other region involved in gene regulation. In some embodiments, the genetic modification is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35 for different amino acids. , causes changes in amino acids such as 40, 45, 50, 100, 200, and 500. In some embodiments, the genetic modification introduces a stop codon. In some embodiments, the genetic modification is external to a protein coding sequence, within RNA, or within regulatory sequences.

Preferably, the genetic modification does not integrate the phage genome or exogenous DNA into the host bacterial chromosome or endogenous plasmid(s). Preferably, the genetic modification does not result in the expression of exogenous proteins from exogenous DNA integrated into the host bacterial chromosome or endogenous plasmid(s). Most preferably, the genetic modification does not involve NHEJ or HR endogenous repair mechanisms of the host bacterium.

Recipient bacterial cell death

In certain embodiments, the predetermined effect is the killing of recipient bacterial cells.

In certain embodiments, the nucleic acid of interest is a gene encoding a nuclease.

In one embodiment, the nucleic acid of interest is a programmable nuclease cycle to be delivered to the targeted bacterium. Such programmable nuclease cycles can mediate sequence-specific clearance in vivo of bacteria containing a target gene of interest (eg, a gene deleterious to humans). Some embodiments of the present disclosure are directed to different CRISPR-Cas system classes and types of Streptococcus pyogenes, such as Type II CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated), as described above. It relates to engineered variants of the system. Other programmable nucleases that may be used include other CRISPR-Cas systems, engineered Transcription Activator-Like Effector nuclease (TALEN) variants, engineered zinc finger nuclease (ZFN) variants, natural, Evolved or engineered meganucleases or recombinase variants, and any combination or hybrid of programmable nucleases. Thus, the programmable nuclease cycles provided herein can be used to selectively cleave DNA encoding a gene of interest, such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene, or a regulatory gene. There are (reference: WO2014124226 and US2015/0064138).

In certain embodiments, the nuclease is a Cpf1 nuclease.

In certain embodiments, the nuclease is a Cas9 nuclease.

In certain embodiments, the nuclease is a Mad4 nuclease, as defined above.

In certain embodiments, the nuclease is a Mad7 nuclease, as defined above.

In certain embodiments, the nuclease is a Cms1 nuclease, as defined above.

In certain embodiments, antibiotic resistant strains are prepared by programming a nuclease to perform DNA cleavage, eg, double-stranded DNA breaks in the antibiotic resistance gene located on the chromosome of the target bacterium or on a plasmid having an intoxication system (toxin/antitoxin). target is killed

Other sequences of interest, preferably programmable, can be delivered to and kill the targeted bacteria. For example, a nucleic acid of interest may encode a possessor or toxin.

In certain embodiments, the nucleic acid of interest also causes the recipient bacterial cell to produce a molecule of interest, as described above, in particular a host regulatory molecule, as described above, eg as a bacterial host, before or immediately after death.

nucleic acid of interest

In the context of the present invention, the nucleic acid of interest may be under the control of a promoter.

As is known to those skilled in the art, promoters can be classified as strong or weak depending on their affinity for RNA polymerase. The strength of a promoter may depend on whether initiation of transcription occurs with high or low frequency at that promoter. Different promoters with different strengths can be used in the present invention to result in different levels of gene/protein expression (e.g., the level of expression initiated from mRNA originating from a weak promoter is higher than that initiated from a strong promoter). lowness).

One skilled in the art will understand that promoter sequences can be selected from a number of known bacterial genes expressed by a variety of bacterial species. In addition, prokaryotic promoter prediction methods exist and can be based on DNA stability analysis as described by Kanhere and Bansal (BMC Bioinformatics 2005, 6:1). Therefore, selection of a promoter for the vector according to the present invention can be made based on the target bacterium.

In some embodiments, a nucleic acid of interest may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid of interest in its natural environment.

Examples of bacterial promoters for use in accordance with the present invention include, but are not limited to, positive regulatory E. E. coli promoters such as the positive regulatory σ70 promoter (e.g., the inducible pBad/araC promoter, the Lux cassette right promoter, the modified lambda Prm promoter, plac Or2-62 (positive), pBad/AraC with redundant REN sites, pBad, P (Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), "s" promoter (eg, Pdps), σ32 promoter (eg, heat shock) and the σ54 promoter (eg, glnAp2); voice control. E. coli promoters such as the negative regulatory σ70 promoter (eg promoter (PRM+), modified lambda Prm promoter, TetR - TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA_DlexO_DLac01, dapAp, FecA , Pspac-hy, pel, plux-cl, plux-lac, CinR, CinL, glucose control, modified Pr, modified Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR_regulation, Betl_regulation, pLac_lux, pTet_Lac, pLac/Mnt, pTet/Mnt, LsrA/cI, pLux/cI, Lacl, LacIQ, pLacIQl, pLas/cI, pLas/Lux, pLux/Las, pRecA with LexA binding site, reverse BBa_R0011, pLacI/ara-1, pLacIq, rrnB PI, cadC, hns, PfhuA, pBad/araC, nhaA, OmpF, RcnR), σS promoter (eg, Lutz-Bujard LacO with alternative sigma factor σ38), σ32 promoter (eg, Lutz-Bujard LacO with alternative sigma factor σ32), σ54 promoter (eg, glnAp2); voice control b. subtilis promoters such as repressive ratio. subtilis σ A promoter (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank), σ promoter, and [Mutalik VK et al (Nature Methods, 2013, 10: 354-360, especially supplement Data reference)], as well as the BioFAB promoter, disclosed on the BioFAB website (http://biofab.synberc.org/data). Other inducible microbial promoters and/or bacterial promoters may be used in accordance with the present invention. Inducible promoters for use according to the present disclosure are susceptible to one or more physiological condition(s), such as pH, temperature, radiation, osmotic pressure, saline gradient, cell surface binding, and changes in the concentration of one or more exogenous or endogenous inducer(s). can be induced (or inhibited) by Exogenous inducers or inducers include, but are not limited to, amino acids and amino acid derivatives, sugars and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum compounds, metal-containing compounds, salts, ions, enzyme substrate analogs, hormones. , or a combination thereof.

Particularly preferred bacterial promoters for use in accordance with the present invention may be selected from constitutive promoters regulated by σ70 such as promoters from the Anderson collection (http://parts.igem.org/Promoters/Catalog/Anderson): BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23108, BBa_J23109, BBa_J23110, BBa_J23111, BBa_J23112, BBa _J23113, BBa_J23114, BBa_J23115, BBa_J23116, BBa_J23117, BBa_J23118, and BBa_J23119.

Other preferred bacterial promoters include TetR, IcaR(A), AmtR, BetI, SrpR, Orf2, BM3R1, ButR, PhlF, PsrA, HlyIIR, AmeR, LmrA, QacR, ScbR, McbR, LitR, HapR, SmcR, TarA, and Incorporated herein by reference, including variants thereof, [Stanton et al. (2014) Nat. Chem. Biol. 10 :99-105]. In certain embodiments, the promoter is SrpR and/or PhlF, or variants thereof.

In some embodiments of the invention, promoters may or may not be used with "enhancers", referred to as ds-acting regulatory sequences, that are involved in the transcriptional activation of nucleic acid sequences downstream of the promoter. Enhancers can be placed at any functional position before or after the promoter.

In some embodiments, a vector may include a terminator sequence, or terminator. As used herein, a “terminator” is a nucleic acid sequence that causes transcription to cease. Terminators can be unidirectional or bidirectional. It contains DNA sequences involved in the specific termination of RNA transcripts by RNA polymerase. The terminator sequence prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters. Thus, in some embodiments, terminators that terminate production of RNA transcripts are contemplated. Terminators may be required in vivo to obtain desired gene/protein expression levels.

The most commonly used type of terminator is the forward terminator. When placed downstream of a normally transcribed nucleic acid of interest, a forward transcription terminator will cause transcription to cease. In some embodiments, a bidirectional transcription terminator is provided, which generally causes transcription to terminate on both the forward and reverse strands. In some embodiments, a reverse transcription terminator is provided, which generally terminates transcription only on the reverse strand. In prokaryotic systems, terminators are generally classified into two categories: (1) rho-independent terminators and (2) rho-dependent terminators. Rho-independent terminators usually consist of a series of uracil bases followed by a palindromic sequence forming a stem loop rich in G-C base pairs.

Terminators for use in accordance with the present invention include any terminators of transcription known to those skilled in the art or described herein. Examples of terminators include, but are not limited to, termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences, such as, for example, the TO terminators, TE terminators, lambda T1 and T1T2 found in bacterial systems. contains the terminator. In some embodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as one resulting from sequence truncation.

Terminators for use according to the present invention are also those disclosed in [Chen YJ et al (2013, Nature Methods, 10: 659-664)], and [Cambray G et al (Nucl Acids Res, 2013, 41(9)). : 5139-5148)].

vector

As used herein, the term “ vector ” refers to a nucleic acid molecule, typically DNA or RNA, that serves to deliver a passenger nucleic acid sequence, ie, DNA or RNA, to a recipient or target cell. A vector may include an origin of replication, a selectable marker, and optionally a suitable site for insertion of a gene such as a multiple cloning site. There are several general types of vectors, including plasmids, bacteriophage genomes, phagemids, phage-plasmids, viral genomes, cosmids, and artificial chromosomes.

In the context of the present invention, a vector may also be referred to as a payload.

A vector used in the context of the present invention may be a plasmid (eg, a conjugative plasmid capable of being transferred into a host cell), a phage, a phagemid or a prophage.

The payload may be a phagemid or phasemid obtained from a natural, evolved or engineered bacteriophage genome. A payload may also consist of only a portion of a phagemid or phagemid obtained from a natural, evolved, or engineered bacteriophage genome.

In some embodiments, the payload is a delivery vehicle as bacteria are naturally competent to take up the payload from the environment on their own.

As used herein, the terms " phagemid " and " phasmid " are equivalent and refer to vectors derived from both plasmids and bacteriophage genomes. The phagemid of the present disclosure includes a phage packaging site and an origin of replication (ori), as described below.

As used herein, the term " packaged phagemid " refers to a phagemid encapsidated in a bacteriophage scaffold, bacterial virus particle or capsid. In particular, it refers to a bacteriophage scaffold, a capsid without bacterial virus particles or a bacteriophage genome. Packaged phagemids can be produced using helper phage strategies well known to those skilled in the art. The helper phage contains all genes encoding structural and functional proteins necessary for the phagemid according to the present invention to be encapsidated. Packaged phagemids can also be produced using satellite virus strategies, known to those skilled in the art. Satellite viruses are subviral sources, consisting of nucleic acids that depend on co-infection of the host cell with helper viruses for all morphogenetic functions, whereas for all their episomal functions (integration and immunity, multicopy plasmid replication), satellite viruses is completely autonomous from the helper. In one embodiment, the satellite gene may encode a protein that promotes the capsid size reduction of the helper phage, as described for the P4 Sid protein that controls the P2 capsid size to fit into its smaller genome.

In certain embodiments, when the vector is a packaged phagemid, the vector does not contain any component derived from the organism from which the conditional origin of replication is derived. In particular, the packaging site of the vector is not derived from the organism from which the conditional origin of replication is derived.

Vectors may include, without limitation, plasmid vectors and recombinant phage vectors. One skilled in the art is familiar with the genetic components that must be present in a vector in order to successfully transform and select host cells comprising any isolated nucleotide or nucleic acid sequence of the present invention.

As used herein, the term " conjugation plasmid " refers to a plasmid that is transferred from one bacterial cell to another during conjugation, and a " donor bacterium " as used herein is a bacterium capable of transferring a conjugation plasmid to another bacterium.

Vectors used in the context of the present invention lack antibiotic resistance markers.

Antibiotic resistance genes are well known in the art and include ampicillin resistance (Amp), chloramphenicol resistance (Cm), tetracycline resistance (Tet), kanamycin resistance (Kan), hygromycin resistance (Qiyg or hph genes), and zeomycin Including immunity (Zeo).

In certain embodiments, vectors used in the context of the present invention include an auxotrophic marker. Bacterial auxotrophic markers are described in, for example, US Pat. Nos. 4,920,048, 5,691,185, 6,291,245, 6,413,768, and 6,752,994; US Patent Application Publication No. 20050186666; [Struhl et al. (1976) PNAS USA 73; 1471-1475]; [MacCormick et al., (1995) FEMS Microbiol. Lett. 127:105-109]; [Dickely et al. (1995) Mol. Microbiol. 15:839-847]; [Sorensen et al. (2000) Appl. Environ. Microbiol 66:1253-1258]; and Fiedler & Skerra (2001) Gene 274: 111 118, all incorporated herein by reference, typically including DapA and ThyA. In certain embodiments, the auxotrophic marker is ThyA.

In certain embodiments, the vector does not contain any restriction sites recognized by restriction enzymes frequently encoded by the targeted recipient bacterial cell. In another specific embodiment, the vector is a 40, 30, 20, 10, 9, 8, 10, 30, 20, 10, 9, 8, or 10, 30, 20, 10, 30, 20, 10, 30, 20, 20, 20, 30, 30, 20, 20, 30, or 20, 30, 30, or 20, 30, 30, 30, 20, 20, 30, 30, 20, 20, 20, 30, 20, 20, 20, 20, 20, 30, 20, 20, 20, 30, 20, 20, 20, 20, 30, 20, 30, or and no more than 7, 6, 5, 4, 3, 2 or 1 restriction site(s).

As used herein, the terms " restriction site " and " restriction enzyme site " are equivalent and refer to a location on a nucleic acid containing a specific sequence of nucleotides recognized by a restriction enzyme. In particular, nucleic acids contain specific sequences to which restriction enzymes bind and cut. Restriction sites are palindromic sequences, usually 4-8 base pairs in length. More precisely, a restriction site refers to a specific sequence and modification state to which a restriction enzyme binds and is cleaved by it. In particular, it refers to a specific unmodified sequence to which a restriction enzyme binds and is cleaved by it. In particular the sequence is not methylated, hydroxymethylated and glucosyl-hydroxymethylated. In this situation, the restriction enzyme is type I, type II, or type III. Alternatively, it may refer to certain modified sequences to which a restriction enzyme binds and cleaves, such as methylated, hydroxymethylated, and glucosyl-hydroxymethylated DNA. In this situation, the restriction enzyme is type IV.

As used herein, “ recognized by” with respect to restriction sites and restriction enzymes means that the restriction site is cleaved by a restriction enzyme.

Sequence N in the restriction site means that the nucleotide can be A, C, G or T; B means that the nucleotide can be C, G or T; Y means that the nucleotide can be C or T; W means that the nucleotide can be A or T; R means that the nucleotide can be A or G; D means A, G or T.

As used herein, the terms “ restriction enzyme ” and “ restriction endonuclease ” are equivalent and refer to enzymes that cleave nucleic acids at or near restriction sites. Restriction enzymes are generally classified into four types (types I to IV). The REBASE database lists restriction enzymes that can be recognized according to the restriction enzymes expressed by a given bacterium.

that at least 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or 99% of bacteria in a group of bacteria of interest " frequently " or "frequently" encode a restriction enzyme. means that

A vector according to the invention, preferably incorporated in a delivery vehicle, preferably in a bacteriophage capsid, preferably comprises no more than 100 restriction sites. In a preferred embodiment, a vector according to the invention, preferably included in a delivery vehicle, comprises no more than 10 restriction sites. In a most preferred embodiment, the vector according to the invention, preferably included in a delivery vehicle, does not contain any restriction sites.

The present invention also relates to a nucleic acid vector, as defined above, for use in the in vivo delivery of a nucleic acid of interest, as defined above, to a targeted recipient bacterial cell, said nucleic acid of interest comprising said targeted recipient bacterial cell. , and the vector is

- the nucleic acid of interest, as defined above, and

- contains a conditional origin of replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell,

The vector lacks antibiotic resistance markers.

conditional origin of replication

The vectors of the present invention contain an origin of conditional replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell.

In the context of the present invention, " conditional origin of replication " means an origin of replication whose functionality can be controlled by the presence of a particular molecule.

In certain embodiments, a conditional origin of replication is an origin of replication whose replication is dependent on the presence of one or more predetermined proteins, peptides, RNAs, nucleic acids, molecules or any combination thereof.

In certain embodiments, replication of the origin of replication may further rely on a process, such as transcription, to activate the replication.

In the context of the present invention, the conditional origin of replication is inactive in the targeted recipient bacterial cell due to the absence of the given protein, peptide, RNA, nucleic acid, molecule or any combination thereof in the recipient bacterial cell.

In certain embodiments, the conditional origin of replication is active in the donor bacterial cell because the donor bacterial cell expresses the predetermined protein, peptide, RNA, nucleic acid, molecule or any combination thereof. In certain embodiments, the protein, peptide, RNA nucleic acid, molecule or any combination thereof is expressed in trans in the donor bacterial cell.

By "in trans" is meant herein that the protein, peptide, RNA, nucleic acid, molecule, or any combination thereof, is not encoded on the same nucleic acid molecule that contains the origin of replication. In certain embodiments, the protein, peptide, RNA, nucleic acid, molecule, or any combination thereof is encoded on a chromosome or plasmid. In certain embodiments, the plasmid contains an antibiotic resistance marker. In an alternative embodiment, the plasmid lacks an antibiotic resistance marker.

Since the conditional origin of replication is inactive in the targeted recipient bacterial cell due to the absence of the given protein, peptide, RNA, nucleic acid, molecule or any combination thereof in the recipient bacterial cell, the conditional origin of replication is the particular recipient bacterial cell to be targeted. can be selected depending on

Conditional origins of replication used in accordance with the present invention may preferably originate from plasmids, bacteriophages or PICIs that share the following characteristics: they contain their origin of replication repeat sequences, or iterons, and the origin of replication specific to them at least one protein that interacts with (i.e., Rep, protein O, protein P, pri).

By way of example, mention may be made of the following plasmids and conditional replication systems of bacteriophages: RK2, R1, pSC101, F, Rts1, RSF1010, P1, P4, lambda, phi82, phi80.

In certain embodiments, the conditional origin of replication is an R6Kλ DNA origin of replication and derivatives, an IncPα oriV origin of replication and derivatives thereof, a ColE1 origin of replication modified to be under an inducible promoter, and a phage-inducible chromosome island (PICI) derived replication Origin and derivatives thereof.

In certain embodiments, the conditional origin of replication is an origin of replication present in less than 50%, or less than 40%, less than 30%, less than 20%, less than 10% or less than 5% of the bacteria of the host microbiome.

In another specific embodiment, the conditional origin of replication is present in bacteria of the host microbiome, in particular in greater than 50%, more particularly greater than 60%, greater than 70%, greater than 80%, greater than 90% or greater than 95% of the host microbiome. less than 80% identical to the sequence of the origin of replication of the bacterium representing, in particular less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or 1 comprises or consists of sequences that are less than % identical.

The term " phage-derived chromosome island " or " PICI " in the context of the present invention is a mobile genetic component with a conserved genetic organization and encodes a pair of branching regulatory genes including a PICI master repressor. Typically, in Gram-positive bacteria, PICI, which is transcribed in the same direction, to the left of rpr, encodes a small set of genes including the integrase (int) gene; To the right of rpr, PICI, transcribed in the opposite direction, is followed by a replication module consisting of an excisional function (xis) and, sometimes fused, a primase homolog (pri) and optionally a replication initiator (rep), followed by these genes , PICI, which encodes an origin of replication (ori) and is also transcribed in the same direction, encodes a gene involved in phage interference, and, optionally, a terminator small subunit homologue (terS).

In certain embodiments, the conditional origin of replication is an origin of replication derived from a phage-derived chromosomal island (PICI).

We have indeed designed a specific conditional origin of replication derived from PICI herein.

The inventors have shown that it is possible to derive novel conditionally replicative plasmids, based in particular on PICI-derived origins of replication and primase-helicases. These origins can be relatively rare in the target strain, and more advantageously, the primase-ori pair can be unique for each PICI, significantly reducing the probability of unwanted recombination or payload spread events. They can be further modified to further restrict recombination opportunities and to remove restriction sites to circumvent the target bacterial defense system.

In certain embodiments, the conditional origin of replication is [Fillol-Salom et al. (2018) The ISME Journal 12 :2114-2128, derived from the PICI-derived origin of replication of Escherichia coli strain CFT073.

In certain embodiments, the conditional origin of replication is primase ori from PICI of Escherichia coli strain CFT073, and is typically the sequence SEQ ID NO:4.

In another specific embodiment, the conditional origin of replication is GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNNNNTGG (SEQ ID NO: 5), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC, RGCCGGYYD, and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, selected from the group consisting of VGCCGGYBD; A PICI-derived primase ori of Escherichia coli strain CFT073 lacking at least 15 or at least 16 restriction site(s).

In certain embodiments, the conditional origin of replication is the PICI-derived primase oir of Escherichia coli strain CFT073, lacking the restriction site GAAABCC. Preferably, said conditional origin of replication is the sequence SEQ ID NO:6.

In certain other embodiments, the conditional origin of replication is GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNNNNTGG (SEQ ID NO: 5), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC, RGCCGGYYD, and It is a PICI-derived primase ori of Escherichia coli strain CFT073 without restriction sites selected from the group consisting of VGCCGGYBD. Preferably, said conditional origin of replication is the sequence SEQ ID NO:7.

In certain embodiments, the conditional origin of replication, wherein the origin of replication is derived from a phage-derived chromosome island (PICI), wherein the donor bacterial cell is a rep protein, in particular a primase-helicase, typically sequence SEQ ID It is active in said donor bacterial cells because it expresses in particular the primase-helicase of the sequence SEQ ID NO: 8, encoded by a nucleic acid comprising or consisting of NO: 9.

The inventors have demonstrated that these specific origins of conditional replication are particularly compatible with lambda-based packaging, resulting in sufficiently high titers (>10 ¹⁰ /mL) required for microbiota-related applications.

In a specific embodiment, a vector of the present invention has the sequence SEQ ID NO: 10. In another specific embodiment, the vector of the invention comprises or consists of the sequence SEQ ID NO: 11.

In certain embodiments, when the vector is a phagemid, the origin of replication may be from a different microorganism than that used to encode structural components of the capsid packaging the phagemid.

transmission of bacteria vehicle

In certain embodiments, the vector is placed inside a bacterial delivery vehicle. Preferably, the vector placed inside the delivery vehicle is a phagemid and the delivery vehicle is a bacterial viral particle or capsid.

As used herein, the term «delivery vehicle» refers to any vehicle that permits delivery of a vector or payload to a bacterium.

Bacteriophage scaffolds, viral scaffolds, bacterial viral particles, chemical-based delivery vehicles (eg, cyclodextrins, calcium phosphate, cationic polymers, cationic liposomes), protein-based or peptide-based delivery vehicles, lipids -based delivery vehicles, nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles (eg, transfection, electroporation, sonoporation, optical transfection), particle-based delivery vehicles (eg, gene guns) There are several types of delivery vehicles encompassed by the present invention, including magnetofection, impalofection, particle bombardment, cell-penetrating peptides) or donor bacteria (conjugation).

Any combination of delivery vehicles is also encompassed by the present invention.

Delivery vehicle may refer to a bacteriophage-derived scaffold and may be obtained from natural, evolved, or engineered capsids.

In some embodiments, the delivery vehicle is a vector or payload as bacteria are naturally competent to take up payloads from the environment on their own.

The present disclosure relates to bacterial delivery vehicles containing a vector or payload as described herein. Bacterial delivery vehicles are typically prepared from bacterial viruses. Bacterial delivery vehicles are typically selected to be able to introduce the vector into the targeted bacteria.

Bacterial viruses from which the bacterial delivery vehicles disclosed herein may be derived include bacteriophages. Optionally, the bacteriophage is based on the taxonomy of [Krupovic et al, Arch Virol, 2015], the Myoviridae family, the Podoviridae family, the Siphoviridae family, and the Ackermannviridae family. ) and is selected from the order Caudovirales, which consists of the family.

Bacteriophages belong to the family Myoviridae (eg, without limitation, the genus Cp220 virus, Cp8 virus, Ea214 virus, Felix1 virus, Muggle virus, Susp virus, Hp1 virus, P2 virus, chi virus, P100 virus, Sylvia virus, Sporo 1virus, Char Bomba virus, Twart virus, Cc31 virus, Jd18 virus, Js98 virus, Kp15 virus, Moon virus, Rb49 virus, Rb69 virus, S16 virus, Schizot4 virus, Sp18 virus, T4 virus, Cr3 virus, Se1 Virus, V5 virus, Arbouovirus, Agate virus, Agrican357 virus, Ap22 virus, Arv1 virus, B4 virus, Bastille virus, Bc431 virus, Bcep78 virus, Bicepmu virus, Bequarta virus, Bxz1 virus, Cd119 virus, Cp51 Virus, Cvm10 Virus, Eah2 Virus, El Virus, Hapuna Virus, Zimmer Virus, Kpp10 Virus, M12 Virus, Machina Virus, Masha Virus, Msw3 Virus, Mu Virus, Miohalovirus, Nit1 Virus, P1 Virus, Park Puna virus, Pbunavirus, Phikzvirus, Rheph4virus, Rsl2virus, Rslunavirus, Secunda5virus, Sep1virus, Spn3virus, Sbunavirus, Tg1virus, Vhmlvirus and Wphvirus).

Bacteriophages belong to the family Poviridae (eg, without limitation, the genus Fri1 virus, Kp32 virus, Kp34 virus, Phikmv virus, Pradovirus, Sp6 virus, T7 virus, Cp1 virus, P68 virus, Phi29 virus, Nona33 virus, Pocj virus, Tl2011 virus, Bcep22 virus, Bpp1 virus, Cba41 virus, Dfl12 virus, Ea92 virus, Epsilon 15 virus, F116 virus, G7c virus, Jalpa virus, Kf1 virus, Kpp25 virus, Lit1 virus, Rouge24 virus, Rouge7 virus, N4 virus, nonanavirus, P22 virus, phage virus, Pieco32 virus, Prtb virus, Sp58 virus, Una961 virus and Vp5 virus).

Bacteriophages belong to the siphoviridae family (eg, without limitation, the genus Camvirus, Lycavirus, R4virus, Arcadianvirus, Coopervirus, Pg1virus, Pipefishvirus, Rosebushvirus, Bruchtavirus, Che9cvirus, Hawkeyevirus , Plot virus, Jersey virus, K1g virus, Sp31 virus, Lmd1 virus, Una4 virus, Bongo virus, Ray virus, Butters virus, Charlie virus, Ready virus, Baxter virus, Nymphadora virus, Vignuz virus, Fishbourne Viruses, Pions Virus, Kp36 Virus, Rogue 1 Virus, Rtp Virus, T1 Virus, Tls Virus, Ab18 Virus, Amigo Virus, Anatole Virus, Andromeda Virus, Artis Virus, Banyard Virus, Bernal 13 Virus, Biceptima Virus, Bronvirus, C2 virus, C5 virus, Cba181 virus, Cbast virus, Cecivirus, Che8 virus, Chi virus, Cjw1 virus, Condok virus, Cronus virus, D3112 virus, D3 virus, Decurovirus, Demosthenes virus, two sets virus, E125 virus, Aau virus, Ff47 virus, Gaia virus, Gils virus, Gordon virus, Gordtnk virus, Harrison virus, Hk578 virus, Hk97 virus, Genst virus, Jwx virus, Kellegiovirus, Coravirus, L5 virus, Lambda virus, Laroyer virus, Lyphi virus, Marvin virus, Mudcat virus, N15 virus, Nonag virus, Np1 virus, Omega virus, P12002 virus, P12024 virus, P23 virus, P70 virus, Pa6 virus, Pamx74 virus, Patience virus , Pbi1 virus, Pepy6 virus, Pfr1 virus, Phic31 virus, Phicbk virus, Pieta virus, Pipel virus, Phijl1 virus, Pis4a virus, Psa virus, Saimuna virus, Rdjl virus, Rer2 virus, Sap6 virus, Send513 virus, Septima 3 virus, soravirus, sextack virus, Sfi11 virus, Sfi21dt1 virus, citara virus, Sk1 virus, slash virus, smoothie virus, soup virus, Sp beta virus, Ssp2 virus, T5 virus, tank virus, Tyn2 virus, Titan virus, Tm4 virus, Tp21 virus, Tp84 virus, Tria virus, Trigintaduo virus, Vegas virus, Vendetta virus, W beta virus, Wildcat virus, Wizard virus, Usu virus, Xp10 virus, Ydn12 virus and Wia virus) can be chosen

The bacteriophage may be selected from the family Ackermanviridae (eg, without limitation, Ag3 virus, Limestone virus, Cba120 virus, and Vi1 virus).

Optionally, the bacteriophage is not part of the order Caudovirales, but belongs to the family of the unspecified order, such as, but not limited to, the family Tectiviridae (such as the genus Alphatectivirus, Betatectivirus), the family Corticoviridae ( e.g. genus Corticovirus), family Inoviridae (e.g. genus Fibrovirus, Harvenivirus, Inovirus, Lineavirus, Plectrovirus, Saetivirus, Vespertiliovirus), Cystoviridae (Cystoviridae) family (eg genus Cystovirus), Leviviridae family (eg genus Allorevivirus, Levivirus), Microviridae family (eg genus Alpha3 microvirus, G4 microvirus, Phix174 microviruses, bdellomicroviruses, chlamydiamicroviruses, spiromicroviruses) and the Plasmaviridae family (such as the genus Plasmaviruses).

Optionally, the bacteriophage targets archaea but is not part of the order Caudovirales and belongs to a family of the order unspecified, such as, without limitation, Ampullaviridae, FuselloViridae, Globuloviridae ), Guttaviridae, Lipothrixviridae, Pleolipoviridae, Rudiviridae, Saltoprovirus and Bicaudaviridae.

A non-limiting list of bacterial genera and their known host-specific bacterial viruses is presented in the following paragraphs. Synonyms and spelling variations are indicated in parentheses. Homonyms are repeated as often as they occur (e.g., D, D, d). Anonymous phages are indicated with "NN" next to their genus and their numbers are provided in parentheses.

Bacteria of the genus Actinomyces can be infected by the following phages: Av-I, Av-2, Av-3, BF307, CTl, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.

Bacteria of the genus Aeromonas can be infected by the following phages: AA-I, Aeh2, N, PMl, TP446, 3, 4, 11, 13, 29, 31, 32, 37, 43, 43 -10T, 51, 54, 55R.1, 56, 56RR2, 57, 58, 59.1, 60, 63, Aehl, F, PM2, 1, 25, 31, 40RR2.8t, (syn= 44R), (syn= 44RR2.8t), 65, PM3, PM4, PM5 and PM6.

Bacteria of the genus Bacillus can be infected by the following phages: A, aizl, Al-K-I, B, BCJAl, BCl, BC2, BLLl, BLl, BP142, BSLl, BSL2, BSl, BS3, BS8, BS15, BS18, BS22, BS26, BS28, BS31, BS104, BS105, BS106, BTB, B1715V1, C, CK-I, Coll, Corl, CP-53, CS-I, CSi, D, D, D, D5, entl, FP8, FP9, FSi, FS2, FS3, FS5, FS8, FS9, G, GH8, GT8, GV-I, GV-2, GT-4, g3, gl2, gl3, gl4, gl6, gl7, g21, g23, g24, g29, H2, kenl, KK-88, Kuml, Kyul, J7W-1, LP52, (syn= LP-52), L7, Mexl, MJ-I, mor2, MP-7, MPlO, MP12, MP14, MP15, Neol, N°2, N5, N6P, PBCl, PBLA, PBPl, P2, S-a, SF2, SF6, Shal, Sill, SP02, (syn= ΦSPP1), SPβ, STI, STi, SU-Il, t, TbI, Tb2, Tb5, TbIO, Tb26, Tb51, Tb53, Tb55, Tb77, Tb97, Tb99, Tb560, Tb595, Td8, Td6, Tdl5, TgI, Tg4, Tg6, Tg7, Tg9, TgIO, TgIl, Tgl3, Tgl5, Tg21, Tinl, Tin7, Tin8, Tinl3, Tm3, Tocl, Togl, toll, TP-I, TP-10vir, TP-15c, TP-16c, TP-17c, TP-19, TP35, TP51, TP-84, Tt4, Tt6, A type, B type, C type, D type, E type, Tφ3, VA-9, W, wx23, wx26, Yunl, α, γ, pll, φmed-2, φT, φμ-4, φ3T , φ75, φlO5, (syn= φlO5), IA, IB, 1-97A, 1-97B, 2, 2, 3, 3, 3, 5, 12, 14, 20, 30, 35, 36, 37, 38 , 41C, 51, 63, 64, 138D, I, II, IV, NN-Bacillus (13), alel, ARl, AR2, AR3, AR7, AR9, Bace-11, (syn= 11), Bastille, BLl, BL2, BL3, BL4, BL5, BL6, BL8, BL9, BP124, BS28, BS80, Ch, CP-51, CP-54, D-5, darl, denl, DP-7, entl, FoSi, FoS2, FS4, FS6, FS7, G, gall, gamma, GEl, GF-2, GSi, GT-I, GT-2, GT-3, GT-4, GT-5, GT-6, GT-7, GV-6, gl5, 19, 110, ISi, K, MP9, MP13, MP21, MP23, MP24, MP28, MP29, MP30, MP32, MP34, MP36, MP37, MP39, MP40, MP41, MP43, MP44, MP45, MP47, MP50, NLP-I, No.l, N17, N19, PBSl, PKl, PMBl, PMB12, PMJl, S, SPOl, SP3, SP5, SP6, SP7, SP8, SP9, SPlO, SP-15, SP50, (syn= SP -50), SP82, SST, subl, SW, Tg8, Tgl2, Tgl3, Tgl4, thul, thuΛ, thuS, Tin4, Tin23, TP-13, TP33, TP50, TSP-I, V type, VI type, V, Vx, β22, φe, φNR2, φ25, φ63, 1, 1, 2, 2C, 3NT, 4, 5, 6, 7, 8, 9, 10, 12, 12, 17, 18, 19, 21, 138, III, 4 (b. Megateriwn (B. megateriwn)), 4 (B. sphaericus) (B. sphaericus)), AR13, BPP-IO, BS32, BS107, Bl, B2, GA-I, GP-IO, GV-3 , GV-5, g8, MP20, MP27, MP49, Nf, PP5, PP6, SF5, Tgl8, TP-I, Versailles, φl5, φ29, 1-97, 837/IV, mi-Bacillus(1), BatlO, BSLlO, BSLIl, BS6, BSIl, BS16, BS23, BSlOl, BS102, gl8, morl, PBLl, SN45, thu2, thu3, TmI, Tm2, TP-20, TP21, TP52, F type, G type, IV type , HN-BacMus(3), BLE, (syn= θc), BS2, BS4, BS5, BS7, BlO, B12, BS20, BS21, F, MJ-4, PBA12, AP50, AP50-04, AP50-11, AP50-23, AP50-26, AP50-27 and Bam35. The following Bacillus-specific phages are defective: DLP10716, DLP-11946, DPB5, DPB12, DPB21, DPB22, DPB23, GA-2, M, No. IM, PBLB, PBSH, PBSV, PBSW, PBSX, PBSY, PBSZ, phi, SPa, type 1 and μ.

Bacteria of the genus Bacteroides can be infected by the following phages: ad I2, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, Fl, βl, φAl , φBrOl, φBrO2, 11, 67.1, 67.3, 68.1, mt-Bacteroides (3), Bf42, Bf71, HN-Bdellovibrio (1) and BF-41.

Bacteria of the genus Bordetella can be infected by the phages: 134 and NN-Bordetella (3).

Bacteria of the genus Borrelia can be infected by the following phages: NN-Borrelia (1) and NN-Borrelia (2).

Bacteria of the genus Brucella can be infected by the following phages: A422, Bk, (syn= Berkeley), BM29, FOi, (syn= FOl), (syn= FQl), D, FP2, (syn= FP2 ), (syn= FD2), Fz, (syn= Fz75/13), (syn= Florence 75/13), (syn= Fi), Fi, (syn= Fl), Fim, (syn= FIm), ( syn= Fim), FiU, (syn= FlU), (syn= FiU), F2, (syn= F2), F3, (syn= F3), F4, (syn= F4), F5, (syn= F5) , F6, F7, (syn= F7), F25, (syn= F25), (syn= ￡25), F25U, (syn= F25u), (syn= F25U), (syn= F25V), F44, (syn - F44), F45, (syn= F45), F48, (syn= F48), I, Im, M, MC/75, M51, (syn= M85), P, (syn= D), S708, R, Tb, (syn= TB), (syn= Tbilisi), W, (syn= Wb), (syn= Waveridge), X, 3, 6, 7, 10/1, (syn= 10), (syn= F8), (syn= F8), 12m, 24/11, (syn= 24), (syn= F9), (syn= F9), 45/111, (syn= 45), 75, 84, 212/XV , (syn = 212), (syn = Fi0), (syn = Fl0), 371/XXIX, (syn = 371), (syn = Fn), (syn = Fl l) and 513.

Bacteria of the genus Burkholderia can be infected by the following phages: CP75, NN-Burkholderia (1) and 42.

Bacteria of the genus Campylobacter can be infected by the following phages: Type C, NTCC12669, NTCC12670, NTCC12671, NTCC12672, NTCC12673, NTCC12674, NTCC12675, NTCC12676, NTCC12677, NTCC12678, NTCC12679, NTCC12680 , NTCC12681, NTCC12682, NTCC12683 , NTCC12684, 32f, 111c, 191, NN-campylobacter (2), Vfi-6, (syn= V19), VfV-3, V2, V3, V8, V16, (syn= Vfi-1), V19, V20 (V45), V45, (syn=V-45) and NN-campylobacter (1).

Bacteria of the genus Chlamydia can be infected by the phage: Chpl.

Bacteria of the genus Clostridium can be infected by destroying phages: CAKl, CA5, Ca7, CEβ, (syn= 1C), CEγ, Cldl, c-n71, c-203 Tox-, DEβ, (syn = ID), (syn= lDt0X+), HM3, KMl, KT, Ms, NAl, (syn= Naltox+), PA135Oe, Pfo, PL73, PL78, PL81, Pl, P50, P5771, P19402, lCt0X+, 2Ct0X\2D3 ( syn= 2Dt0X+), 3C, (syn= 3Ctox+), 4C, (syn= 4Ct0X+), 56, III-l, NN-Clostridium (61), NBlt0X+, αl, CAl, HMT, HM2, PFl5 P-23 , P-46, Q-05, Q-oe, Q-16, Q-21, Q-26, Q-40, Q-46, S111, SA02, WA01, WA03, Wm, W523, 80, C, CA2 , CA3, CPTl, CPT4, cl, c4, c5, HM7, H11/A1, H18/Ax, FWS23, Hi58ZA1, K2ZA1, K21ZS23, ML, NA2t0X; Pf2, Pf3, Pf4, S9ZS3, S41ZA1, S44ZS23, α2, 41, 112ZS23, 214/S23, 233/Ai, 234/S23, 235/S23, II-l, II-2, II-3, NN-Clost Iridium (12), CAl, Fl, K, S2, 1, 5 and NN-Clostridium (8).

Bacteria of the genus Corynebacterium can be infected by the following phages: CGKl (defective), A, A2, A3, AlOl, A128, A133, A137, A139, A155, A182, B, BF, B17 , B18, B51, B271, B275, B276, B277, B279, B282, C, capi, CCl, CGl, CG2, CG33, CL31, Cog, (syn= CG5), D, E, F, H, H-I, hqi , hq2, 11ZH33, Ii/31, J, K, K, (syn= Ktox"), L, L, (syn= Ltox+), M, MC-I, MC-2, MC-3, MC-4, MLMa, N, O, ovi, ov2, ov3, P, P, R, RP6, RS29, S, T, U, UB1, ub2, UH1, UH3, uh3, uh5, uh6, β, (syn= βtox+), βhv64, βvir, γ, (syn= γtoχ-), γl9, δ, (syn= δ'ox+), p, (syn= ptoχ-), Φ9, φ984, ω, IA, 1/1180, 2, 2/ 1180, 5/1180, 5ad/9717, 7/4465, 8/4465, 8ad/10269, 10/9253, 13Z9253, 15/3148, 21/9253, 28, 29, 55, 2747, 2893, 4498 and 5848.

Bacteria of the genus Enterococcus can be infected by the following phages: DF78, Fl, F2, 1, 2, 4, 14, 41, 867, Dl, SB24, 2BV, 182, 225, C2, C2F, E3, E62, DS96, H24, M35, P3, P9, SBlOl, S2, 2BII, 5, 182a, 705, 873, 881, 940, 1051, 1057, 21096C, NN-Enterococcus (1), PEl, Fl , F3, F4, VD13, 1, 200, 235 and 341.

Bacteria of the genus Erysipelothrix can be infected by the phage: NN-Acipelotrix (1).

Bacteria of the genus Escherichia can be infected by the following phages: BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, (syn= mu), (syn= MuI), (syn= Mu-I), (syn= MU-I), (syn= MuI), (syn= μ), 025, PhI- 5, Pk, PSP3, Pl, PlD, P2, P4 (defective), Sl, Wφ, φK13, φR73 (defective), φl, φ2, φ7, φ92, ψ (defective), 7 A, 8φ, 9φ , 15 (defective), 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM, C4, C16, DD-VI, (syn= Dd-Vi), (syn= DDVI ), (syn= DDVi), E4, E7, E28, FIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND- 7, Ox-I (syn= OXl), (syn= HF), Ox-2 (syn= 0x2), (syn= 0X2), Ox-3, Ox-4, Ox-5, (syn= 0X5), Ox-6, (syn= 66F), (syn= φ66t), (syn= φ66t-)5 0111, PhI-I, RB42, RB43, RB49, RB69, S, SaI-I, Sal-2, Sal-3 , Sal-4, Sal-5, Sal-6, TC23, TC45, TuII*-6, (syn= TuII*), TuIP-24, TuII*46, TuIP-60, T2, (syn= ganuTia), ( syn= γ), (syn= PC), (syn= P.C.), (syn= T-2), (syn= T2), (syn= P4), T4, (syn= T-4), (syn= T4), T6, T35, αl, 1, IA, 3, (syn= Ac3), 3A, 3T+, (syn= 3), (syn= Ml), 5φ, (syn= φ5), 9266Q, CFO103, HK620 , J, K, KlF, m59, no. A, no. E, no. 3, no. 9, N4, sd, (syn= Sd), (syn= SD), (syn= Sa)3 (syn= sd), (syn= SD), (syn= CD), T3, (syn= T-3 ), (syn= T3), T7, (syn= T-7), (syn= T7), WPK, W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, φ04-CF, φ05, φ06, φ07, φl, φl.2, φ20, φ95, φ263, φlO92, φl, φll, (syn=φW), Ω8, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W, NN-Escherichia (1), Esc-7-11, AC30, CVX-5, Cl, DDUP, ECl, EC2, E21, E29, Fl, F26S, F27S, Hi , HK022, HK97, (syn= ΦHK97), HK139, HK253, HK256, K7, ND-I, no.D, PA-2, q, S2, Tl, (syn= α), (syn= P28), ( syn= T-I), (syn= Tx), T3C, T5, (syn= T-5), (syn= T5), UC-I, w, β4, γ2, λ (syn= lambda), (syn= Φλ ), φD326, φγ, φ06, φ7, φ10, φ80, χ, (syn= χi), (syn= φχ), (syn= φχi), 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, KlO, ZG/3A, 5, 5A, 21EL, H19-J and 933H.

Bacteria of the genus Fusobacterium can be infected by the following phages: NN-Fusobacterium (2), fv83-554/3, fv88-531/2, 227, fv2377, fv2527 and fv8501.

Bacteria of the genus Haemophilus can be infected by the following phages: HPl, S2 and N3.

Bacteria of the genus Helicobacter can be infected by the following phages: HPl and ^^-Helicobacter (1).

Bacteria of the genus Klebsiella can be infected by the following phages: AIO-2, KI4B, Kl6B, Kl9, (syn=K19), Kl14, Kl15, Kl21, Kl28, Kl29, KI32, Kl33, Kl35, Kl106B, Kl171B, Kl181B, Kl832B, AIO-I, AO-I, AO-2, AO-3, FC3-10, K, Kl1, (syn= KIl), Kl2, (syn= K12), Kl3, (syn = K13), (syn= Kl 70/11), Kl4, (syn= K14), Kl5, (syn= K15), Kl6, (syn= K16), Kl7, (syn= K17), Kl8, (syn= Kl18), Kl19, (syn= K19), Kl27, (syn= K127), Kl31, (syn= K131), Kl35, Kl171B, II, VI, IX, CI-I, Kl4B, Kl8, Kl11, Kl12, Kl13 , Kl16, Kl17, Kl18, Kl20, Kl22, Kl23, Kl24, Kl26, Kl30, Kl34, Kl106B, KIi65B, Kl328B, KLXI, K328, P5046, 11, 380, III, IV, VII, VIII, FC3-11, Kl2B , (syn= K12B), Kl25, (syn= K125), Kl42B, (syn= K142), (syn= K142B), Kl181B, (syn= K181), (syn= K1181B), Kl765/!, (syn = K1765/1), Kl842B, (syn= K1832B), Kl937B, (syn= K1937B), Ll, φ28, 7, 231, 483, 490, 632 and 864/100.

Bacteria of the genus Lepitospira can be infected by the following phages: LEl, LE3, LE4 and ~NN-Leptospira (1).

Bacteria of the genus Listeria can be infected by the following phages: A511, 01761, 4211, 4286, (syn= BO54), A005, A006, A020, A500, A502, A511, Al 18, A620, A640, B012 ; H107, H108, HI lO, H163/84, H312, H340, H387, H391/73, H684/74, H924A, PSA, U153, φMLUP5, (syn= P35), 00241, 00611, 02971A, 02971C, 5/476 5/911, 5/939, 5/11302, 5/11605, 5/11704, 184, 575, 633, 699/694, 744, 900, 1090, 1317, 1444, 1652, 1806, 1807, 1921/959 ; 4277, 4292, 4477, 5337, 5348 /11363, 5348/11646, 5348/12430, 5348/12434, 10072, 11355C, 11711A, 12029, 12981, 13441, 90666, 90816, 93253, 907515, 91071 6 and NN-Listeria (15).

Bacteria of the genus Morganella can be infected by the following phage: 47.

Bacteria of the genus Mycobacterium can be infected by the following phages: 13, AGl, ALi, ATCC 11759, A2, B.C3, BG2, BKl, BK5, Butyricum, B-I, B5, B7, B30 , B35, Clark, Cl, C2, DNAIII, DSP1, D4, D29, GS4E, (syn= GS4E), GS7, (syn= GS-7), (syn= GS7), IPa, Lacticola, Legendre, Leo , L5, (syn= ΦL-5), MC-I, MC-3, MC-4, Minetti, MTPHI l, Mx4, MyF3P/59a, phlei, (syn= phlei 1), phlei 4, Polonus II , Rabinowitz, Smegmatis, TM4, TM9, TMlO, TM20, Y7, YlO, φ630, IB, IF, IH, 1/1, 67, 106, 1430, Bl, (syn=Bol), B24, D , D29, F-K, F-S, HP, Polonus I, Roy, Rl, (syn= Rl-Myb), (syn= Ri), 11, 31, 40, 50, 103a, 103b, 128, 3111-D, 3215 -D and NN-mycobacterium (1).

Bacteria of the genus Neisseria can be infected by the following phages: Group I, Group II and NPl.

Bacteria of the genus Nocardia can be infected by the following phages: MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.

Bacteria of the genus Proteus can be infected by the following phages: Pm5, 13vir, 2/44, 4/545, 6/1004, 13/807, 20/826, 57, 67b, 78, 107/69, 121, 9/0, 22/608, 30/680, PmI, Pm3, Pm4, Pm6, Pm7, Pm9, PmIO, PmI l, Pv2, πl, φm, 7/549, 9B/2, 10A/31, 12 /55, 14, 15, 16/789, 17/971, 19A/653, 23/532, 25/909, 26/219, 27/953, 32A/909, 33/971, 34/13, 65, 5006M , 7480b, VI, 13/3a, Clichy 12, π2600, φχ7, 1/1004, 5/742, 9, 12, 14, 22, 24/860, 2600/D52, Pm8 and 24/2514.

Bacteria of the genus Providencia can be infected by the following phages: PL25, PL26, PL37, 9211/9295, 9213/921 Ib, 9248, 7/R49, 7476/322, 7478/325, 7479, 7480 , 9000/9402 and 9213/921 Ia.

Bacteria of the genus Pseudomonas can be infected by the following phages: PfI, (syn= Pf-I), Pf2, Pf3, PP7, PRRl, 7s, im-Pseudomonas (1), AI-I, AI-2 , B 17, B89, CB3, Col 2, Col 11, Col 18, Col 21, C154, C163, C167, C2121, E79, F8, ga, gb, H22, K1, M4, N2, Nu, PB-I, (syn= PBl), pfl6, PMN17, PPl, PP8, Psal, PsPl, PsP2, PsP3, PsP4, PsP5, PS3, PS17, PTB80, PX4, PX7, PYOl, PYO2, PYO5, PYO6, PYO9, PYOlO, PYO13, PYO14, PYO16, PYO18, PYO19, PYO20, PYO29, PYO32, PYO33, PYO35, PYO36, PYO37, PYO38, PYO39, PYO41, PYO42, PYO45, PYO47, PYO48, PYO64, PYO69, PYO103, PlK, SLPl, SL2, S2, UNL-I, wy, Yai, Ya4, Yan, φBE, φCTX, φC17, φKZ, (syn=φKZ), φ-LT, φmu78, φNZ, φPLS-1, φST-1, φW-14, φ-2, 1/72, 2/79, 3, 3/DO, 4/237, 5/406, 6C, 6/6660, 7, 7v, 7/184, 8/280, 9/95, 10/502, 11/ DE, 12/100, 12S, 16, 21, 24, 25F, 27, 31, 44, 68, 71, 95, 109, 188, 337, 352, 1214, NN-Pseudomonas (23), A856, B26, CI -I, CI-2, C5, D, gh-1, Fl 16, HF, H90, K5, K6, Kl 04, K109, K166, K267, N4, N5, O6N-25P, PE69, Pf, PPN25, PPN35 , PPN89, PPN91, PP2, PP3, PP4, PP6, PP7, PP8, PP56, PP87, PPl 14, PP206, PP207, PP306, PP651, Psp231a, Pssy401, Pssy9220, psi, PTB2, PTB20, PTB42, PXl, PX3, PXlO, PX12, PX14, PYO70, PYO71, R, SH6, SH133, tf, Ya5, Ya7, φBS, φKf77, φ-MC, φmnF82, φPLS27, φPLS743, φS-1, 1, 2, 2, 3, 4, 5, 6, 7, 7, 8, 9, 10, 11, 12, 12B, 13, 14, 15, 14, 15, 16, 17, 18, 19, 20, 20, 21, 21, 22, 23, 23, 24, 25, 31, 53, 73, 119x, 145, 147, 170, 267, 284, 308, 525, NN-Pseudomonas (5), af, A7, B3, B33, B39, BI-I, C22 , D3, D37, D40, D62, D3112, F7, FlO, g, gd, ge, gξHwl2, Jb 19, KFl, L°, OXN-32P, O6N-52P, PCH-I, PC13-1, PC35-1 , PH2, PH51, PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PMl 13, PM681, PM682, PO4, PPl, PP4, PP5, PP64, PP65, PP66, PP71, PP86, PP88, PP92, PP401, PP711, PP891, Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4, PS-IO, Pz, SDl, SLl, SL3, SL5, SM, φC5, φCll, φCll-1, φC13, φC15, φMO, φX, φO4, φll, φ240, 2, 2F, 5, 7m, 11, 13, 13/441, 14, 20, 24, 40, 45, 49, 61, 73, 148, 160, 198, 218, 222, 236, 242, 246, 249, 258, 269, 295, 297, 309, 318, 342, 350, 351, 357-1, 400-1, HN-Pseudomonas (6), GlOl, M6, M6a , Ll, PB2, Pssyl5, Pssy4210, Pssy4220, PYO12, PYO34, PYO49, PYO50, PYO51, PYO52, PYO53, PYO57, PYO59, PYO200, PX2, PX5, SL4, φO3, φO6 and 1214.

Bacteria of the genus Rickettsia can be infected by the phage: NN-Rickettsia.

Bacteria of the genus Salmonella can be infected by the following phages: b, Beccles, CT, d, Dundee, f, FeIs 2, GI, GUI, GVI, GVIII, k, K, i, j, L, 01, (syn= 0-1), (syn= O1), (syn= O-I), (syn= 7), 02, 03, P3, P9a, PlO, Sab3, Sab5, SanlS, Sanl7, SI, Towntone , ViI, (syn= ViI), 9, im Salmonella (1), N-I, N-5, N-IO, N-17, N-22, 11, 12, 16-19, 20.2, 36, 449C/C178 , 966A/C259, a, B.A.O.R., e, G4, GUI, L, LP7, M, MG40, N-18, PSA68, P4, P9c, P22, (syn= P22), (syn= PLT22), (syn= PLT22), P22al, P22-4, P22-7, P22-11, SNT-I, SNT-2, SP6, Villi, ViIV, ViV, ViVI, ViVII, Worksop, Sj5, ε34, 1,37, 1( 40), (syn= φl[40]), 1,422, 2, 2.5, 3b, 4, 5, 6,14(18), 8, 14(6,7), 10, 27, 28B, 30, 31, 32, 33, 34, 36, 37, 39, 1412, SNT-3, 7-11, 40.3, c, C236, C557, C625, C966N, g, GV, G5, Gl 73, h, IRA, Jersey, MB78 , P22-1, P22-3, P22-12, Sabl, Sab2, Sab2, Sab4, Sanl, San2, San3, San4, San6, San7, San8, San9, Sanl3, Sanl4, Sanl6, Sanl8, Sanl9, San20, San21 , San22, San23, San24, San25, San26, SasLl, SasL2, SasL3, SasL4, SasL5, SlBL, SII, ViII, φl, 1, 2, 3a, 3al, 1010, Ym-salmonella (1), N-4, SasL6 and 27.

Bacteria of the genus Serratia can be infected by the following phages: A2P, PS20, SMB3, SMP, SMP5, SM2, V40, V56, ic, ΦCP-3, ΦCP-6, 3M, 10/la, 20A , 34CC, 34H, 38T, 345G, 345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232, L2232, L34, L.228, SLP, SMPA, V.43, σ, φCWl , ΦCP6-1, ΦCP6-2, ΦCP6-5, 3T, 5, 8, 9F, 10/1, 2OE, 32/6, 34B, 34CT, 34P, 37, 41, 56, 56D, 56P, 6OP, 61 /6, 74/6, 76/4, 101/8900, 226, 227, 228, 229F, 286, 289, 290F, 512, 764a, 2847/10, 2847/1Oa, L.359 and SMBl.

Bacteria of the genus Shigella can be infected by the following phages: Fsa, (syn=a), FSD2d, (syn= D2d), (syn= W2d), FSD2E, (syn= W2e), fv, F6 , f7.8, H-Sh, PE5, P90, SfII, Sh, SHm, SHrv, (syn= HIV), SHvi, (syn= HVI), SHVvm, (syn= HVIII), SKγ66, (syn= gamma 66 ), (syn=yββ), (syn=γ66b), SKm, (syn=SIIIb)5 (syn=UI), SKw, (syn=Siva), (syn=IV), SIC™, (syn=SIVA. ), (syn= IVA), SKvi, (syn= KVI), (syn= Svi), (syn= VI), SKvm, (syn= Svm), (syn= VIII), SKVÐIA, (syn= SvmA), (syn= VIIIA), STvi, STK, STx1, STxn, S66, W2, (syn= D2c), (syn= D20), φl, φIVb 3-SO-R, 8368-SO-R, F7, (syn= FS7), (syn= K29), FlO, (syn= FSlO), (syn= K31), I1, (syn= alpha), (syn= FSa), (syn= Kl 8), (syn= α), I2, (syn= a), (syn= K19), SG33, (syn= G35), (syn= SO-35/G), SG35, (syn= SO-55/G), SG3201, (syn= SO -3201/G), SHn, (syn= HII), SHv, (syn= SHV), SHx, SHX, SKn, (syn= K2), (syn= KII), (syn= Sn), (syn= SsII ), (syn= II), SKrv, (syn= Sm), (syn= SsIV), (syn= IV), SK1Va, (syn= Swab), (syn= SsIVa), (syn= IVa), SKV, (syn= K4), (syn= KV), (syn= SV), (syn= SsV), (syn= V), SKx, (syn= K9), (syn= KX), (syn= SX), (syn= SsX), (syn= X), STV, (syn= T35), (syn= 35-50-R), STvm, (syn= T8345), (syn= 8345-SO-S-R), W1, (syn= D8), (syn= FSD8), W2a, (syn= D2A), (syn= FS2a), DD-2, Sf6, FSi, (syn= Fl), SF6, (syn= F6), SG42, (syn= SO-42/G), SG3203, (syn= SO-3203/G), SKF12, (syn= SsF12), (syn= F12), (syn= F12), STn, (syn= 1881-SO -R), γ66, (syn= gamma 66a), (syn= Ssγ66), φ2, BIl, DDVII, (syn= DD7), FSD2b, (syn= W2B), FS2, (syn= F2), (syn= F2), FS4, (syn= F4), (syn= F4), FS5, (syn= F5), (syn= F5), FS9, (syn= F9), (syn= F9), FI l, P2- S0-S, SG36, (syn= SO-36/G), (syn= G36), SG3204, (syn= SO-3204/G), SG3244, (syn= SO-3244/G), SHi, (syn = HI), SHvπ, (syn= HVII), SHK, (syn= HIX), SHx1, SHxπ, (syn= HXn), SKI, KI, (syn= S1), (syn= SsI), SKVII, (syn = KVII), (syn= Svπ), (syn= SsVII), SKIX, (syn= KIX), (syn= S1x), (syn= SsIX), SKXII, (syn= KXII), (syn= Sxn), (syn= SsXII), STi, STffl, STrv, STVi, STvπ, S70, S206, U2-S0-S, 3210-SO-S, 3859-SO-S, 4020-SO-S, φ3, φ5, φ7, φ8, φ9, φlO, φl l, φl3, φl4, φl8, SHm, (syn= Hði), SHχi, (syn= HXt) and SKxI, (syn= KXI), (syn= Sχi), (syn= SsXI) , (syn = XI).

Bacteria of the genus Staphylococcus can be infected by the following phages: A, EW, K, Ph5, Ph9, PhIO, Phl3, Pl, P2, P3, P4, P8, P9, PlO, RG, SB -i, (syn= Sb-I), S3K, twort, φSK311, φ812, 06, 40, 58, 119, 130, 131, 200, 1623, STCl, (syn=stcl), STC2, (syn=stc2 ), 44AHJD, 68, ACl, AC2, A6"C", A9"C", b581, CA-I, CA-2, CA-3, CA-4, CA-5, DI l, L39x35, L54a, M42 , Nl, N2, N3, N4, N5, N7, N8, NlO, Ni l, N12, N13, N14, N16, Ph6, Phl2, Phl4, UC-18, U4, U15, Sl, S2, S3, S4, S5, X2, Z1, φB5-2, φD, ω, 11, (syn= φl l), (syn= P11-M15), 15, 28, 28A, 29, 31, 31B, 37, 42D, (syn= P42D), 44A, 48, 51, 52, 52A, (syn= P52A), 52B, 53, 55, 69, 71, (syn= P71), 71A, 72, 75, 76, 77, 79, 80, 80α , 82, 82A, 83 A, 84, 85, 86, 88, 88A, 89, 90, 92, 95, 96, 102, 107, 108, 111, 129-26, 130, 130A, 155, 157, 157A, 165, 187, 275, 275A, 275B, 356, 456, 459, 471, 471A, 489, 581, 676, 898, 1139, 1154A, 1259, 1314, 1380, 1405, 1563, 2148, 2638A, 2638B, 2638C, 2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841, 12100, AC3, A8, AlO, A13, b594n, D, HK2, N9, N15, P52, P87, Sl, S6, Z4, φRE, 3A, 3B, 3C, 6, 7, 16, 21, 42B, 42C, 42E, 44, 47, 47A5 47C, 51, 54, 54x1, 70, 73, 75, 78, 81, 82, 88, 93, 94, 101 , 105, 110, 115, 129/16, 174, 594n, 1363/14, 2460 and mS-Staphylococcus (1).

Bacteria of the genus Streptococcus can be infected by the following phages: EJ-I, NN-Streptococcus (1), a, Cl, FL0Ths, H39, Cp-I, Cρ-5, Cp-7 , Cp-9, Cp-IO, AT298, A5, alO/Jl, alO/J2, alO/J5, alO/J9, A25, BTIl, b6, CAl, c20-l, c20-2, DP-I, Dp-4, DTl, ET42, elO, FA101, FEThs, Fκ, FKKIOI, FKLIO, FKP74, FKH, FLOThs, FyIOl, fl, F10, F20140/76, g, GT-234, HB3, (syn= HB-3 ), HB-623, HB-746, M102, O1205, φO1205, PST, PO, Pl, P2, P3, P5, P6, P8, P9, P9, P12, P13, P14, P49, P50, P51, P52, P53, P54, P55, P56, P57, P58, P59, P64, P67, P69, P71, P73, P75, P76, P77, P82, P83, P88, sc, sch, sf, SfIl 1, (syn= SFiI l ), (syn= φSFill), (syn= φSfil l), (syn= φSfil l), sfil9, (syn= SFil9), (syn= φSFil9), (syn= φSfil9), Sfi21, (syn= SFi21), (syn= φSFi21), (syn= φSfi21), ST0, STX, st2, ST2, ST4, S3, (syn= φS3), s265, φ17, φ42, φ57, φ80, φ81, φ82, φ83, φ84, φ85, φ86, φ87, φ88, φ89, φ90, φ91, φ92, φ93, φ94, φ95, φ96, φ97, φ98, φ99, φlOO, φlOl, φlO2, φ227, φ7201, ωl, ω2, ω3, ω4, ω5, ω 6, ω8, ωIO, 1, 6, 9, 1OF, 12/12, 14, 17SR, 19S, 24, 50/33, 50/34, 55/14, 55/15, 70/35, 70/36, 71/ ST15, 71/45, 71/46, 74F, 79/37, 79/38, 80/J4, 80/J9, 80/ST16, 80/15, 80/47, 80/48, 101, 103/39, 103/40, 121/41, 121/42, 123/43, 123/44, 124/44, 337/ST17 and mStreptococcus (34).

Bacteria of the genus Treponema can be infected by the phage: NN-Treponema (1).

Bacteria of Vibrio can be infected by the following phages: CTXΦ, fs, (syn=si), fs2, Ivpf5, Vfl2, Vf33, VPIΦ, VSK, v6, 493, CP-Tl, ET25, kappa, K139, Lavol), XN-69P, OXN-86, O6N-21P, PB-I, P147, rp-1, SE3, VA-I, (syn= VcA-I), VcA-2, VPl, VP2, VP4 ; ΦHAWI-7, XHAWI-8, ΦHAWI-9, ΦHAWI-10, ΦHCl-1, ΦHC1-2, ΦHC1-3, ΦHC1-4, ΦHC2-1, >HC2-2, ΦHC2-3, ΦHC2-4, ΦHC3 -1, ΦHC3-2, ΦHC3-3, ΦHD1S-1, ΦHD1S-2, ΦHD2S-1, ΦHD2S-2, ΦHD2S-3, ΦHD2S-4, ΦHD2S-5, ΦHDO-1, ΦHDO-2, ΦHDO-3 , ΦHDO-4, ΦHDO-5, ΦHDO-6, ΦKL-33, ΦKL-34, ΦKL-35, ΦKL-36, ΦKWH-2, ΦKWH-3, ΦKWH-4, ΦMARQ-1, ΦMARQ-2, ΦMARQ -3, ΦMOAT-1, ΦO139, ΦPEL1A-1, ΦPEL1A-2, ΦPEL8A-1, ΦPEL8A-2, ΦPEL8A-3, ΦPEL8C-1, ΦPEL8C-2, ΦPEL13A-1, ΦPEL13B-1, ΦPEL13B-2, ΦPEL13B -3, φPEL13B-4, φPEL13B-5, φPEL13B-6, φPEL13B-7, φPEL13B-8, φPEL13B-9, φPEL13B-10, φVP143, φVP253, φ16, φl38, 1-II, 5, 13, 14, 16 , 24, 32, 493, 6214, 7050, 7227, II, (syn=group II), (syn== φ2), V, VIII, ~m-Vibrio (13), KVP20, KVP40, nt-1, O6N -22P, P68, el, e2, e3, e4, e5, FK, G, I, K, nt-6, Nl, N2, N3, N4, N5, O6N-34P, OXN-72P, OXN-85P, OXN -100P, P, Ph-I, PL163/10, Q, S, T, φ92, 1-9, 37, 51, 57, 70A-8, 72A-4, 72A-10, 110A-4, 333, 4996 , I (syn = group I), III (syn = group III), VI, (syn = A-Saratov), VII, IX, X, HN-Vibrio (6), pAl, 7, 7-8, 70A -2, 71A-6, 72A-5, 72A-8, 108A-10, 109A-6, 109A-8, lLOA-1, 110A-5, 110A-7, hv-1, OXN-52P, P13, P38, P53, P65, P108, Pill, TPl3 VP3, VP6, VP12, VP13, 70A-3, 70A-4, 70A-10, 72A-1, 108A-3, 109-B1, 110A-2, 149, ( syn=φl49), IV, (syn=group IV), NN-Vibrio (22), VP5, VPIl, VP15, VP16, αl, α2, α3a, α3b, 353B and HN-Vibrio (7).

Bacteria of the genus Yersinia can be infected by the following phages: H, HI, H-2, H-3, H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7, YerA20, YerA41, 3/M64-76, 5/G394-76, 6/C753-76 , 8/C239-76, 9/F18167, 1701, 1710, PST, 1/F2852-76, Thererell, EV, H, Kotljarova, PTB, R, Y, YerA41, φYerO3-12, 3, 4/C1324-76 , 7/F783-76, 903, 1/M6176 and Yer2AT.

In one embodiment, the bacteriophage is selected from the group consisting of Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya virus RC2014, Escherichia virus CBA120, Escherichia virus PhaxI, Salmonella virus 38 , Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus ECML4, Salmonella virus Marshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonella virus STML131, Salmonella virus ViI, Erwinia virus Ea2809, Klebsiella virus 0507KN21, Serratia virus IME250, Serratia virus MAM1, Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virus CPt10, Campylobacter virus IBB35, Campylobacter Virus CP81, Campylobacter virus CP30A, Campylobacter virus CPX, Campylobacter virus NCTC12673, Awinia virus Ea214, Irwinia virus M7, Escherichia virus AYO145A, Escherichia virus EC6, Escherichia virus HY02, S Escherichia Virus JH2, Escherichia Virus TP1, Escherichia Virus VpaE1, Escherichia Virus wV8, Salmonella Virus FelixO1, Salmonella Virus HB2014, Salmonella Virus Mushroom, Salmonella Virus UAB87, Citrobacter Virus Moogle, Citrobacter Virus Mor Dean, Escherichia virus SUSP1, Escherichia virus SUSP2, Aeromonas virus phiO18P, Haemophilus virus HP1, Haemophilus virus HP2, Pasteurella virus F108, Vibrio virus K139, Vibrio virus kappa, Burkholderia Virus phi52237, Burkholderia virus phiE122, Burkholderia virus phiE202, Escherichia virus 186, Escherichia virus P4, Escherichia virus P2, Escherichia virus Wphi, Mannheimia virus PHL101, Pseudomonas virus phiCTX, Rall Stonia Virus RSA1, Salmonella Virus Fels2, Salmonella Virus PsP3, Salmonella Virus SopEphi, Yersinia Virus L413C, Staphylococcus Virus G1, Staphylococcus Virus G15, Staphylococcus Virus JD7, Staphylococcus Virus K, Staphylococcus Virus MCE2014, Staphylococcus Virus P108, Staphylococcus Virus Rodi, Staphylococcus Virus S253, Staphylococcus Virus S25-4, Staphylococcus Virus SA12, Listeria Virus A511, Listeria Virus P100, Staphylococcus Virus Remus, Staphylococcus Virus SA11, Staphylococcus Virus Stau2, Bacillus Virus Camphawk, Bacillus Virus SPO1, Bacillus Virus BCP78, Bacillus Char Bomba, Staphylococcus Virus Twert, Entero Coccus virus phiEC24C, Lactobacillus virus Lb338-1, Lactobacillus virus LP65, Enterobacter virus PG7, Escherichia virus CC31, Klebsiella virus JD18, Klebsiella virus PKO111, Escherichia virus Bp7, Escherichia virus IME08, Escherichia virus JS10, Escherichia virus JS98, Escherichia virus QL01, Escherichia virus VR5, Enterobacter virus Eap3, Klebsiella virus KP15, Klebsiella virus KP27, Klebsiella virus matis, Kleb Ciella virus Miro, Citrobacter virus Merlin, Citrobacter virus phylum, Escherichia virus JSE, Escherichia virus phi1, Escherichia virus RB49, Escherichia virus HX01, Escherichia virus JS09, Escherichia Virus RB69, Shigella virus UTAM, Salmonella virus S16, Salmonella virus STML198, Vibrio virus KVP40, Vibrio virus nt1, Vibrio virus ValKK3, Escherichia virus VR7, Escherichia virus VR20, Escherichia virus VR25, Escherichia virus VR26, Shigella virus SP18, Escherichia virus AR1, Escherichia virus C40, Escherichia virus E112, Escherichia virus ECML134, Escherichia virus HY01, Escherichia virus Ime09, Escherichia virus RB3, S Kericchia virus RB14, Escherichia virus T4, Shigella virus Pss1, Shigella virus Shfl2, Yersinia virus D1, Yersinia virus PST, Acinetobacter virus 133, Aeromonas virus 65, Aeromonas virus Aeh1 , Escherichia virus RB16, Escherichia virus RB32, Escherichia virus RB43, Pseudomonas virus 42, Chronobacter virus CR3, Chronobacter virus CR8, Chronobacter virus CR9, Chronobacter virus PBES02, Pectobacterium virus phiTE, Chronobacter virus GAP31, Escherichia virus 4MG, Salmonella virus SE1, Salmonella virus SSE121, Escherichia virus FFH2, Escherichia virus FV3, Escherichia virus JES2013, Escherichia virus V5, Brevibacillus virus Abouo, Brevibacillus Davis, Bacillus Virus Agate, Bacillus Virus Bobb, Bacillus Virus Bp8pC, Irwinia Virus Deimos, Irwinia Virus Ea35-70, Irwinia Virus RAY, Irwinia Virus Simmy50, Irwinia Virus Special G, Acinetobacter Virus AB1, Acinetobacter virus AB2, Acinetobacter virus AbC62, Acinetobacter virus AP22, Atrobacter virus ArV1, Atrobacter virus trina, Bacillus virus Abesobmore, Bacillus virus B4, Bacillus virus bigbusha, Bacillus virus Lily, Bacillus Virus Spoke, Bacillus Virus Troll, Bacillus Virus Bastille, Bacillus Virus CAM003, Bacillus Virus Bc431, Bacillus Virus Bcp1, Bacillus Virus BCP82, Bacillus Virus BM15, Bacillus Virus Deep Blue, Bacillus Virus JBP901, Burkholderia Virus Bcep1, Burkholde Ria virus Bcep43, Burkholderia virus Bcep781, Burkholderia virus BcepNY3, Xanthomonas virus OP2, Burkholderia virus BcepMu, Burkholderia virus phiE255, Aeromonas virus 44RR2, Mycobacterium virus Alice, Mycobacter Mycobacterium Virus Bxz1, Mycobacterium Virus Dandelion, Mycobacterium Virus HyRo, Mycobacterium Virus I3, Mycobacterium Virus Tapi, Mycobacterium Virus Sebata, Clostridium Virus phiC2, Clostridium Virus phiCD27, Clostridium virus phiCD119, Bacillus virus CP51, Bacillus virus JL, Bacillus virus Shanette, Escherichia virus CVM10, Escherichia virus ep3, Irwinia virus Asecino, Irwinia virus EaH2, Pseudomonas virus EL, Halomonas Virus HAP1, Vibrio virus VP882, Brevibacillus virus Zimmer, Brevibacillus virus Osiris, Pseudomonas virus Ab03, Pseudomonas virus KPP10, Pseudomonas virus PAKP3, Cynorizobium virus M7, Cynorizobium virus M12, Cynorizobium virus N3, U Winia Virus Machina, Atrobacter Virus Brent, Atrobacter Virus Jonsky, Atrobacter Virus Marsha, Atrobacter Virus Sonni, Edwardsiella Virus MSW3, Edwardsiella Virus PEi21, Escherichia Virus Mu, Shigella Virus SfMu, Halobacterium virus phiH, Bacillus virus grass, Bacillus virus NIT1, Bacillus virus SPG24, Aeromonas virus 43, Escherichia virus P1, Pseudomonas virus CAb1, Pseudomonas virus CAb02, Pseudomonas virus JG004, Pseudomonas virus PAKP1, Pseudomonas Virus PAKP4, Pseudomonas virus PaP1, Burkholderia virus BcepF1, Pseudomonas virus 141, Pseudomonas virus Ab28, Pseudomonas virus DL60, Pseudomonas virus DL68, Pseudomonas virus F8, Pseudomonas virus JG024, Pseudomonas virus KPP12, Pseudomonas virus LBL3, Pseudomonas virus LMA2 , Pseudomonas Virus PB1, Pseudomonas virus SN, Pseudomonas virus PA7, Pseudomonas virus phiKZ, Rhizobium virus RHEph4, Ralstonia virus RSF1, Ralstonia virus RSL2, Ralstonia virus RSL1, Aeromonas virus 25, Aeromonas virus 31, Aeromonas Virus Aes12, Aeromonas Virus Aes508, Aeromonas Virus AS4, Stenotropomonas Virus IME13, Staphylococcus Virus IPLAC1C, Staphylococcus Virus SEP1, Salmonella Virus SPN3US, Bacillus Virus 1, Geobacillus virus GBSV1, Yersinia virus R1RT, Yersinia virus TG1, Bacillus virus G, Bacillus virus PBS1, Microcystis virus Ma-LMM01, Vibrio virus MAR, Vibrio virus VHML, Vibrio virus VP585, Bacillus virus BPS13, Bacillus virus Hakuna, Bacillus virus megatron, Bacillus virus Wph, Acinetobacter virus AB3, Acinetobacter virus Abp1, Acinetobacter virus Fri1, Acinetobacter virus IME200, Acinetobacter virus PD6A3, Acinetobacter virus PDAB9, Acinetobacter native virus phiAB1, Escherichia virus K30, Klebsiella virus K5, Klebsiella virus K11, Klebsiella virus Kp1, Klebsiella virus KP32, Klebsiella virus KpV289, Klebsiella virus F19, Klebsiella Virus K244, Klebsiella virus Kp2, Klebsiella virus KP34, Klebsiella virus KpV41, Klebsiella virus KpV71, Klebsiella virus KpV475, Klebsiella virus SU503, Klebsiella virus SU552A, Pantoea virus lime Light, Pantoea Virus Lyme Zero, Pseudomonas Virus LKA1, Pseudomonas Virus phiKMV, Xanthomonas Virus f20, Xanthomonas Virus f30, Xyllella Virus Prado, Awinia Virus Era103, Escherichia Virus K5, Escherichia Virus K1-5 , Escherichia virus K1E, Salmonella virus SP6, Escherichia virus T7, Cluyvera virus Kvp1, Pseudomonas virus gh1, Prochlorococcus virus PSSP7, Synecococcus virus P60, Synecococcus virus Syn5, Streptococcus virus Cp1 , Streptococcus virus Cp7, Staphylococcus virus 44AHJD, Streptococcal virus C1, Bacillus virus B103, Bacillus virus GA1, Bacillus virus phi29, Curtia virus 6, Actinomyces virus Av1, Mycoplasma virus P1, S Escherichia Virus 24B, Escherichia Virus 933W, Escherichia Virus Min27, Escherichia Virus PA28, Escherichia Virus Stx2 II, Shigella Virus 7502Stx, Shigella Virus POCJ13, Escherichia Virus 191, Escherichia Virus PA2, Escherichia Virus TL2011, Shigella Virus VASD, Burkholderia Virus Bcep22, Burkholderia Virus Bcepil02, Burkholderia Virus Bcepmigl, Burkholderia Virus DC1, Bordetella Virus BPP1, Burkholderia Virus BcepC6B, Cellulite Rophaga virus Cba41, Cellulophaga virus Cba172, Dinoroseobacter virus DFL12, Irwinia virus Ea9-2, Irwinia virus Frozen, Escherichia virus phiV10, Salmonella virus Epsilon15, Salmonella virus SPN1S, Pseudomonas virus F116 , Pseudomonas virus H66, Escherichia virus APEC5, Escherichia virus APEC7, Escherichia virus Bp4, Escherichia virus EC1UPM, Escherichia virus ECBP1, Escherichia virus G7C, Escherichia virus IME11, Shigella virus Sb1, Achromobacter virus Axp3, Acromobacter virus JWAlpha, Edwardsiella virus KF1, Pseudomonas virus KPP25, Pseudomonas virus R18, Pseudomonas virus Ab09, Pseudomonas virus LIT1, Pseudomonas virus PA26, Pseudomonas virus Ab22, Pseudomonas virus CHU, Pseudomonas virus LUZ24, Pseudomonas virus PAA2, Pseudomonas virus PaP3, Pseudomonas virus PaP4, Pseudomonas virus TL, Pseudomonas virus KPP21, Pseudomonas virus LUZ7, Escherichia virus N4, Salmonella virus 9NA, Salmonella virus SP069, Salmonella virus BTP1, Salmonella virus HK620, Salmonella virus P22, Salmonella virus ST64T, Shigella virus Sf6, Bacillus virus phage, Bacillus virus Palmer, Bacillus virus Pascal, Bacillus virus Pony, Bacillus virus Puki, Escherichia virus 172-1, Escherichia virus ECB2, Escherichia virus NJ01 , Escherichia Virus phiEco32, Escherichia Virus Septima 11, Escherichia Virus SU10, Brucella Virus Pr, Brucella Virus Tb, Escherichia Virus Pollock, Salmonella Virus FSL SP-058, Salmonella Virus FSL SP-076, Helicobacter Virus 1961P, Helicobacter Virus KHP30, Helicobacter Virus KHP40, Hamiltonella Virus APSE1, Lactococcus Virus KSY1, Formidium Virus WMP3, Formidium Virus WMP4, Pseudomonas Virus 119X, Roseobacter Virus SIO1, Vibrio Virus VpV262, Vibrio Virus VC8 , Vibrio virus VP2, Vibrio virus VP5, Streptomyces virus Amella, Streptomyces virus phiCAM, Streptomyces virus Aronocolus, Streptomyces virus Caliburn, Streptomyces virus complex or Streptomyces virus Hydra, Streptomyces virus Idge, Streptomyces virus Lannister, Streptomyces virus rica, Streptomyces virus resinidase, Streptomyces virus Gemlia, Streptomyces virus ELB20, Streptomyces virus R4, Streptomyces virus phiHau3, Mycobacterium Virus Arcadian, Mycobacterium Virus Baee, Mycobacterium Virus Reprobate, Mycobacterium Virus Adawi, Mycobacterium Virus Bane1, Mycobacterium Virus BrownCNA, Mycobac Therium Virus Crismitch, Mycobacterium Virus Cooper, Mycobacterium Virus Jamal, Mycobacterium Virus Nigel, Mycobacterium Virus Stinger, Mycobacterium Virus Vincenzo, Mycobacterium Virus Gemanar, Mycobacterium Virus Cobacterium Virus Apigeum, Mycobacterium Virus Manad, Mycobacterium Virus Olin, Mycobacterium Virus Osmaximus, Mycobacterium Virus Pg1, Mycobacterium Virus Soto, Mycobacterium Virus Count Polk, Mycobacterium Virus Athena, Mycobacterium Virus Bernardo, Mycobacterium Virus Gadget, Mycobacterium Virus Pipefish, Mycobacterium Virus Godines, Mycobacterium Virus Rosebusi, Mycobacter Mycobacterium virus Bobsiella, Mycobacterium virus Bruchita, Mycobacterium virus Che9c, Mycobacterium virus Sbash, Mycobacterium virus Hawkeye, Mycobacterium virus Plot, Salmonella virus AG11, Salmonella virus Ent1, Salmonella virus f18SE, Salmonella virus inhibitor, Salmonella virus L13, Salmonella virus LSPA1, Salmonella virus SE2, Salmonella virus SETP3, Salmonella virus SETP7, Salmonella virus SETP13, Salmonella virus SP101, Salmonella virus SS3e, Salmonella virus wksl3, Escherichia virus K1G, Escherichia virus K1H, Escherichia virus K1ind1, Escherichia virus K1ind2, Salmonella virus SP31, Leuconostoc virus Lmd1, Leuconostoc virus LN03, Leuconostoc virus LN04, Leuconostoc virus LN12, Leuconostoc virus LN6B, Leuconostoc virus P793, Leuconostoc virus 1A4, Leuconostoc virus Ln8, Leuconostoc virus Ln9, Leuconostoc virus LN25, Leuconostoc virus LN34, Leuconostoc virus LNTR3, Mycobacterium virus Bongo , Mycobacterium Virus Ray, Mycobacterium Virus Butters, Mycobacterium Virus Micelle, Mycobacterium Virus Charlie, Mycobacterium Virus Phipsquix, Mycobacterium Virus Xeno, Mycobacterium Virus Panchito, Mycobacterium Virus Pran, Mycobacterium Virus Ready, Mycobacterium Virus Skinnip, Gordonia Virus Baxter Fox, Gordonia Virus Easy, Gordonia Virus Kita, Gordonia Virus Zirinca, Cordonia Virus Nymphadora, Mycobacterium Virus Bignus, Mycobacterium Virus Brusacoram, Mycobacterium Virus Donovan, Mycobacterium Virus Fishburn, Mycobacterium Virus Jebex, Mycobacterium Virus Mali T, Mycobacterium Virus Phyons, Enterobacter Virus F20, Klebsiella Virus 1513, Klebsiella Virus KLPN1, Klebsiella Virus KP36, Klebsiella Virus PKP126, Klebsiella Virus Susi, Escherichia Virus AHP42 , Escherichia virus AHS24, Escherichia virus AKS96, Escherichia virus C119, Escherichia virus E41c, Escherichia virus Eb49, Escherichia virus Jk06, Escherichia virus KP26, Escherichia virus Rogue1, S Escherichia virus ACGM12, Escherichia virus Rtp, Escherichia virus ADB2, Escherichia virus JMPW1, Escherichia virus JMPW2, Escherichia virus T1, Shigella virus PSf2, Shigella virus Shfl1, Citrobacter virus Stevie , Escherichia virus TLS, Salmonella virus SP126, Chronobacter virus Esp2949-1, Pseudomonas virus Ab18, Pseudomonas virus Ab19, Pseudomonas virus PaMx11, Artrobacter virus amigo, Propionibacterium virus anatole, Propionibacterium virus B3 , Bacillus Virus Andromeda, Bacillus Virus Blastoid, Bacillus Virus Curly, Bacillus Virus Eogan, Bacillus Virus Pin, Bacillus Virus Glittering, Bacillus Virus Rigi, Bacillus Virus Taylor, Gordonia Virus Artis, Mycobacterium Virus Barnyard, My Cobacterium Virus Constantin, Mycobacterium Virus Predator, Mycobacterium Virus Bernal 13, Staphylococcus Virus 13, Staphylococcus Virus 77, Staphylococcus Virus 108PVL, Mycobacterium Virus Bron, Mycobacterium Virus Cobacterium virus Pate 1, Mycobacterium virus Joudt, Mycobacterium virus Rumpelstiltskin, Lactococcus virus bIL67, Lactococcus virus c2, Lactobacillus virus c5, Lactobacillus virus Ld3, Lactobacillus virus Ld17, Lactobacillus virus Ld25A, Lactobacillus virus LLKu, Lactobacillus virus phiLdb, Cellulophaga virus Cba121, Cellulophaga virus Cba171, Cellulophaga virus Cba181, Cellulophaga virus ST, Bacillus virus 250, Bacillus Virus IEBH, Mycobacterium Virus Ardmore, Mycobacterium Virus Avani, Mycobacterium Virus Boomer, Mycobacterium Virus Che8, Mycobacterium Virus Che9d, Mycobacterium Virus Deadp, Mycobac Therium Virus Delan, Mycobacterium Virus Dorothy, Mycobacterium Virus Dot Product, Mycobacterium Virus Drago, Mycobacterium Virus Fruit Loop, Mycobacterium Virus Gumbi, Mycobacterium Virus Iphuvesi, Mycobacterium Virus Llij, Mycobacterium Virus Moji, Mycobacterium Virus Mutaforma 13, Mycobacterium Virus Pacc40, Mycobacterium Virus PMC, Mycobacterium Virus Ramsay, Mycobacterium Virus Rocky Horror, Mycobacterium Virus SG4, Mycobacterium Virus Shauna 1, Mycobacterium Virus Silan, Mycobacterium Virus Spartacus, Mycobacterium Virus Taj, Mycobacterium Virus Tweety, Mycobac Therium virus Wee, Mycobacterium virus Yoshi, Salmonella virus Chi, Salmonella virus FSLSP030, Salmonella virus FSLSP088, Salmonella virus iEPS5, Salmonella virus SPN19, Mycobacterium virus 244, Mycobacterium virus Bask21, Mycobacterium Virus CJW1, Mycobacterium virus Eureka, Mycobacterium virus Kostya, Mycobacterium virus Polky, Mycobacterium virus Pumpkin, Mycobacterium virus Sirduracel, Mycobacterium virus Toto, Mycobacterium virus Cobacterium Virus Condok, Mycobacterium Virus Firecracker, Rhodobacter Virus RcCronus, Pseudomonas Virus D3112, Pseudomonas Virus DMS3, Pseudomonas Virus FHA0480, Pseudomonas Virus LPB1, Pseudomonas Virus MP22, Pseudomonas Virus MP29, Pseudomonas Virus MP38, Pseudomonas Virus PA1KOR , Pseudomonas virus D3, Pseudomonas virus PMG1, Atrobacter virus decouro, Gordonia virus demosthenes, Gordonia virus catiusa, Gordonia virus kvote, Propionibacterium virus B22, Propionibacterium virus two sets, Propioni Bacterium virus E6, Propionibacterium virus G4, Burkholderia virus phi6442, Burkholderia virus phi1026b, Burkholderia virus phiE125, Edwardsiella virus eiAU, Mycobacterium virus Ff47, Mycobacterium virus Mudi, Mycobacterium Virus Gaia, Mycobacterium Virus Gills, Atrobacter Virus Capten Murica, Atrobacter Virus Gordon, Gordonia Virus GordTnk2, Paenibacillus Virus Harrison, Escherichia Virus EK99P1, Escherichia Virus HK578 , Escherichia virus JL1, Escherichia virus SSL2009a, Escherichia virus YD2008s, Shigella virus EP23, Sodalis virus SO1, Escherichia virus HK022, Escherichia virus HK75, Escherichia virus HK97, Escherichia Virus HK106, Escherichia Virus HK446, Escherichia Virus HK542, Escherichia Virus HK544, Escherichia Virus HK633, Escherichia Virus mEp234, Escherichia Virus mEp235, Escherichia Virus mEpX1, Escherichia Virus mEpX2 Escherichia virus mEp043, Escherichia virus mEp213, Escherichia virus mEp237, Escherichia virus mEp390, Escherichia virus mEp460, Escherichia virus mEp505, Escherichia virus mEp506, Brevibacillus virus Genst, Acromobacter virus 83-24, Acromobacter virus JWX, Atrobacter virus Kellegio, Atrobacter virus KitKat, Atrobacter virus Benny, Artrobacter virus Dr. Robert, Artrobacter virus Glenn, Atrobacter virus Hunterdal, Atrobacter Virus Joan, Atrobacter Virus Cora, Atrobacter Virus Preamble, Atrobacter Virus Fumancal, Atrobacter Virus Wayne, Mycobacterium Virus Alma, Mycobacterium Virus Arturo, Mycobacterium Virus Astro, Mycobacterium Virus Baekyardigan, Mycobacterium Virus BBPiebs31, Mycobacterium Virus Benedict, Mycobacterium Virus Bethlehem, Mycobacterium Virus Billknuckles, Mycobacterium Virus Bruns, Mycobacterium Virus Bxb1, Mycobacterium Virus Bxz2, Mycobacterium Virus Che12, Mycobacterium Virus Cuco, Mycobacterium Virus D29, Mycobacterium Virus Doom, Mycobacterium Virus Ericb, Mycobacterium Virus Euphoria, Mycobacterium Virus George, Mycobacterium Virus Gladiator, Mycobacterium Virus Goose, Mycobacterium Virus Hammer, Mycobacterium Virus Heldan, Mycobacterium Virus Jasper, Mycobacterium Virus JC27, Mycobacterium Virus Jappa Bunny, Mycobacterium Virus JHC117, Mycobacterium Virus KBG, Mycobacterium Virus Kssjeb, Mycobacterium Virus Kugel, Mycobacterium Virus L5, Mycobacterium virus Resedi, Mycobacterium virus LHTSCC, Mycobacterium virus Lockley, Mycobacterium virus Marcel, Mycobacterium virus Microwolf, Mycobacterium virus Mr. Gordo, Mycobac Therium Virus Museum, Mycobacterium Virus Nepal, Mycobacterium Virus Paksman, Mycobacterium Virus Peaches, Mycobacterium Virus Perseus, Mycobacterium Virus Pukovnik, Mycobacterium Virus Rebecca, Mai Cobacterium Virus Redlock, Mycobacterium Virus Ridgecb, Mycobacterium Virus Rockstar, Mycobacterium Virus Saintus, Mycobacterium Virus Schiphol, Mycobacterium Virus Solon, Mycobacterium Virus Sweeney Me, mycobacterium virus SWU1, mycobacterium virus Ta17a, mycobacterium virus tiger, mycobacterium virus thymcell, mycobacterium virus trixie, mycobacterium virus turbido, mycobacterium virus Twister, Mycobacterium Virus U2, Mycobacterium Virus Violet, Mycobacterium Virus Wonder, Escherichia Virus DE3, Escherichia Virus HK629, Escherichia Virus HK630, Escherichia Virus Lambda, Atrobacter Virus Raroye, Mycobacterium Virus Halo, Mycobacterium Virus Liepie, Mycobacterium Virus Marvin, Mycobacterium Virus Mos Morris, Arthrobacter Virus Circum, Arthrobacter Virus Mudcat, Escheri Chia Virus N15, Escherichia Virus 9g, Escherichia Virus JenK1, Escherichia Virus JenP1, Escherichia Virus JenP2, Pseudomonas Virus NP1, Pseudomonas Virus PaMx25, Mycobacterium Virus Vaca, Mycobacterium Virus Courthouse , Mycobacterium Virus Littlely, Mycobacterium Virus Omega, Mycobacterium Virus Optimus, Mycobacterium Virus Tibalt, Polaribacter Virus P12002L, Polaribacter Virus P12002S, Nonravens Virus P12024L, Nonravens Virus P12024S, Thermus Virus P23-45, Thermus Virus P74-26, Listeria Virus LP26, Listeria Virus LP37, Listeria Virus LP110, Listeria Virus LP114, Listeria Virus P70, Propionibacterium Virus ATCC29399BC, Propionibacterium Virus ATCC29399BT, Propionibacterium Virus Atacne, Propionibacterium Virus Keiki, Propionibacterium Virus Kuved, Propionibacterium Virus Rauchelli, Propionibacterium Virus MrAK, Propionibacterium Virus Ouroboros, Propionibacterium Virus P91, Propionibacterium virus P105, Propionibacterium virus P144, Propionibacterium virus P1001, Propionibacterium virus P1.1, Propionibacterium virus P100A, Propionibacterium virus P100D, Propionibacter Propionibacterium virus P101A, Propionibacterium virus P104A, Propionibacterium virus PA6, Propionibacterium virus Pacnes201215, Propionibacterium virus PAD20, Propionibacterium virus PAS50, Propionibacterium virus PHL009M11, Propionibacterium Virus PHL025M00, Propionibacterium virus PHL037M02, Propionibacterium virus PHL041M10, Propionibacterium virus PHL060L00, Propionibacterium virus PHL067M01, Propionibacterium virus PHL070N00, Propionibacterium virus PHL071N05, Propionibacterium virus PHL082M03, Propionibacterium virus PHL092M00, Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01, Propionibacterium virus PHL112N00, Propionibacterium virus PHL113M01, Propionibacterium virus PHL114L00, Propionibacterium virus PHL116M00 , Propionibacterium virus PHL117M00, Propionibacterium virus PHL117M01, Propionibacterium virus PHL132N00, Propionibacterium virus PHL141N00, Propionibacterium virus PHL151M00, Propionibacterium virus PHL151N00, Propionibacterium virus PHL152M00, Propionibacterium virus PHL163M00, Propionibacterium virus PHL171M01, Propionibacterium virus PHL179M00, Propionibacterium virus PHL194M00, Propionibacterium virus PHL199M00, Propionibacterium virus PHL301M00, Propionibacterium virus PHL308M00, Propionibacterium Onibacterium Virus Pyrate, Propionibacterium Virus Procras1, Propionibacterium Virus SKKY, Propionibacterium Virus Solid, Propionibacterium Virus Stormbone, Propionibacterium Virus Fake, Pseudomonas Virus PaMx28, Pseudomonas Virus PaMx74, Mycobacterium virus Patience, Mycobacterium virus PBI1, Rhodococcus virus Pepy6, Rhodococcus virus Poco6, Propionibacterium virus PFR1, Streptomyces virus phiBT1, Streptomyces virus phiC31, Streptomyces Myses virus TG1, Caulobacter virus Karma, Caulobacter virus Magneto, Caulobacter virus phiCbK, Caulobacter virus log, Caulobacter virus Swift, Staphylococcus virus 11, Staphylococcus virus 29, Star Staphylococcus Virus 37, Staphylococcus Virus 53, Staphylococcus Virus 55, Staphylococcus Virus 69, Staphylococcus Virus 71, Staphylococcus Virus 80, Staphylococcus Virus 85, Staphylococcus Virus Coccus Virus 88, Staphylococcus Virus 92, Staphylococcus Virus 96, Staphylococcus Virus 187, Staphylococcus Virus 52a, Staphylococcus Virus 80alpha, Staphylococcus Virus CNPH82, Staphylococcus Virus Coccus Virus EW, Staphylococcus Virus IPLA5, Staphylococcus Virus IPLA7, Staphylococcus Virus IPLA88, Staphylococcus Virus PH15, Staphylococcus Virus phiETA, Staphylococcus Virus phiETA2, Staphylococcus Staphylococcus virus phiETA3, Staphylococcus virus phiMR11, Staphylococcus virus phiMR25, Staphylococcus virus phiNM1, Staphylococcus virus phiNM2, Staphylococcus virus phiNM4, Staphylococcus virus SAP26, Staphylococcus Virus X2, Enterococcus virus FL1, Enterococcus virus FL2, Enterococcus virus FL3, Lactobacillus virus ATCC8014, Lactobacillus virus phiJL1, Pediococcus virus cIP1, Aeromonas virus pIS4A, Listeria virus LP302, Listeria virus PSA, Methanobacterium Virus psiM1, Roseobacter Virus RDJL1, Roseobacter Virus RDJL2, Rhodococcus Virus RER2, Enterococcus Virus BC611, Enterococcus Virus IMEEF1, Enterococcus Virus SAP6, Enterococcus Virus VD13 , Streptococcus Virus SPQS1, Mycobacterium Virus Papyrus, Mycobacterium Virus Send513, Burkholderia Virus KL1, Pseudomonas Virus 73, Pseudomonas Virus Ab26, Pseudomonas Virus Kaheti25, Escherichia Virus Kazan, Escheri Chia Virus Seura, Staphylococcus Virus SEP9, Staphylococcus Virus Sextag, Streptococcal Virus 858, Streptococcal Virus 2972, Streptococcal Virus ALQ132, Streptococcal Virus O1205, Streptococcal Virus Sfi11, Streptococcal Virus Coccus Virus 7201, Streptococcus Virus DT1, Streptococcal Virus phiAbc2, Streptococcal Virus Sfi19, Streptococcal Virus Sfi21, Paenibacillus Virus Diva, Paenibacillus Virus Hb10c2, Paenibacillus Virus Rani, Paenibacillus Virus Celli, Paenibacillus Virus Sitara, Paenibacillus Virus Willow, Lactococcus Virus 712, Lactococcus Virus ASCC191, Lactococcus Virus ASCC273, Lactococcus Virus ASCC281, Lactococcus Virus ASCC465, Lactococcus Virus ASCC532, lactococcal virus Bibb29, lactococcal virus bIL170, lactococcal virus CB13, lactococcal virus CB14, lactococcal virus CB19, lactococcal virus CB20, lactococcal virus jj50, lactococcal virus P2, Lactococcus virus P008, Lactococcus virus sk1, Lactococcus virus Sl4, Bacillus virus slash, Bacillus virus Stahl, Bacillus virus Stali, Bacillus virus stills, Gordonia virus Bachita, Gordonia virus ClubL, Gordonia Virus One Up, Gordonia Virus Smoothie, Gordonia Virus Soup, Bacillus Virus SP Beta, Vibrio Virus MAR10, Vibrio Virus SSP002, Escherichia Virus AKFV33, Escherichia Virus BF23, Escherichia Virus DT57C, Escherichia Virus EPS7 , Escherichia virus FFH1, Escherichia virus H8, Escherichia virus slur09, Escherichia virus T5, Salmonella virus 118970sal2, Salmonella virus sivani, Salmonella virus SPC35, Salmonella virus stitch, Atrobacter virus tank, Chuka Murella Virus TIN2, Chuka Murella Virus TIN3, Chucam Murella Virus TIN4, Rhodobacter Virus RcSpartan, Rhodobacter Virus RcTitan, Mycobacterium Virus Anaya, Mycobacterium Virus Angelica, Mycobacterium Virus Creamed, Mycobacterium Virus Cobacterium Virus Pionbat, Mycobacterium Virus Joss, Mycobacterium Virus Raba, Mycobacterium Virus Mcncheese, Mycobacterium Virus Pixie, Mycobacterium Virus TM4, Bacillus Virus BMBtp2, Bacillus Virus TP21 , Geobacillus virus Tp84, Staphylococcus virus 47, Staphylococcus virus 3a, Staphylococcus virus 42e, Staphylococcus virus IPLA35, Staphylococcus virus phi12, Staphylococcus virus phiSLT, Mycobacteria Therium Virus 32HC, Rhococcus Virus RGL3, Paenibacillus Virus Vegas, Gordonia Virus Vendetta, Bacillus Virus W Beta, Mycobacterium Virus Wildcat, Gordonia Virus Twister 6, Gordonia Virus Wizard, Gordonia Virus Hoto Robo, Gordonia virus Monti, Gordonia virus Us, Xanthomonas virus CP1, Xanthomonas virus OP1, Xanthomonas virus phil7, Xanthomonas virus Xop411, Xanthomonas virus Xp10, Streptomyces virus TP1604, Streptomyces virus YDN12, Alpha Proteobacteria virus phiJl001, Pseudomonas virus LKO4, Pseudomonas virus M6, Pseudomonas virus MP1412, Pseudomonas virus PAE1, Pseudomonas virus infant, Pseudoalteromonas virus PM2, Pseudomonas virus phi6, Pseudomonas virus phi8, Pseudomonas virus phi12, Pseudomonas virus phi13, Pseudomonas s Virus phi2954, Pseudomonas virus phiNN, Pseudomonas virus phiYY, Vibrio virus fs1, Vibrio virus VGJ, Ralstonia virus RS603, Ralstonia virus RSM1, Ralstonia virus RSM3, Escherichia virus M13, Escherichia virus I22, Salmonella Virus IKe, Acoleplasma virus L51, Vibrio virus fs2, Vibrio virus VFJ, Escherichia virus If1, Propionibacterium virus B5, Pseudomonas virus Pf1, Pseudomonas virus Pf3, Ralstonia virus PE226, Ralstonia virus RSS1, Spiroplasma virus SVTS2, Stenotropomonas virus PSH1, Stenotropomonas virus SMA6, Stenotropomonas virus SMA7, Stenotropomonas virus SMA9, Vibrio virus CTXphi, Vibrio virus KSF1, Vibrio virus VCY, Vibrio Virus Vf33, Vibrio virus VfO3K6, Xanthomonas virus Cf1c, Spiroplasma virus C74, Spiroplasma virus R8A2B, Spiroplasma virus SkV1CR23x, Escherichia virus FI, Escherichia virus Qbeta, Escherichia virus BZ13, Escherichia virus MS2, Escherichia Virus Alpha3, Escherichia Virus ID21, Escherichia Virus ID32, Escherichia Virus ID62, Escherichia Virus NC28, Escherichia Virus NC29, Escherichia Virus NC35, Escherichia Virus phiK , Escherichia Virus St1, Escherichia Virus WA45, Escherichia Virus G4, Escherichia Virus ID52, Escherichia Virus Thalmos, Escherichia Virus phiX174, Bdellovibrio Virus MAC1, Bdellovibrio Virus MH2K, Chlamydia virus Chp1, Chlamydia virus Chp2, Chlamydia virus CPAR39, Chlamydia virus CPG1, Spiroplasma virus SpV4, Acoleplasma virus L2, Pseudomonas virus PR4, Pseudomonas virus PRD1, Bacillus virus AP50, Bacillus virus Bam35, Bacillus virus GIL16, Bacillus virus Wip1 , Escherichia virus phi80, Escherichia virus RB42, Escherichia virus T2, Escherichia virus T3, Escherichia virus T6, Escherichia virus VT2-Sa, Escherichia virus VT1-Sakai, Escherichia Virus VT2-Sakai, Escherichia Virus CP-933V, Escherichia Virus P27, Escherichia Virus Stx2phi-I, Escherichia Virus Stx1phi, Escherichia Virus Stx2phi-II, Escherichia Virus CP-1639, S Escherichia Virus BP-4795 Based, Escherichia Virus 86, Escherichia Virus Min27, Escherichia Virus 2851, Escherichia Virus 1717, Escherichia Virus YYZ-2008, Escherichia Virus EC026_P06, Escherichia Virus ECO103_P15, Escherichia virus ECO103_P12, Escherichia virus ECO111_P16, Escherichia virus ECO111_P11, Escherichia virus VT2phi_272, Escherichia virus TL-2011c, Escherichia virus P13374, Escherichia virus Sp5.

In one embodiment, the bacterial viral particle is typically E. coli. E. coli, BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, 025, PhI-5, Pk , PSP3, Pl, PlD, P2, P4, Sl, Wφ, φK13, φl, φ2, φ7, φ92, 7 A, 8φ, 9φ, 18, 28-1, 186, 299, HH-Escherichia (2) , AB48, CM, C4, C16, DD-VI, E4, E7, E28, FIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5 , ND6, ND-7, Ox-I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, PhI-I, RB42, RB43, RB49, RB69, S, SaI-I, Sal -2, Sal-3, Sal-4, Sal-5, Sal-6, TC23, TC45, TuII*-6, TuIP-24, TuII*46, TuIP-60, T2, T4, T6, T35, αl, 1, IA, 3, 3A, 3T+, 5φ, 9266Q, CFO103, HK620, J, K, KlF, m59, no. A, no. E, no. 3, no. 9, N4, sd, T3, T7, WPK, W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, φ04-CF, φ05, φ06, φ07, φl, φl.2, φ20, φ95, φ263, φlO92, φl, φll, Ω8, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W, NN-Escherichia (1), Esc -7-11, AC30, CVX-5, Cl, DDUP, ECl, EC2, E21, E29, Fl, F26S, F27S, Hi, HK022, HK97, HK139, HK253, HK256, K7, ND-I, PA-2 , q, S2, Tl), T3C, T5, UC-I, w, β4, γ2, λ, φD326, φγ, φ06, φ7, φ10, φ80, χ, 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, KlO, ZG / 3A, 5, 5A, 21EL, a bacteriophage selected from the group consisting of H19-J and 933H contains the capsid of

Accordingly, the present invention also relates to a bacterial delivery vehicle, as defined above, for use in the in vivo delivery of a nucleic acid of interest to a targeted recipient bacterial cell, as defined above, said bacterial delivery vehicle comprising a vector of the present invention. includes

donor bacterial cells

In the context of this application, the term "donor bacterial cell" means a bacterial cell hosting a vector or plasmid, a production cell line, or a bacterium capable of transferring a conjugating plasmid to another bacterium.

The invention also relates to a donor bacterial cell comprising a vector of the invention or producing a bacterial delivery vehicle of the invention, wherein the donor bacterial cell stably contains the vector of the invention and is capable of replicating the vector.

In certain embodiments, when the conditional origin of replication of the vector is an origin of replication whose replication is dependent on the presence of a given protein, peptide, nucleic acid, RNA, molecule or any combination thereof, the donor bacterial cell is dependent on the protein, Expresses a peptide, nucleic acid, RNA, molecule or any combination thereof. Preferably, said protein, peptide, nucleic acid, RNA, molecule or any combination thereof is expressed in trans, as defined in the "Conditional Origin of Replication" section above.

In certain embodiments, the donor bacterial cell stably contains a nucleic acid encoding the protein, peptide, nucleic acid, RNA, molecule or any combination thereof.

In certain embodiments, when the origin of replication is derived from a phage-derived chromosome island (PICI), the conditional origin of replication is active in the donor bacterial cell, wherein the donor bacterial cell is rep protein, in particular primase-heli expresses a case, in particular a primase-helicase of sequence SEQ ID NO: 8.

In certain embodiments, the donor bacterial cell stably comprises a nucleic acid encoding the rep protein, in particular the primase-helicase, which typically comprises or consists of the sequence SEQ ID NO: 9.

In certain embodiments, the donor bacterial cell is a production cell line, in particular a cell line that produces a packaged phagemid comprising a vector of the invention.

Production of packaged phagemids and bacteriophage particles by production cell lines is a routine technique well known to those skilled in the art. In one embodiment, satellite phages and/or helper phages can be used to facilitate packaging of vectors in the delivery vehicles disclosed herein. Helper phages provide functions in trans and are well known to those skilled in the art. The helper phage contains all genes encoding structural and functional proteins essential for packaging the phagemid (ie, the helper phage provides all gene products essential for assembly of the delivery vehicle). The helper phage may contain a defective origin of replication or packaging signal, or completely lack the latter, and for that reason will not be able to self-package, so only bacterial transfer particles bearing the vector or plasmid will be produced. Helper phages can be selected such that they cannot induce lysis of the bacterial cells used to produce the delivery particle. One skilled in the art will understand that some bacteriophages are deficient and require helper phages for payload packaging. Thus, depending on the bacteriophage selected to produce the bacterial delivery particle, the skilled person will know whether a helper phage is required or not. Sequences encoding one or more proteins or regulatory processes essential for assembly or production of the packaged payload may be supplied in trans. For example, the STF, gpJ and gpH proteins can be provided on a plasmid or constitutively expressed under the control of an inducible promoter. In this case, the phage wild-type sequence may or may not contain a deletion of the gene or sequence supplied in trans. In addition, chimeric or modified phage sequences encoding new functions, such as engineered STF, gpJ or gpH proteins, can be directly inserted into desired locations in the helper's genome, thus the need to provide modified sequences in trans. can avoid Methods for generating direct genomic insertions, modifications and mutations, including methods for supplying sequences or proteins in trans in the form of plasmids, are well known to those skilled in the art.

In certain embodiments, the helper phage comprises a nucleic acid sequence encoding a chimeric STF comprising or consisting of the sequence SEQ ID NO: 12, wherein the nucleic acid sequence typically comprises or consists of the sequence SEQ ID NO: 13 wherein the helper phage optionally further comprises a nucleic acid sequence encoding a chimeric gpJ variant comprising or consisting of the sequence SEQ ID NO: 14, wherein the nucleic acid sequence typically comprises or consists of the sequence SEQ ID NO: 15 It is done.

In certain embodiments, the helper phage is a lambda prophage, and (i) the nucleic acid encoding the wild-type STF protein has been replaced with a nucleic acid sequence encoding a chimeric STF comprising or consisting of the sequence SEQ ID NO: 12, wherein the nucleic acid The sequence typically comprises or consists of the sequence SEQ ID NO: 13, and (ii) the nucleic acid encoding the wild-type gpJ protein is a nucleic acid sequence encoding a chimeric gpJ variant comprising or consisting of the sequence SEQ ID NO: 14. substituted, the nucleic acid sequence typically comprises or consists of the sequence SEQ ID NO: 15, (iii) the Cos site has been removed, and optionally (iv) the helper prophage is mutated to prevent spontaneous cell lysis, such as Sam7 and (v) the helper prophage contains the thermosensitive form of the master cI suppressor, such as the cI857 form.

In certain embodiments, the donor bacterial cell of the invention comprises a helper phage as defined above.

Treatment of disease - cosmetic therapy

Vectors used in the control methods of the present invention may be administered by themselves, either through a donor bacterial cell that delivers the vector to the recipient bacterial cell, or as a bacterial delivery vehicle. The vector, bacterial delivery vehicle or donor bacterial cells may more particularly be administered in the form of a pharmaceutical or cosmetic composition comprising the vector, bacterial delivery vehicle or donor bacterial cells and a pharmaceutically acceptable carrier.

Generally, for pharmaceutical or cosmetic use, the vector, bacterial delivery vehicle or donor bacterial cell comprises at least one vector, bacterial delivery vehicle or donor bacterial cell, and at least one pharmaceutically or cosmetically acceptable carrier, diluent or excipients, and optionally one or more additional pharmaceutically or cosmetically active compounds. Such formulations may be in a form suitable for oral administration, parenteral administration (eg intravenous, intramuscular or subcutaneous injection or intravenous infusion), topical administration, inhalation administration, skin patches, implants, suppositories and the like. In certain embodiments, the composition is for oral administration. Such dosage forms may be solid, semi-solid, or liquid, depending on the mode and route of administration. For example, formulations for oral administration may be provided with an enteric coating that will allow the vector, bacterial delivery vehicle, or donor bacterial cells in the formulation to survive the gastric environment and pass into the intestine. More generally, vector preparations, bacterial delivery vehicle preparations or donor bacterial cell preparations for oral administration may be suitably formulated for delivery to any desired portion of the stomach. In addition, suitable suppositories may be used for delivery to the gastrointestinal tract. A variety of pharmaceutically or cosmetically acceptable carriers, diluents, and excipients useful in pharmaceutical or veterinary or cosmetic compositions are known to those skilled in the art.

A pharmaceutical or veterinary composition according to the present invention may further comprise a pharmaceutically acceptable vehicle. The cosmetic composition of the present invention may further comprise a cosmetically acceptable vehicle. Solid pharmaceutically or cosmetically acceptable vehicles include flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, lubricants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. It can include one or more substances that can also act as Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrins, starches, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins.

Pharmaceutical or veterinary or cosmetic compositions may be prepared as sterile solid compositions which can be suspended for administration using sterile water, saline, or other suitable sterile injectable medium. The pharmaceutical or veterinary or cosmetic composition of the present invention may contain other solutes or suspending agents (eg, sufficient saline or glucose to render the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (the oleate ester of sorbitol and its anhydride copolymerized with ethylene oxide) and the like. Particles according to the present disclosure may also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for enteral administration include sterile solutions, emulsions, and suspensions.

A vector, bacterial delivery vehicle or donor bacterial cell disclosed herein may be dissolved in a pharmaceutically or cosmetically acceptable liquid vehicle such as water, an organic solvent, or a mixture of both or pharmaceutically acceptable oils or fats, or can be suspended. Liquid vehicles may contain other suitable pharmaceutical or cosmetic additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, coloring agents, viscosity regulators, stabilizers, or osmo-regulators. Suitable examples of liquid vehicles for oral and enteral administration include water (partially containing such additives as eg cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (monohydric and polyhydric alcohols such as glycols). ) and their derivatives, and oils (eg, fractionated coconut oil and peanut oil). For parenteral administration, the vehicle may also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful for sterile liquid form compositions for enteral administration. Liquid vehicles for pressurized compositions may be halogenated hydrocarbons or other pharmaceutically or cosmetically acceptable propellants.

In some embodiments, the present invention encompasses pharmaceutical or veterinary or cosmetic compositions formulated for delayed or progressive intestinal release. In a preferred embodiment, the formulations or pharmaceutical or cosmetic preparations of the present invention are formulated for translocation of vectors into the distal small intestine and/or colon. The preparation can allow the vector to pass through gastric acid and pancreatic enzymes and bile, and reach the distal small intestine and colon intact to survive.

In some embodiments, the pharmaceutical or veterinary or cosmetic composition is micro-encapsulated, formed into a tablet, and/or placed in a capsule, preferably a purple coated capsule.

In some embodiments, the pharmaceutical or veterinary or cosmetic composition is formulated for delayed or progressive enteral release using cellulose acetate (CA) and polyethylene glycol (PEG). In some embodiments, the pharmaceutical or veterinary or cosmetic composition is formulated for delayed or progressive enteral release using hydroxypropylmethylcellulose (HPMC), microcrystalline cellulose (MCC) and magnesium stearate. Pharmaceutical or veterinary compositions may be formulated using, for example, poly(meth)acrylates such as methacrylic acid copolymer B, methyl methacrylate and/or methacrylic acid esters, or polyvinylpyrrolidone (PVP). It is formulated for delayed or gradual intestinal release.

In some embodiments, the pharmaceutical or veterinary or cosmetic composition comprises a release-retarding matrix material such as: acrylic polymers, cellulose, waxes, fatty acids, shellac, zein, hydrogenated vegetable oils, hydrogenated castor oil, polyvinylpyrrolidone, vinyl acetate copolymers. , vinyl alcohol copolymer, polyethylene oxide, acrylic acid and methacrylic acid copolymer, methyl methacrylate copolymer, ethoxyethyl methacrylate polymer, cyanoethyl methacrylate polymer, aminoalkyl methacrylate copolymer, poly (acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acid anhydride), methyl methacrylate polymer, polymethacrylate, poly( methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymer, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxymethyl cellulose, Hydroxyethyl cellulose, hydroxypropyl cellulose, cross-linked sodium carboxymethylcellulose, cross-linked hydroxypropylcellulose, natural waxes, synthetic waxes, fatty alcohols, fatty acids, fatty acid esters, fatty acid glycerides, hydrogenated fats, hydrocarbon waxes, stearic acid, stearyl Alcohol, beeswax, glycowax, castor wax, carnauba wax, polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, carboxymethyl starch, potassium methacrylate/divinylbenzene copolymer, cross-linked polyvinylpyrroly It is formulated for delayed or progressive intestinal release using money, polyvinyl alcohol, polyvinyl alcohol copolymer, polyethylene glycol, non-crosslinked polyvinylpyrrolidone, polyvinyl acetate, polyvinyl acetate copolymer, or any combination thereof.

In some embodiments, the pharmaceutical or veterinary composition is formulated for delayed or progressive enteral release as described in US Patent Application Publication No. 20110218216, which describes an extended release pharmaceutical composition for oral administration, hydrophilic polymers, hydrophobic materials and hydrophobic polymers or mixtures thereof and microenvironmental pH modifiers. The hydrophobic polymer may be ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, methacrylic acid-acrylic acid copolymer, or mixtures thereof. The hydrophilic polymer may be polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acid copolymer or mixtures thereof. The hydrophobic material may be hydrogenated vegetable oil, hydrogenated castor oil, carnauba wax, candelia wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or mixtures thereof. Microenvironmental pH modifiers can be inorganic acids, amino acids, organic acids or mixtures thereof. Alternatively, the microenvironmental pH modifier may be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid; glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, salicylic acid, tosylic acid, mesylic acid or malic acid or mixtures thereof.

In some embodiments, the pharmaceutical or veterinary or cosmetic composition is a powder that can be incorporated into a tablet or suppository. In an alternative embodiment, an agent or pharmaceutical or cosmetic preparation of the present invention may be a 'powder for reconstitution' as a liquid to be drunk or otherwise administered.

In some embodiments, the pharmaceutical or veterinary or cosmetic composition may be administered as a cream, gel, lotion, liquid, feed, or aerosol spray. In some embodiments, bacteriophages are produced using any material known in the art and any technique known in the art, including, without limitation, the technique outlined in U.S. Patent No. 7,482,115, incorporated herein by reference. It is immobilized on a solid surface using the immobilization of bacteriophages on polymeric beads. Phage are immobilized on polymeric beads of appropriate size so that the coated beads can be added to aerosols, creams, gels or liquids. The size of the polymeric beads may be between about 0.1 μm and 500 μm, for example between 50 μm and 100 μm. The coated polymeric beads can be incorporated into animal feed, including pelleted feed and any other form of feed, incorporated into any other edible device used to provide phages to animals, and containers provided to animals. It is added to the water contained in the bottle and provided to the animals through the water supply system. In some embodiments, the composition is used to treat superficial wounds and other superficial infections using creams, gels, aerosol sprays, and the like.

In some embodiments, the pharmaceutical or veterinary or cosmetic composition is administered by inhalation, in the form of a suppository or pessary, topically (eg in the form of a lotion, liquid, cream, ointment or dusting), epidermally or transdermally ( eg, using a skin patch), orally (eg, as a tablet which may contain excipients such as starch or lactose), capsules, vaginal suppositories, elixirs, solutions, or suspensions, each optionally flavoring, coloring and/or containing excipients), or they can be injected parenterally (eg intravenously, intramuscularly or subcutaneously). For parenteral administration, the composition may be used in the form of a sterile aqueous solution which may contain other substances, for example, sufficient salts or monomers to render the solution isotonic with blood. For buccal or sublingual administration, the composition may be administered in the form of a tablet or lozenge that may be formulated in a conventional manner. In a preferred embodiment, the bacteriophages and/or polypeptides of the invention are administered topically, either as a sole agent or in combination with other antibiotic treatments, as described herein or known in the art.

In some embodiments, pharmaceutical or veterinary or cosmetic compositions may also be administered dermally or intranasally. For topical application to the skin, pharmaceutical or veterinary or cosmetic compositions may include aqueous liquids, alcohol-based liquids, water-soluble gels, lotions, ointments, non-aqueous liquid bases, mineral oil bases, blends of mineral oil and petrolatum, lanolin, liposomes, proteins. Carriers such as serum albumin or gelatin, powdered cellulose camel, and combinations thereof may include, but are not limited to, one carrier or combination of carriers. Topical delivery modes may include smears, sprays, bandages, time-release patches, liquid-absorbing wipes, and combinations thereof. The pharmaceutical or veterinary or cosmetic composition may be applied directly or in a carrier(s) to patches, wipes, bandages, and the like. The patch, wipe, bandage, etc. can be moist or dry, wherein the phage and/or polypeptide (eg, lysin) is in lyophilized form on the patch. Carriers of topical compositions can include polymeric thickeners, water, preservatives, active surfactants, or emulsifiers, antioxidants, sunscreens, and semi-solid and gel-like vehicles, including solvents or mixed solvent systems. U.S. Patent No. 5,863,560 discloses a number of different carrier combinations that can aid skin exposure to drugs, the contents of which are incorporated herein.

For intranasal or inhalation administration, the pharmaceutical or veterinary or cosmetic composition may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes such as 1,1,1, 2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane, carbon dioxide, or other suitable gas in the form of an aerosol spray present from a pressurized container, pump, spray, or nebulizer; or It is conveniently delivered in the form of a dry powder inhaler. In the case of pressurized aerosols, dosage units may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebulizer may additionally contain a lubricant, such as sorbitan trioleate, for example, a solution or suspension of the active compound using a mixture of a propellant and ethanol as a solvent. may contain Capsules and cartridges (eg, made of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a bacteriophage and/or polypeptide of the invention and a suitable powder base such as lactose or starch.

For administration in the form of a suppository or pessary, the pharmaceutical or veterinary composition may be topically applied in the form of a gel, hydrogel, lotion, solution, cream, ointment, or dusting. Compositions of the present invention may also be administered via the ocular route. For ophthalmic use, the compositions of the present invention are prepared as a finely divided suspension in isotonic, pH-adjusted, sterile saline, or preferably in isotonic, pH-adjusted, sterile saline, optionally in combination with a preservative such as benzylalkonium chloride. It can be formulated as a solution. Alternatively, they may be formulated in an ointment such as petrolatum.

Pharmaceutical and Veterinary Compositions of the Invention Doses and preferred drug concentrations of the compositions may vary depending on the particular use. Determination of an appropriate dose or route of administration is within the skill of the general practitioner. Animal studies can provide reliable guidelines for determining effective doses in human therapy.

For transdermal administration, the pharmaceutical or veterinary composition may be formulated in the form of an ointment, cream, or gel, and appropriate penetrants or detergents such as dimethyl sulfoxide, dimethyl acetamide, and dimethylformamide may be used to promote penetration. can

For transmucosal administration, nasal sprays, rectal or vaginal suppositories may be used. The active compound may be incorporated into any known suppository base by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycol (carbowax), polyethylene sorbitan monostearate, and mixtures thereof with other compatible materials to modify the melting point or dissolution rate.

In certain embodiments, a composition of the present invention comprises at least one additional active ingredient, such as a prebiotic and/or probiotic and/or antibiotic, and/or other antibacterial or antibiofilm agent, and/or It may further include any agent that enhances targeting of the vector to the bacteria and/or delivery of the vector to the bacteria.

As used herein, “prebiotic” refers to a component that allows specific changes in both the activity and/or composition of a gastrointestinal microbiota that can confer a benefit to the host. A prebiotic can be an edible food or beverage or ingredient thereof. A prebiotic can be a component that is selectively fermented. Prebiotics can include complex carbohydrates, amino acids, peptides, minerals, or other essential nutritional components for the survival of the bacterial composition. Prebiotics include amino acids, biotin, fructo-oligosaccharides, galacto-oligosaccharides, hemicelluloses (e.g., arabinosilanes, silanes, syloglucans, and glucomannans), insulin, chitin, lactulose, mannan oligosaccharides, oligofruc Tose-enriched inulin, gums (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-gal Lacttooligosaccharide, pectin (e.g., silogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soybean fiber, beet fiber, pea fiber, corn bran) , and oat fiber) and xylooligosaccharides.

As used herein, "probiotic" refers to dietary supplements based on live microorganisms that, when consumed in adequate amounts, have beneficial effects on the host organism by enhancing the intestinal ecosystem. A probiotic can include a non-pathogenic bacterial or fungal population, eg, an immunomodulatory bacterial population, such as an anti-inflammatory bacterial population, with or without one or more prebiotics. They contain a sufficiently high number of viable and active probiotic microorganisms that can exert a balancing action on the intestinal flora through direct colonization. For purposes of this description, the term "probiotic" preferably includes, but is not limited to, lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria ( propionibacteria or saccharomycetes and also other microorganisms constituting the normal intestinal flora, or also any biologically active form of probiotics, including fragments of the bacterial wall or DNA of these microorganisms. I understand. These compositions are advantageously suitable for safe administration to human and other mammalian subjects and are effective for the treatment, prevention of diseases or disorders caused by bacteria such as bacterial infections. Probiotics include lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria, saccharomycetes, and lactobacilli. lactobacilli, bifidobacteria, or proteobacteria, but are not limited thereto.

In certain embodiments, the probiotic is unaffected by the vectors of the invention. In certain embodiments, when the vector is included in a bacterial delivery vehicle, the vehicle is capable of binding the probiotic, but the probiotic is unaffected by the vector. In an alternative embodiment, when the vector is included in a bacterial delivery vehicle, the vehicle does not bind the probiotic and the probiotic is unaffected by the vector.

In certain embodiments, the effect of the vector induces or increases synergy with the effect of the additional active ingredient. In a more specific embodiment, the vector is capable of enabling the probiotic to engraft into the host organism.

Antibiotics include penicillins such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, pibampicillin, hetacillin , bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; Cephalosporins such as cefasettril, cefadroxil, cephalexin, cephaloglycin, cephalonium, cephaloridine, cephalotin, cepapyrin, cefathrizine, cefazafluor, cefazedone, cefazolin, cefra Dean, ceproxadine, ceftesol, cefaclor, cefoniside, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef, cefbuperazone, cefminox, cefotetan, cefoxitin, sefotiam, cefcapen, cefdaloxime, cefdinier, cefditoren, cefetamet, cefixim, cefmenoxime, cefodizim, cefotaxime, cefbecin, ceffimizole, cef Podoxim, Cefteram, Ceftamer, Ceftibutene, Cefthiopur, Ceftiolene, Ceftizoxim, Ceftriaxone, Seefoperazone, Ceftagidim, Ratamoxef, Cefclidine, Cefepime, Cefclidine fluprenam, sefocellis, sefozofran, cefpyrom, cefquinom, flomoxef, ceftobiprole, ceftaroline, ceftolozan, cephaloram, cefharol, cefcanel, cefedrol; cefempidone, cefetrizole, cefibitril, cefmatylene, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cefuracetim, and nitrocepin; polymyxins such as polysporin, neosporin, polymyxin B, and polymyxin E, rifampicins such as rifampicin, rifapentine, and rifaximin; fidaxomycin; Quinolones such as cinoxacin, nalidic acid, oxolinic acid, pyromidic acid, pipemidic acid, losoxacin, ciprofloxacin, enoxacin, fleoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin reaper, lufloxacin, balofloxacin, grepafloxacin, levofloxacin, pajufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, squarenoxacin, and zabofloxacin; Sulfonamides such as sulfafurazole, sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole, sulfisomidine, sulfadoxine, sulfamethoxazole, sulfamoxol, sulfanitran, sulfadimethoxine, sulfamethoxypyr minced, sulfamethoxydiazine, sulfadoxine, sulfamethopyrazine, and terepthyl; macrolides such as azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josomycin, kitasamycin, midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, and roxithromycin; ketolides such as telithromycin, and setromycin; fluoroketolides such as solithromycin; lincosamides such as lincomycin, clindamycin, and pilimycin; tetramycins such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline; aminoglycosides such as amikacin, dibecacin, gentamicin, kanamycin, neomycin, netylmicin, sisomicin, tobramycin, paromomycin, and streptomycin; ansamycins such as geldanamycin, herbimycin, and rifaximin; carbacephem such as laurakarbef; carbapenems such as eltapenem, doripenem, imipenem (or cilastatin), and meropenem; glycopeptides such as teicoplanin, vancomycin, telavancin, dalba reply, and orita reply; lincosamides such as clindamycin and lincomycin; lipopeptides such as daptomycin; monobactams such as aztreonam; nitrofurans such as furazolidone, and nitrofurantoin; oxazolidinones such as linezolid, pocizolide, radezolide, and torezolide; Teixobactin, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, alsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin , platensimycin, quinupristine (or dalfopristine), thiamphenicol, tigecycline, tinidazole, trimethoprim, alatrofloxacin, fidaxomicin, nalidixic acid, rifampin, derivatives and combinations thereof can be selected from.

In certain embodiments, the modulating methods of the present invention are for treating and/or preventing a disease in said host subject.

In certain embodiments, the disease is caused or mediated by bacteria.

Diseases or disorders caused or mediated by bacteria include chronic inflammation of the skin such as acne (acne vulgaris), progressive macular hypopigmentation, abdominal cramps, acute epiglottitis, arthritis, bacteremia, hemorrhage, botulism, brucellosis, brain abscess, myocardium Diseases, soft sexually transmitted diseases, chlamydia, Crohn's disease, conjunctivitis, cholecystitis, colorectal cancer, polyposis, colony collapse, Lyme disease, diarrhea, diphtheria, duodenal ulcer, endocarditis, erythema, enteric fever, fever, glomerulonephritis, gastroenteritis, gastric ulcer, Guillain- Barré syndrome, tetanus, gonorrhea, gingivitis, inflammatory bowel disease, irritable bowel syndrome, leptospirosis, leprosy, listeriosis, tuberculosis, Lady Windermere syndrome, legionnaires' disease, meningitis, mucous conjunctivitis, multidrug-resistant bacterial infection, multidrug-resistant bacterial carrier , myocarditis, myonecrosis-gas gangrene, mycobacterium avium complex, neonatal necrotizing enteritis, nocardiosis, nosocomial infection, otitis, periodontitis, pharyngitis, pneumonia, peritonitis, purpura, Rocky Mountain spot fever, dysentery, Syphilis, sinusitis, sigmoiditis, sepsis, subcutaneous abscess, tularemia, tracheobronchitis, tonsillitis, typhoid fever, ulcerative colitis, urinary tract infection, whooping cough, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) It may be selected from the group consisting of

Infections caused by bacteria are infections, preferably intestinal infections such as esophagitis, gastritis, enteritis, colitis, sigmoiditis, proctitis, and peritonitis, urinary tract infection, vaginal infection, female upper genital tract infection such as salpingitis, endometritis, oophoritis, uterus Myositis, parametritis and pelvic peritoneal infections, respiratory infections such as pneumonia, intra-amniotic infections, odontogenic infections, root canal infections, fibrosis, meningitis, bloodstream infections, nosocomial infections such as catheter-associated infections, nosocomial infectious pneumonia, postpartum infections, nosocomial infectious gastroenteritis, hospital infectious urinary tract infection, or a combination thereof. Preferably, infections according to the present invention are caused by bacteria that exhibit antibiotic resistance. In certain embodiments, the infection is caused by bacteria as listed above in the targeted bacteria.

The present disclosure also relates to pharmaceutical or veterinary compositions of the present invention for the treatment of metabolic disorders, including, for example, obesity, type 2 diabetes and non-alcoholic fatty liver disease. Indeed, emerging evidence suggests that these disorders are characterized by altered gut microbiota composition and its metabolites (Tilg et al., Nature Reviews Immunology, volume 20, pages 40-54, 2020). Thus, a pharmaceutical or veterinary composition may be used to improve the composition of the intestinal microbiota or its metabolism (e.g. by inducing the expression, overexpression or secretion of some molecules by said bacteria, for example molecules with a beneficial role against metabolic inflammation). It can be used to deliver nucleic acids of interest that can alter the product in some enteric bacteria. The present disclosure also relates to the use of the pharmaceutical or veterinary compositions of the present invention for the manufacture of a medicament for the treatment of metabolic disorders including, for example, obesity, type 2 diabetes and non-alcoholic fatty liver disease. Also directed to methods for treating metabolic disorders, including, for example, obesity, type 2 diabetes and non-alcoholic fatty liver disease, wherein a provided pharmaceutical or veterinary composition, in particular a therapeutically effective amount of a provided pharmaceutical or veterinary composition, is used to treat It includes the step of administering to a subject having a metabolic disorder in need of.

In certain embodiments, the present invention relates to pharmaceutical or veterinary compositions for use in the treatment of conditions involving bacteria of the human microbiome, such as inflammatory and auto-immune diseases, cancers, infections or brain disorders. Indeed, some bacteria in the microbiome can secrete molecules that will induce and/or enhance the development of inflammatory or autoimmune diseases or cancers without triggering any infection. More particularly, the present invention also relates to immunotherapies based on Tregs (regulatory T cells), also known as, for example, CAR-T (chimeric antigen receptor T) cells, TIL (tumor infiltrating lymphocytes) and suppressor T cells. Modulation of microbiome composition to improve efficacy. Modulation of microbiome composition to improve the efficacy of immunotherapy is also well known in the art, including immune checkpoint inhibitors such as, without limitation, PD-1 (programmed cell death protein 1) inhibitors, PD-L1 (programmed cell death protein 1) inhibitors, death ligand 1) inhibitors and CTLA-4 (cytotoxic T lymphocyte-associated protein 4).

In an alternative embodiment, the disease is not caused by bacteria.

In some embodiments, the condition to be treated is breast cancer (eg, triple negative breast cancer, ER+ breast cancer, or ER- breast cancer), basal cell carcinoma, skin cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, medulloblastoma, neuroblastoma. Gliomas (including glioblastoma, oligodendroglioma, astrocytoma, ependymoma), neuroblastoma, colorectal cancer, ovarian cancer, liver cancer, pancreatic cancer (eg, carcinoma, angiosarcoma, adenosarcoma), stomach cancer, gastroesophageal junction cancer, prostate cancer , cervical cancer, bladder cancer, head and neck cancer, lymphoma (eg, mantle cell lymphoma, diffuse large B-cell lymphoma), solid tumors that cannot be surgically removed, locally advanced solid tumors, metastatic solid tumors, leukemia (eg For example, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia (CML)), or a cancer or proliferative disorder including, but not limited to, a recurrent or refractory tumor.

In one embodiment, conditions to be treated include, but are not limited to: systemic anaphylaxis and hypersensitivity disorders, atopic dermatitis, urticaria, drug allergies, insect sting allergies, food allergies (including celiac disease, etc.), and obesity. inflammatory or allergic diseases, including cytoma; inflammatory bowel disease, including Crohn's disease, ulcerative colitis, ileitis, and enteritis; vasculitis and Behcet's syndrome; Viral skin, including dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, psoriasis and inflammatory dermatoses, including urticaria, those of human papillomavirus, HIV or RLV infection, bacterial, fungal, and other parasitic skin pathologies pathology, and cutaneous lupus erythematosus; asthma and respiratory allergic diseases, including allergic asthma, exercise-induced asthma, allergic rhinitis, otitis media, allergic conjunctivitis, hyperactive pulmonary disease, and chronic obstructive pulmonary disease; autoimmune diseases, including arthritis (including rheumatoid and psoriasis), systemic lupus erythematosus, type I diabetes, myasthenia gravis, multiple sclerosis, Graves' disease, and glomerulonephritis; transplant rejection (including allograft rejection and graft versus host disease), eg, skin transplant rejection, solid organ transplant rejection, bone marrow transplant rejection; Fever; cardiovascular disorders, including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, cardiomyopathy, congestive heart failure, atherosclerosis, coronary artery disease, restenosis and vascular stenosis; cerebrovascular disorders including traumatic brain injury, stroke, ischemic reperfusion injury and aneurysm; genital and reproductive conditions including fibrosis, connective tissue disease and sarcoidosis, erectile dysfunction; gastrointestinal disorders, including gastritis, ulcers, nausea, pancreatitis, and vomiting; neurological disorders, including Alzheimer's disease; sleep disorders, including insomnia, narcolepsy, sleep apnea syndrome, and Pickwick syndrome; ache; renal failure; eye disorders, including glaucoma; and non-bacterial infectious diseases including HIV.

In some embodiments, the disease to be treated is an autoimmune disease such as autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmunopenia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis , recurrent polychondritis, rheumatoid heart disease, glomerulonephritis, multiple sclerosis, neuritis, ocular uveitis, polyendocrinopathy, purpura, Reiter's disease, ankylosing-person syndrome, autoimmune lung inflammation, myocarditis, IgA glomerulonephritis, dense deposits , rheumatic heart disease, Guillain-Barré syndrome, insulin-dependent diabetes mellitus, autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism, systemic lupus erythematosus, discoid lupus, Goodpasture syndrome, pemphigus, Graves' disease, myasthenia gravis, Insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis, Bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, asthma or adrenergic drug resistance with cystic fibrosis, chronic active hepatitis, primary biliary cirrhosis, endocrine insufficiency, vitiligo, vasculitis, post-MI, myocardial infarction syndrome, urticaria, atopic dermatitis , asthma, inflammatory myopathy, inflammatory disorders, granulomatous disorders, atrophic disorders, or alloimmune diseases.

The subject to be treated may be diagnosed with, or may be at risk of developing, an infection, disorder and/or disease, preferably caused by bacteria. Methods for diagnosing such infections, disorders, and/or diseases are well known to those skilled in the art.

In certain embodiments, the infection, disorder, and/or disease exhibits resistance to treatment, preferably the infection, disorder, or disease exhibits antibiotic resistance.

In a specific embodiment, the subject is a vector according to the present invention, in particular a vector packaged in a delivery vehicle according to the present invention, preferably a plasmid or phagemid packaged as a bacterial viral particle according to the present invention, or a pharmaceutical or veterinary medicine according to the present invention No treatment was received prior to administration of the composition.

In a specific embodiment, the subject is a vector according to the present invention, in particular a vector packaged in a delivery vehicle according to the present invention, preferably a plasmid or phagemid packaged as a bacterial viral particle according to the present invention, or a pharmaceutical or veterinary medicine according to the present invention Prior to administration of the composition, it has already received at least one treatment line, preferably several treatment lines.

Preferably, the treatment is administered regularly, preferably between daily and monthly, more preferably between daily and every two weeks, more preferably between daily and weekly, even more preferably the treatment is administered daily. In certain embodiments, the treatment is administered several times per day, preferably twice or three times per day, and even more preferably three times per day.

Duration of treatment with a vector according to the invention, in particular a vector packaged in a delivery vehicle according to the invention, preferably a plasmid or phagemid packaged into bacterial viral particles according to the invention, or a pharmaceutical or veterinary composition according to the invention. preferably comprises between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, still more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks. In certain embodiments, the duration of treatment is about 1 week. Alternatively, treatment may continue as long as the infection, disorder and/or persists.

A vector according to the present invention, in particular a vector packaged with a delivery vehicle according to the present invention, preferably a plasmid or phagemid packaged as a bacterial virus particle according to the present invention, or a pharmaceutical or veterinary composition according to the present invention Route of administration, dosage , and the form of the pharmaceutical or veterinary composition depends on the type and severity of the infection (e.g., depending on the disease, disorder, and/or bacterial species involved in the infection and its localization within the patient or subject body), and the patient or subject, In particular, it can be adjusted by a person skilled in the art according to its age, weight, sex, and general physical condition.

In particular, the amount of a vector according to the invention to be administered, in particular a vector packaged with a delivery vehicle according to the invention, preferably a plasmid or phagemid packaged with bacterial viral particles according to the invention, or a pharmaceutical or veterinary composition according to the invention It should be determined according to standard procedures well known to those skilled in the art. The subject's or patient's physiological data (eg, age, size, and weight) and route of administration should be considered in determining the appropriate dose so that a therapeutically effective amount is administered to the patient or subject.

For example, for each administration, the total amount of a vector, in particular a vector packaged with a delivery vehicle according to the present invention, preferably a plasmid or phagemid packaged in a bacterial viral particle according to the present invention, is between 10 ⁴ and 10 ¹⁵ delivery Includes vehicle.

In another specific embodiment, the modulating method of the present invention is for cosmetic treatment of said host subject.

Phytotherapy and other applications

In another specific embodiment, the host organism is a plant, and the control methods of the present invention are for agronomic, prophylactic or phytotherapeutic treatment of said host plant.

In certain embodiments, the control method is for improving the growth of the host plant, preventing a disease, or treating a disease affecting the host plant.

The present invention also relates to a method for modulating the microbiome from the environment ex vivo, comprising collecting targeted recipient bacterial cells from the environment and delivering a nucleic acid of interest to the targeted recipient bacterial cells of the microbiome. and wherein the nucleic acid of interest produces a desired effect, as described above, on the targeted recipient bacterial cell;

The method comprises contacting a nucleic acid vector containing the nucleic acid of interest with the microbiome,

wherein the vector further comprises an origin of conditional replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell, wherein the vector is free of an antibiotic resistance marker;

thereby delivering the nucleic acid of interest to a targeted recipient bacterial cell, and

Upon delivery to the targeted recipient bacterial cell, the nucleic acid of interest produces the desired effect on the targeted recipient bacterial cell without the vector being replicated in the targeted recipient bacterial cell.

By “environment” we mean herein all the components that surround a species and some of which directly or indirectly contribute to the survival of that species. In certain embodiments, the environment is not an animal. In an alternative embodiment, the environment is an animal, particularly a human.

In certain embodiments, the environment can be any medium in which the microbiome lives, such as a solid or semi-solid surface or a liquid medium, such as water, particularly wastewater.

In certain embodiments, the ex vivo method is for protecting a surface against biofouling. In another specific embodiment, the ex vivo method is for decontamination of water.

The invention is further illustrated through the following figures and examples.

Brief description of the drawing

Figure 1 : BLAST analysis of the origin of PICI-CFT073 in Escherichia coli.

Figure 2 : BLAST analysis of the modified p15a origin of replication of sequence SEQ ID NO: 4 in Escherichia coli.

Figure 3 : BLAST analysis of PICI-CFT073 origin in domain bacteria.

Figure 4: BLAST analysis of modified p15a origins in domain bacteria.

Figure 5 : Transformation of a 2.3 kb payload containing a primase-origin of replication (p1319) in a production strain carrying an inducible primase RBS library in trans.

Figure 6 : Comparison of packaged phagemid titers obtained using plasmids containing primase-ori in production strains (p1319) tested against 7 different primase RBS. Black, right column, control plasmid with p15a-derived origin of replication (p1220). Titration values indicated are after 10X concentration.

7 : Comparison of cells transduced with primase-ori plasmid (top row, p1319) and p15a-based packaged phagemid (p1220) on LB agar supplemented with 25 μg/mL chloramphenicol. RBS 3 is SEQ ID NO: 20.

Figure 8 : E. coli transduced with a nuclease cycle targeting the lacZ gene. Comparison of killing activity of E. coli MG1655 in the absence of antibiotic selection. black line, primase-ori (conditional duplication, p1322); Gray line, modified p15a-ori, replicative (p780).

Figure 9 : Packaged phagemid titers obtained with -12 kb plasmids carrying mutated primase-ori in production strains containing primase RBS 3. Titration values indicated are after 10X concentration. Left bar, lacZ target (p1326); Right bar, 4stx target (p1327).

Figure 10 : Differential effects mediated by targeting lacZ by transduction of a packaged phagemid carrying the conditional origin of replication, payload p1326 (gray line c, strain O157 lacking the lacZ gene served as a non-kill control). Nuclease-mediated killing of strain 0157.

Figure 11 : Nuclease-mediated killing of four O157 strains mediated by stx targeting after transduction of a packaged phagemid carrying a conditional origin of replication (payload p1327).

Figure 12 : Changes in colonization in colonized mice administered orally with neutralization buffer alone ('+ buffer') or 10 ¹² particles of packaged phagemid in neutralization buffer ('+ EB'). Changes in colonization between T0 and T8h are reported for each animal, and the median and quartiles for each experimental group are graphed. ****p < 0.0001 by unpaired t-test.

Figure 13 : E. coli after phagemid transduction in vitro using a payload comprising a conditional origin of replication of the sequence SEQ ID NO: 7. Adenine base editing of β-lactamase on the E. coli genome. 96 individual colonies for each MOI were spotted on LB and LB (Carbenicillin) plates and base editing efficiencies were calculated.

Brief description of sequence

Example

Packaged phagemids are used to deliver DNA payloads to target bacteria with high efficiency. A required property for phagemid packaging is the presence of a functional origin of replication and packaging site in the producer cell line.

The use of a constitutive origin of replication to produce packaged phagemids has several advantages, among others:

• Can be stably maintained in production strains, simplifying manipulation, production and manufacturing processes.

• Some homeostatic ORIs compatible with lambda-based packaging lead to sufficiently high titers (>10 ¹⁰ /mL) required for microbiota-related applications (killing, delivery of therapeutic payloads, etc.).

● Since the payload will be replicated in the target strain once injected, the effect of expression of the gene of interest can last long enough to have the desired result, for example, killing efficiency is greater when delivering a CRISPR-cas system targeted to a chromosomal sequence. The residence time is increased, as it can be higher, since it will be much more difficult to remove the payload by cleavage in the case of the target strain.

Since phages have precise tropism for the same or closely related species from which they are produced, packaged phagemids derived from these phages, when their payloads are delivered to the target bacteria, allow the phages to infect/infect new groups of bacteria. If it has not been manipulated to inject, it will keep replicas.

However, having a homeostatic origin of replication for phagemids can have some risks when used in clinical, industrial, or non-containing settings:

• As the payload is replicative, some injection event will spread the plasmid.

• In addition, when the payload is based on a common origin of replication present in many enterobacteria (eg, a ColE-type origin), there may be a high risk of recombination with plasmids already present in the target bacterial strain. For regulatory purposes, this is problematic because transduced cells are considered GMOs and then replicating GMOs pose a containment risk that must be assessed accordingly.

For all these reasons, the present inventors aimed to develop a conditional replication system that encompasses all of the above mentioned advantages while reducing the risk of proliferation and recombination. Such a system needs to have the following characteristics:

● Replication of the payload should occur only in the production strain, and the payload should be easy to maintain and stable.

• The system must yield a sufficiently high titer (>10 ¹⁰ /mL) to be relevant in an industrial environment.

• The system must be capable of sequence changes if it is necessary to remove a restriction site.

• The system needs to be sufficiently rare in potential target strains to reduce the risk of spread and recombination.

• Finally, the system should allow the gene of interest to be expressed and produce the desired outcome (eg, killing of the target strain at a similar MOI as when using a replicative payload).

In the examples below, we have developed a PICI-based conditional origin of replication.

First, they tested how common the region of origin is in the bacterial genome, in order to assess the probability of unwanted recombination or payload spread events.

Second, they tested in trans primases and ori (on chromosomes or bacteria) to assess whether replication is dependent on the presence of primases and indeed conditional, and to validate titres obtained when these systems are used to package DNA payloads. We developed a system with the ori on the phagemid-primase gene) on another plasmid carried by

Thirdly, they are. In vitro killing of E. coli was tested and compared to that of the current generation of replicative payloads.

Finally, they evaluated whether primase-derived could remove unwanted restriction sites.

In the following examples,

• We show for the first time that phagemids can be packaged with conditional ORIs at high titers.

• We show for the first time that phagemids can be packaged at high titers with conditional ORIs that have oris and proteins necessary for replication in trans.

• We show additional advantages of using the ORI system found in the PICI genome, in contrast to other systems based on plasmid-derived ORI (of bacterial origin), which significantly limit the risk of spread. Furthermore, even if the ORI system is indeed present in the transduced bacteria, it means that native PICI with the same ORI system is present in the bacteria, and must be active against the introduced phagemid to replicate (in the lytic cycle), and the PICI This is because the primase gene is inactive if it is not found in the induced (soluble) state. This is completely different from the bacterial ORI, as it is meant to be naturally and constitutively active.

Example One

this. ori region blasting for frequent evaluation in E. coli and other bacteria

A 282 bp region immediately after the stop codon of PICI-CFT073 primase (SEQ ID NO: 4) was used for BLAST against all sequenced Escherichia coli genomes and filtered to give a maximum of 20,000 hits.

As shown in Figure 1, all sequenced E. Among the E. coli genomes, there are only 90 hits, meaning that these specific primase-ori are very rare, thus significantly reducing the risk of recombination and duplication in target and non-target strains.

Also under normal circumstances, the primase of PICI is inactive, so even if an injection occurs in a strain containing this specific PICI, it will not replicate unless the cells are under phage-induced conditions, which is an undesirable effect on the introduced payload. It is also worth noting that it means further reducing the chance of duplication.

As a comparison, performing a BLAST analysis with the non-conditionally modified p15a-based origin of replication returned the hits shown in FIG. 2 .

884 sequences were identified. It is also worth noting that when sequencing strains, plasmids can be excluded from assembly if they are small (e.g., pOSAK found in strain wSTEC O157), resulting in a higher number of hits.

Next, we performed the same search, but this time with a different B-E. Domain Bacteria was used to assess the presence of PICI-ori in E. coli species: 165 hits were found for PICI origins, while more than 2000 hits were found for p15a-based origins (see Figures 3 and 4).

In conclusion, we showed that primase-ori was a good candidate for reducing the risk of recombination and unwanted replication in target and non-target bacteria, based on BLAST analysis, its presence compared to p15a-based origins. This is because it is 10 to 20 times lower, and for effective replication, cells injected with the payload need to proceed with active phage production against the PICI primase present.

Example 2

phagemid packaging and commercially available transro primase - ori development of a system with

Next, we sought to develop a system in which the payload contains a 282-bp primase origin and the primase protein is supplied in trans (SEQ ID NO: 8 and SEQ ID NO: 9). To simplify the manipulation process, the PICI primase gene was used in E. coli. It was extracted from the genome of E. coli CFT073 and cloned into a plasmid under the control of an inducible system to generate a ribosome-binding site (RBS) library. This series of plasmids was cloned into the lambda production strain s1965. Next, we constructed a small payload bearing a primase-ori instead of a p15a-based origin of replication to yield the 2.3 kb payload p1319 (SEQ ID NO: 16). Since these plasmids are principally non-replicating, competent cells of s1965 carrying an RBS library of inducible primase constructs were created, the p1319 plasmid was transformed into them and expression of the primase was induced in trans with the inducer DAPG ( in the presence of) on LB agar + kanamycin and chloramphenicol. The next day, we observed that the plate contained hundreds of colonies, suggesting that the primase-based system was operated in trans (Fig. 5).

Several clones were sequenced to verify that the p1319 plasmids did not contain a p15a-based origin and that they also contained the intact primase gene with RBS from the library.

Subsequently, 7 of these clones were grown overnight and lambda production was performed in the presence of kanamycin, chloramphenicol and DAPG. As a control, we included an original 2.8 kb plasmid containing a derivative of the p15a origin of replication (p1220, SEQ ID NO: 17) to compare titres.

To verify the sequence of the obtained RBS variants, plasmids encoding the inducible primase in the 7 clones tested were miniprepped and sequenced (SEQ ID NOs: 18 to 24). They were also transformed into MG1655 cells (s003), and these strains were used to validate the titers obtained, as the payload should not be replicable in the absence of the primase protein supplied in trans.

As can be seen in FIG. 6 , as measured in MG1655 containing the primase plasmid in trans, the titer of 5 out of 7 primase-containing samples was the same as that of the packaged phagemid originally having a modified p15a origin.

Finally, we tested whether the primase-ori containing payload could be replicated in trans in the MG1655 strain without the primase plasmid. To this end, serial 5X dilutions of primase-ori containing plasmids derived from production strains with different primase RBS, plus a p15a-derived control, were transduced into dense cultures (OD600 ∼0.8) of MG1655; Plated on LB agar plates containing chloramphenicol. As can be seen in Figure 7, the p15a-derived control shows healthy colonies up to the last dilution, indicating active plasmid replication, while the sample containing the primase-containing payload shows colonies only at high MOI, as the strain is not ready for division. Because division is stopped at high cell densities, these droplets containing high numbers of transduced bacteria appear as dense specks, and as the MOI decreases, the specks become increasingly transparent and single Colonies are indistinguishable, meaning cells that die due to loss of plasmid and exposure to antibiotics. This also implies a burst of expression of the chloramphenicol acetyltransferase gene upon transduction, which, in the absence of active replication, will dilute over time, which reduces the level of chloramphenicol acetyltransferase below a critical level to support growth in antibiotic-supplemented media. It will allow the recipient cells to survive for some amount of time until the intracellular concentration drops.

In conclusion, the PICI primase and origin can be stably maintained in the production strain, are compatible with lambda-based phagemid packaging judged by titres obtained, and the payload has its potential for active replication and maintenance in the target strain. dependent on the presence of the cognate primase.

Example 3

Those who used the conditional origin of replication. In vitro killing of E. coli

Next, the inventors tested whether sequence-specific killing mediated by Cpf1 nuclease still occurs in cells transduced by the packaged phagemid. As cells lose plasmids by division, the initial burst of expression of the nuclease cycle was ignored if it was still sufficient to obtain killing at an MOI similar to that observed in the homeostatic origin of replication.

To this end, we constructed a large plasmid plasmid (∼12 kb) in which the p15a-based origin of replication was exchanged for the primase origin. This plasmid targeted the lacZ gene (p1322, SEQ ID NO: 25) and also contains a chloramphenicol marker. Since the RBS strength was ignored if necessary to be modified to replicate the large plasmid, we tested this with the production strain s1965, which carries an inducible primase RBS library in trans, as was done earlier with smaller payloads. The plasmid was transformed. The next day, we observed that the plate contained hundreds of colonies. One of these colonies was selected and sequenced to verify that the payload contained the primase-ori, the RBS of the primase in trans (SEQ ID NO: 26) was sequenced and a packaged phagemid was produced. As a control, we produced the same phagemid containing a p15a-based origin of replication (p780, SEQ ID NO: 27) from the same production strain.

In this case, since the payload targets the MG1655 strain, we verified the titre of production in a derivative of MG1655 lacking the lacZ gene (s248) and containing the primase RBS 3 plasmid (p1321) in trans.

Titers of both packaged phagemids whose payloads contain both constitutive and conditional origins of replication are indistinguishable after 10X concentration, approximately 1.5 x 10 ⁸ /μL, suggesting that this approach is also feasible for larger payloads. do.

Next, we tested whether killing of the target strain by the packaged phagemid is possible in the absence of selection and active replication of the payload, as we have already demonstrated in a p15a-based origin. For this, this. A culture of E. coli MG1655 was grown in LB+CaCl ₂ to an OD600 of about 0.8 and diluted in LB+CaCl ₂ to an OD=0.025. Packaged phagemids targeting lacZ and containing p15a-based origin (control) or primase origin were serially diluted 3X; This approach allowed testing of different MOIs. 90 μL of cells were added to each well containing the packaged phagemid dilution. After 30 min-incubation at 37°C, a 10X dilution of each reaction was performed and 10 μL was plated onto LB agar plates and incubated overnight at 37°C.

As can be seen in Figure 8, the behavior of the p15a-containing nuclease payload was indistinguishable from payloads containing the primase conditional origin: about 2-log kill at an MOI of 10.

In conclusion, a conditional origin of replication based on PICI allows for high titer production and selection of large payloads (∼12 kb) and nuclease-mediated killing of target strains in the absence of primase proteins.

Example 4

pici -removal of restriction sites from the derived origin of replication

Finally, we tested whether the PICI origin of replication is suitable for the removal of restriction sites present in certain target strains, since the presence of such sites will result in the payload being degraded in the target strain prior to expression of the nuclease gene. Nuclease-specific killing may be completely abolished.

To this end, we analyzed the 282-bp PICI origin and found that it contained the O157 restriction site GAAABCC (GAAAGCC). We modified this site in the origin and obtained the sequence GAAAGCa (small cap means introduced mutation) which is not conscious by strain O157. The modified PICI origin (SEQ ID NO: 6) is a Cpf1 nuclease cycle (p1326, SEQ ID NO: 28) targeting the lacZ gene as mentioned in Example 3 and a quadruplex crRNA targeting the stx1 and stx2 genes Cloned into a -12 kb payload containing guide (p1327, SEQ ID NO: 29).

We have previously designed a bacterial cell line that produces an engineered lambda-based capsid containing chimeric 1A2 gpJ protein and chimeric STF-V10 [Helix], which can be efficiently injected into strain O157 (s15816) and , so these two plasmids were transformed in trans into this production strain containing the primase RBS 3.

Colonies were readily obtained, suggesting that the mutation introduced into the origin does not affect the ability of PICI primase to recognize and replicate. Sequencing results verified the presence of a modified, deltaGAAABCC primase origin of replication.

Packaged phagemids were produced from these two strains and titrated against variants of MG1566 recognized by this special packaged phagemid, supplemented with a plasmid encoding primase RBS variant 3 (s18241).

As can be seen in Figure 9, the titre is equivalent to either the p15a-containing origin or the non-mutated PICI origin (>1x10 ⁸ /μL after 10X concentration).

Finally, two killing experiments were performed on the 0157 strain as described above for MG1655:

• Killing using the lacZ target in two O157-delta-stx strains (s2185 and s17465). As a control for non-specific killing, the packaged phagemid was also transduced into strain s11983, a derivative of the O157 H10dstx strain lacking the lacZ gene.

● Killing using a quadruplex crRNA guide targeting the stx target in four wild-type O157 strains (s13861, s13862, s13863, s13864).

Briefly, cell cultures were brought to OD600 = 0.025, and packaged phagemids were serially diluted 1:3. 90 μL of cell culture was added to the packaged phagemid dilution, incubated for 30 min at 37° C., and serial 10× dilutions were performed to allow for cytometry. 10 μL of each dilution was spread on LB agar.

As can be seen in Figures 10 and 11, both packaged phagemids targeting the lacZ or stx genes were effective, and the MOI required for killing was equivalent to that obtained with packaged phagemids containing homeostatic replication origins in the absence of antibiotic selection. do. Strains that do not contain the target (s11983) are, as expected, not killed at all, suggesting little to no non-specific killing. Additionally, when plated on selective medium (LB agar containing chloramphenicol), the non-targeted strain shows a profile similar to that identified for MG1655: dense specks at high MOI and low dilution (cells are actively cannot divide and lose plasmid) At low MOI and high dilution, weak density spots are translucent, indicating cell death due to exposure to antibiotics.

Example 5

having a conditional origin of replication as payload in vivo decolonization

This example demonstrates efficient decolonization in vivo by specifically killing bacteria carrying the six gene using a packaged phagemid having a conditional origin of replication.

Materials and Methods

Streptomycin-treated mice have either a target bacterial strain (hereinafter referred to as 'target strain') or a gene of particular interest, the stx gene (hereinafter referred to as 'non-target strain'), in order to establish robust intestinal colonization of these bacterial strains. Mutants of the same bacterial strain that were deleted were administered orally.

The plasmid of sequence SEQ ID NO: 10, which has the conditional origin of replication of sequence SEQ ID NO: 7 and encodes a nuclease and its guide targeting the aforementioned stx gene, contains chimeric 1A2 gpJ protein and chimeric STF- V10[Helix] (1A2-V10 packaged phagemid), which is packaged in an engineered lambda-based capsid.

Mice colonized with both strains were given 100 μl of packaged phagemid (approximately 10 ¹² particles) and 100 μl of buffer (sucrose and bicarbonate in water) for the purpose of temporarily neutralizing gastric pH. A separate group of mice colonized with the target strain received buffer only, to account for natural variations in colonization levels over the time course of the experiment.

Bacterial colonization levels were determined non-invasively by plating agar plates of dilutions of feces recovered from each animal individually.

These levels were compared before treatment was initiated (referred to as 'T0') and 8 hours after treatment (referred to as 'T8h'), and the change in colonization between T8h and T0 was calculated for each animal, as the log change. indicated (see Figure 12).

result

The pH-neutralizing buffer alone had no effect on the level of target strain colonization, but the packaged phagemid resulted in a 3.5-log reduction in the bacterial burden recovered from feces at 8 hours after oral administration. As expected, the packaged phagemid had no effect on the level of colonization of non-target strains, demonstrating the specificity of the packaged phagemid for their target sequences.

Thus, these results demonstrate that efficient in vivo killing of a targeted bacterium can be obtained by delivering to the targeted bacterium a packaged phagemid having a conditional origin of replication that is inactive in the targeted bacterium, and that the phagemid is unable to replicate in the targeted bacteria.

Example 6

having a conditional origin of replication payload using in vitro phagemid After transduction E. coli β- on the genome lactamase Adenine base editing.

This example is based on primase-helicase, after phagemid transduction in vitro using a payload comprising a conditional origin of replication of the sequence SEQ ID NO: 7 E. coli. A method for base editing of a nucleic acid sequence encoding β-lactamase (SEQ ID NO: 30) on the E. coli MG1655 genome is provided.

The non-replicating payload includes an adenine base editor (ABE8e), a transcribed guideRNA targeting the active site of the β-lactamase gene (K71E) on the genome, a lambda packaging sequence, a chloramphenicol resistance marker, and the sequence SEQ ID NO: 7 contains the conditional origin of replication of this. Production of lambda phagemid, packaged inside a bacterial delivery vehicle containing A8 gpJ protein and EB6 STF protein for delivery to E. coli MG1655, resulted in a titer of 6.7 Х 10 ⁶ transducing units per μl (tu/μl).

Transduced cells were plated on LB plates 2 hours after transduction at different multiplicities of infection (MOIs). The next day, 96 individual colonies for each MOI were spotted on LB and LB (Carbenicillin) to assay for base editing efficiency.

As shown in Figure 13, the efficiency of adenine base editing targeting the active site of the β-lactamase gene (K71E) on the genome was multiplicity of infection (MOI)-dependent. A base editing efficiency of -63% of the bacterial population was obtained at a high MOI using a payload containing a conditional origin of replication.

<110> ELIGO BIOSCIENCE <120> MICROBIOME MODULATION OF A HOST BY DELIVERY OF DNA PAYLOADS WITH MINIMIZED SPREAD <130> EB2020-06 <150> US63/132,190 <151> 2020-12-30 <150> US63/132,090 <151> 2020-12-30 <150> PCT/EP2020/088043 <151> 2020-12-30 <150> US17/138084 <151> 2020-12-30 <150> US63/137,989 <151> 2021-01-15 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> PRT <213> artificial sequence <220> <223> insulin B9-25 epitope <400> 1 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe 1 5 10 15 Phe <210> 2 <211> 15 <212> PRT <213> artificial sequence <220> <223> T cell (Beta2GPI) epitope <400> 2 Lys Val Ser Phe Phe Cys Lys Asn Lys Glu Lys Lys Cys Ser Tyr 1 5 10 15 <210> 3 <211> 11 <212> PRT <213> artificial sequence <220> <223> B cell epitope <400> 3 Val Ser Arg Gly Gly Met Arg Lys Phe Ile Cys 1 5 10 <210> 4 <211> 282 <212> DNA <213> artificial sequence <220> <223> primase ori from the PICI of the Escherichia coli strain CFT073 <400> 4 tttgttgcaa tggctgtcta ccctgtctac ctgagtaaag aaaaatacat ttaattcagt 60 acattaactt gggtagacag ccttttttta ctgtctacct actatctacc ctctctacct 120 gattttacct gaatcagaca gggaggtaga tacggggtag atagtggata aaagcactct 180 accccactga aagccgcgcc attactggca tggtggccag taaggtagat aaggtagaca 240 aggggaggca caactcaaaa ctttttaaac gagggggtaa aa 282 <210> 5 <211> 13 <212> DNA <213> artificial sequence <220> <223> Restriction site <220> <221> misc_feature <222> (5) <223> n may be any nucleotide <220> <221> misc_feature <222> (6) <223> n may be any nucleotide <220> <221> misc_feature <222> (7) <223> n may be any nucleotide <220> <221> misc_feature <222> (8) <223> n may be any nucleotide <220> <221> misc_feature <222> (9) <223> n may be any nucleotide <220> <221> misc_feature <222> (10) <223> n may be any nucleotide <400> 5 twcannnnnn tgg 13 <210> 6 <211> 282 <212> DNA <213> artificial sequence <220> <223> Primase ori deltaGAAABCC <400> 6 tttgttgcaa tggctgtcta ccctgtctac ctgagtaaag aaaaatacat ttaattcagt 60 acattaactt gggtagacag ccttttttta ctgtctacct actatctacc ctctctacct 120 gattttacct gaatcagaca gggaggtaga tacggggtag atagtggata aaagcactct 180 accccactga aagcagcgcc attactggca tggtggccag taaggtagat aaggtagaca 240 aggggaggca caactcaaaa ctttttaaac gagggggtaa aa 282 <210> 7 <211> 282 <212> DNA <213> artificial sequence <220> <223> Primase ori devoid of restriction sites <400> 7 tttgttgcaa tggctgtcta ccctgtctac ctgagtaaag aaaaatacat ttaattcagt 60 atattaactt gggtagacag ccttttttta ctgtctacct tctgtctacc ctctctacct 120 gattttacct gaatcagaca gggaggtaga cacggggtag acagtggata aaagcactct 180 accccactga aagcagtgcc attactggca tggttgccag taaggttgat aaggtagaca 240 aggggaggga caactcaaaa ctttttaaac gagggggtaa aa 282 <210> 8 <211> 584 <212> PRT <213> artificial sequence <220> <223> PICI primase-helicase <400> 8 Met Lys Leu Ala Pro Asn Val Lys Gln Gln Ser Arg Gly Ile Lys His 1 5 10 15 Lys Glu Thr Glu Val Ile Ile Phe Ala Gly Ser Asp Ala Trp Ser His 20 25 30 Ala Lys Gln Trp Gln Glu His Asp Ala Arg Met Ala Gly Asp Asn Glu 35 40 45 Pro Pro Val Trp Leu Gly Glu Gln Gln Leu Ser Glu Leu Asp Lys Leu 50 55 60 Gln Ile Val Pro Glu Gly Arg Lys Ser Val Arg Ile Phe Arg Ala Gly 65 70 75 80 Tyr Leu Ala Pro Val Met Ile Lys Ala Ile Gly Gln Lys Leu Ala Ala 85 90 95 Ala Gly Val Gln Asp Ala Asn Phe Tyr Pro Asp Gly Met His Gly Gln 100 105 110 Lys Val Glu Asn Trp Arg Glu Tyr Leu Ala Arg Glu Arg Gln Asn Leu 115 120 125 Ser Asp Gly Leu Val Ile Glu Leu Pro Val Lys Gln Lys Ala Gln Leu 130 135 140 Ser Gln Met Ala Asp Ser Glu Arg Ala Gln Leu Leu Ala Asp Arg Phe 145 150 155 160 Asp Gly Val Cys Val His Pro Glu Ser Glu Ile Val His Val Trp Cys 165 170 175 Gly Gly Val Trp Cys Pro Val Ser Thr Met Glu Leu Ser Arg Glu Met 180 185 190 Val Ala Ile Tyr Ser Glu His Arg Ala Thr Phe Ser Lys Arg Val Ile 195 200 205 Asn Asn Ala Val Glu Ala Leu Lys Val Ile Ala Glu Pro Met Gly Glu 210 215 220 Pro Ser Gly Asp Leu Leu Pro Phe Ala Asn Gly Ala Leu Asp Leu Lys 225 230 235 240 Thr Gly Glu Phe Ser Pro His Thr Pro Glu Asn Trp Ile Thr Thr His 245 250 255 Asn Gly Ile Glu Tyr Thr Pro Pro Ala Pro Gly Glu Asn Ile Arg Asp 260 265 270 Asn Ala Pro Asn Phe His Lys Trp Leu Glu His Ala Ala Gly Lys Asp 275 280 285 Pro Arg Lys Met Met Arg Ile Cys Ala Ala Leu Tyr Met Ile Met Ala 290 295 300 Asn Arg Tyr Asp Trp Gln Met Phe Ile Glu Ala Thr Gly Asp Gly Gly 305 310 315 320 Ser Gly Lys Ser Thr Phe Thr His Ile Ala Ser Leu Leu Ala Gly Lys 325 330 335 Gln Asn Thr Val Ser Ala Glu Met Thr Ser Leu Asp Asp Ala Gly Gly 340 345 350 Arg Ala Gln Val Val Gly Ser Arg Leu Ile Val Leu Ala Asp Gln Pro 355 360 365 Lys Tyr Thr Gly Glu Gly Thr Gly Ile Lys Lys Ile Thr Gly Gly Asp 370 375 380 Pro Val Glu Ile Asn Pro Lys Tyr Glu Lys Arg Phe Thr Ala Val Ile 385 390 395 400 Arg Ala Val Val Leu Ala Thr Asn Asn Asn Pro Met Ile Phe Thr Glu 405 410 415 Arg Ala Gly Gly Val Ala Arg Arg Arg Val Ile Phe Arg Phe Asp Asn 420 425 430 Ile Val Ser Glu Ala Glu Lys Asp Arg Glu Leu Pro Glu Lys Ile Ala 435 440 445 Ala Glu Ile Pro Val Ile Ile Arg Arg Leu Leu Ala Asn Phe Ala Asp 450 455 460 Pro Glu Lys Ala Arg Ala Leu Leu Ile Glu Gln Arg Asp Gly Asp Glu 465 470 475 480 Ala Leu Ala Ile Lys Gln Gln Thr Asp Pro Val Ile Glu Phe Cys Gln 485 490 495 Phe Leu Asn Phe Leu Glu Glu Ala Arg Gly Leu Met Met Gly Gly Gly 500 505 510 Gly Asp Ser Val Lys Tyr Thr Thr Arg Asn Ser Leu Tyr Arg Val Tyr 515 520 525 Leu Ala Phe Met Ala Tyr Ala Gly Arg Ser Lys Pro Leu Asn Val Asn 530 535 540 Asp Phe Gly Lys Ala Met Lys Pro Ala Ala Lys Val Tyr Gly His Glu 545 550 555 560 Tyr Ile Thr Arg Lys Val Lys Gly Val Thr Gln Thr Asn Ala Ile Thr 565 570 575 Thr Asp Asp Cys Asp Ala Phe Leu 580 <210> 9 <211> 1752 <212> DNA <213> artificial sequence <220> <223> PICI primase-helicase <400> 9 atgaaactgg caccgaacgt aaaacagcag tcacgcggca taaaacacaa agaaacagaa 60 gtcattattt ttgcgggtag tgatgcctgg tcacacgcaa aacaatggca ggaacatgac 120 gcgcgtatgg ccggagataa tgagcctcct gtgtggcttg gggagcagca gttatccgaa 180 ctggataagc tgcaaattgt gccggaaggc agaaaatccg tgcgcatatt cagggccgga 240 tatcttgcgc cagtaatgat aaaggcgatt ggtcagaagc tggcggcggc aggcgtacag 300 gatgcaaatt tttaccctga tggtatgcac ggtcagaagg tggagaactg gcgcgaatat 360 ctggcccgtg agcgccagaa tctttctgat ggtctggtca ttgagcttcc ggtaaagcaa 420 aaggcgcaac tttcgcagat ggcggacagt gagcgcgcgc agctgcttgc cgatcgcttt 480 gatggcgttt gcgtacatcc tgaaagtgaa atcgttcacg tatggtgcgg cggggtatgg 540 tgtccggtca gcacaatgga gctgagccgc gaaatggtgg cgatctattc agagcacagg 600 gccactttca gcaagcgcgt aatcaataac gccgtggaag cgttaaaagt tattgccgaa 660 ccaatgggcg agccgtccgg cgatttgctg ccgttcgcca atggtgcgct tgacctgaaa 720 acgggggaat tttccccgca cacgccggag aactggatca ccacgcacaa cggcattgag 780 tacacgccac cagcacccgg ggagaacatc cgcgataacg cgccaaactt tcataaatgg 840 cttgagcacg cagccggaaa agacccgcgc aagatgatgc gtatatgtgc cgcgctgtac 900 atgattatgg cgaaccggta cgactggcag atgtttattg aggccaccgg agacggcggg 960 agcggtaaaa gtacattcac acacatagcc agccttctgg cagggaaaca aaacacggta 1020 agcgctgaaa tgacatcgct tgatgatgct ggtgggcgtg cgcaggttgt cgggagtcgt 1080 cttatcgtcc tggcagacca gccgaaatat acaggcgaag gaacgggcat caagaaaatc 1140 acgggcggcg accccgtgga aattaacccg aaatatgaaa agcgttttac ggcggtaatc 1200 aggggcggtgg tgctggcaac caataacaat ccgatgatat tcaccgaacg ggccggaggt 1260 gtggcacgtc gtcgggtgat attccggttc gataacatcg taagcgaggc agaaaaagac 1320 agggagctac cggaaaagat cgcggctgaa atccctgtca ttatccgccg cttgctggcg 1380 aactttgccg accctgaaaa ggcacgggct ttactcattg aacagcgtga cggtgatgaa 1440 gcactggcaa taaagcaaca gacggatccg ggtattgagt tttgccagtt cctgaatttt 1500 ctggaggaag cacgcggcct gatgatggggc ggcggtggcg attcagtgaa gtacacgacc 1560 agaaacagcc tttaccgcgt ctatctggcg tttatggcgt acgcaggcag gagcaaaccg 1620 ctaaacgtaa atgactttgg caaggctatg aagccagccg cgaaagttta cggacatgaa 1680 tatattacgc ggaaagttaa aggagtaacg cagactaacg caataacaac agacgattgc 1740 gacgcgtttt ta 1752 <210> 10 <211> 11615 <212> DNA <213> artificial sequence <220> <223> payload p1392 plasmid <400> 10 gtttgcaata agggacaagt tacgagtgta gacacgcaga attatccagc ctttagtctt 60 taggaaggca aagctattgt acgcggtagc cgtcgtagca atttaccaac tgtagaatta 120 ttggacacac gtaacaaggg cttacagttg aagtttaata aggtcacacg caaaaccgct 180 aaggaataat cgcaccgtta gcgaaagaat atttcagagc ggttagtaaa ggttgagtaa 240 agtgagattc caaagtgagc ctttataaaa agtaaagagc tataataaaa ccgtcgatcg 300 gaaaacaatc gcctgaaatc tcaagcacgt tgccctttct aacgtcgcta aggtttcgta 360 aacccgtttg attaggaaga agaataagta acccgattag gtttgagatc gcgggttatc 420 ggtttggatt aaaagtggat accagcggag tcaacgccga cgcaaacgta cagtgatcca 480 atcctgttcc acggtcaagc acaatcagct agcaagatct tggaatagag tcgttgcacc 540 gctttgattt acatgctctc cattgcacaa cattccggaa ggactggctt ctctgccatg 600 atcggataat gaaaaacatc agtatgccct gtcatttttc tttgggtgtc ctcaaataat 660 tgccctcacg ttatcgtatg tgacgcgctc atctatgctc gaagtattcc ttgttctccc 720 atcttttaat agaaagtctt taatgaacgt gtcgttacgc agtgtatgaa ctcttgtttt 780 atagggcaga ctttggcgtg gcctaagtgt gttcgataag aaggcaagga caactagctg 840 acgcgctgta atacggatat tatggcacgg ttgatacaaa cgctgatatc ctgatttgct 900 aatgtgccca acactttagt tgagtgccac gttccgacta caagttgctt caagagggga 960 atttggattt ggcaatagcc ccccgtttct acctcaagag gcgacgagta ttaaccgcgc 1020 cagctttcgg cacaagggcc aaagaagatt ccaatttctt attcccgaat aacctccgaa 1080 tccctgcggg aaaatcaccg accgaatagc ctagaagcaa gggggaacag ataggtataa 1140 ttagcttaag agagtaccag ccgtgacaac accgtagtaa ccacaaactt acgctggggc 1200 ttctttggcg gatttttaca gatactaaca aggtgatttg aagtacctta gttgaggatt 1260 taaacgcgct atccggtagt ctacaaattg ggaaataccg ttcaaagagg gctagaatta 1320 cttaaaagcc ttcacaccgc ctgcgctata cgcgcccact ctcccgttta tccgtccaag 1380 cggaagcagg gcgaacttcc gctaagatat tcttacgtgt aacgtagcta agtatcccaa 1440 atagctggcg tacgcgttga acaccgccta gaggatcggg agtcgccgga cgagcgtgtt 1500 attggggact tacgccagcg tagactacaa cgcgcccaga ttaaccctgc acgtattgcc 1560 ttgaataacg tactaatctc tccggctctc gacaatctat cgagcgactc gattatcaac 1620 gggtgtcttg cagttctaat ctcttgcccc cgcccgtaat agcctccaag tgattcaaga 1680 tagtaaaggg caagagctta ttcggcgttg aaggatagcg gactttcggt caaccacaat 1740 tccccactcg acaaaaccag ccgtgcgaag aactctgaaa gtacaagcaa cccaagaggg 1800 ctgagcctaa actcagctaa ttcctaagtg agctaaagac tcgaagtgac agctattaat 1860 aaatagagcg ggaacgtcga acggtcgtga aagtaatagt acaacgggta ttaacttact 1920 gaggatattg cttgaagctg taccgtttta ttgggtgaac gaataagatc cagcaattca 1980 gccaaagaag ctaccaattt ttagtttaag agtgtcacgt ctgacctcgc gggtggatag 2040 ccgaacgtag agcttacgag ccagcggaaa cagtagccgc aggataagta aggggagtaa 2100 gtgatcgaac gaatcagaag tgacaatata cttaggctgg atctcgtccc gtgaatccca 2160 accctcacca actacgagat aagaggtaag ccagaaatcg gcatggtggc gaccaacgac 2220 tgttcccccc ctgtaactaa tcgttccgtc aaaacctgac ttacttcaag gccaattcca 2280 agcgcaaaca ataccgtcct agttcttcgg ttaagtttcc gaagtaggag tgagcctacc 2340 tccgtttgcg tcttgttacc actgacccag ctatttactt tgtattgcct gcaatcgaat 2400 ttctgaactc tcagatagtg gggataacgg gaaagttcct atatttgcga actaacttag 2460 ccgtccacct cgaagctacc tactcacacc caccccgcgc ggggtaaata aggcactaat 2520 cccagcttag agcttgcgta gcacttagcc acaagttaat taacagttgt ctggtagttt 2580 ggcggtatta gcgagatcct agaagcaagg cagagttagt tctaacctaa agccacaaat 2640 aagacaggtt gccaaagccc gccggaaatt aaatcttgct cagttcggta acggagtttc 2700 cctcccgcgt acttaattcc caataagaaa cgcgcccaag tcctatcagg caaaattcag 2760 ccccttcccg tgttagaacg agggtaaaaa tacaagccga ttgaacaagg gttgggggct 2820 tcaaatcgtc gtttacccca ctttacaacg gagggtaagt agttcaccct atagtacgaa 2880 gcagaactat ttcgaggggc gtgcaataat cgaatcttct gcggttgact taacacgcta 2940 gggacgtgcc ctcgattcag tcgcaggtac tcctactcag actgcctcac acccagctag 3000 tcactgagcg ataaaattga cccgccctct aaggtagcga gtacgtccca aagggctccg 3060 gacagggcta tataggagag tttgatctcg ccccgacaac tgcaaccctc aactccctta 3120 gataatattg ttagccgaag ttgcacgacc cgccgtccac ggactgctct tagggtgtgg 3180 ctccttaatc tgacaacgtg caacccctat cgagggcgat tgtttctgcg aaaggtgttg 3240 tcctaatagt cgcgacattt ggcccttgta ggtgtgaaac cacttagctt cgcgccgtag 3300 tcctaaaggc ccacctattg actttgtttc gggtagcact aggaatctta acaatttgaa 3360 tttggacgtg gaacgcgtac accttgatct tcgaataatt ctagggattt ggaagtcctc 3420 tacgttgaca cacctacaat gctccaagta aatatacgaa taacgcgggc ctcgcggagc 3480 cgttccgaat cgtcacgtgt tcgtttactg ttaattggtg gcaaataagc aatatcgtag 3540 tccgtcaggc ccagccctgt tatccacggc gttatttgtc aaattgcgta gaactggatt 3600 gactgcctga caatacctaa ttatcggtac gaagtccccg aatctgtccg gctatttcac 3660 taatactttc caaacgcccc gtatccaaga agaacgaatt tatccacgct cccgtctttg 3720 ggacgaatac cgctacaagt ggacagagga tcggtacggg cctctaataa atccaacact 3780 ctacgccctc ttcaagagct agaagaacag ggtgcagttg gaaagggaat tatttcgtaa 3840 ggcgagccaa taccgtaatt aattcggaag agttaacacg attggaagta ggaatagttt 3900 ctaaccacgg tactaatcc taataacgga acgctgtctg atagattagt gtcagcgctc 3960 actaccaaag aaaaataaaa agacgctgaa aagcgtcttt ttatttttcg gtccagtgta 4020 actcaggcaa aagcacgtaa tattcgtact caccaaacga aactcatccg gcgcatcgcg 4080 cttcttcctc cgtaagcgtc acccccatta cttaaagagt gcatgtgcat attttgttat 4140 caataaaaaa ggccgcgatt tgcggcctta ttgttcgtct tgccggatta gatagctacc 4200 ggtgctttaa tacccggatg cggatcatag ccttcgattt cgaagtcctc aaaacgataa 4260 tcgaagatgc tttccggttt gcgtttgata atcagtttcg ggagcgggcg tggctcacgg 4320 cttaattgta aatgcgtctg atccatgtga tttgagtaca ggtgagtatc cccaccagtc 4380 caaacaaagt caccaacttc cagatcacac tgctgtgcca tcatatgaac taataaggcg 4440 taggaggcaa tgttaaacgg taagcccaga aacacgtcgc aagaacgctg gtacagttgg 4500 cacgataact taccatccgc aacatagaat tgaaagaagg catgacacgg tgctaaagcc 4560 attttgtcta attcccccac gttccatgcg gacacgataa tccggcgaga gtccggatca 4620 tttttcagtt ggttaagaac ggtagtgatc tgatcaatat gccgaccatc cggcgtaggc 4680 catgcacgcc attgcttacc atacactggc cctaagtcac cgttttcatc tgcccactca 4740 tcccagatgg taacgttatt ctcgtgcagg tacgcaatgt tcgtatcgcc ttgcagaaac 4800 cataataact cgtgaataat agaacggagg tggcaacgct tggtagtgac cagcgggaaa 4860 ccgtcttgca ggttgaaacg catctgatga ccaaagatag acagcgtacc agtgccagta 4920 cgatcattct tctgagtgcc ttcgtccagc actttttgca tcagttccag atactgtttc 4980 attttagctt ccttagcttg cgaaatctcg ataactcaaa aaatagtagt gatcttattt 5040 cattatggtg aaagttgtct tacgtgcaac attttcgcaa aaagttggcg ctttatcaac 5100 actgtccgaa tgacaaatgg ttacaattat tgaacaccct tcggggtgtt tttttgtttc 5160 tggtttcccg aggccgaact tttgttgcaa tggctgtcta ccctgtctac ctgagtaaag 5220 aaaaatacat ttaattcagt atattaactt gggtagacag ccttttttta ctgtctacct 5280 tctgtctacc ctctctacct gattttacct gaatcagaca gggaggtaga cacggggtag 5340 acagtggata aaagcactct accccactga aagcagtgcc attactggca tggttgccag 5400 taaggttgat aaggtagaca agggggaggga caactcaaaa ctttttaaac gagggggtaa 5460 aacgcagatc aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga 5520 tttcagtgca atttatctct tcaaatgtag caccggcgcg ccgtgaccaa ttattgaagg 5580 ccgctaacgc ggcctttttt tgtttctggt ttcccgaata gagcgacttc tccccaaaaa 5640 gcctcgcttt cagcacctgt cgtttccttt cttttcagag ggtattttaa ataaaacat 5700 taagttatga cgaagaagaa cggaaacgcc ttaaaccgga aaattttcat aaatagcgaa 5760 aacccgcgag gtcgccgccc cgtaacctgt cggatcaccg gaaagaacct gtaaagtgat 5820 aatgattatc atctacatat cacaacgtgc gtaaagggta agtatgaagg tcgtgtactc 5880 catcgctacc aaattccaga aaacagacgc tttcgagcgt cttttttcgt tttggtcacg 5940 acgtacggtg gaagattcgt taccaattga cagctagctc agtcctaggt atatacatac 6000 atgcttgttt gtttgtaaac tactgttttc attaaagagg agaaaggaag ccatgtccat 6060 ctatcaggag tttgttaaca agtattccct gtctaaaacc ctgcgttttg aactgatccc 6120 gcagggcaaa actttggaaa acattaaagc gcgtggcctg attctggatg acgaaaaacg 6180 tgcaaaggat tacaagaaag ctaaacagat catcgacaaa tatcaccagt tctttatcga 6240 agaaattctg tcctcggtgt gcatcagtga ggatctgtta cagaattatt ctgatgtata 6300 ctttaaactt aaaaagtccg atgacgataa tctgcaaaaa gatttcaagt cagccaaaga 6360 taccatcaag aaacagatct cagaatatat taaagatagc gaaaagttca aaaacctgtt 6420 taaccaaaac ctcattgatg ctaagaaagg ccaagaatct gacctgatct tatggctgaa 6480 acagagcaaa gataacggca ttgaactgtt caaagctaat agcgacatca ccgatattga 6540 tgaagcgctc gaaatcatca agtctttcaa aggctggacg acgtatttca aaggttttca 6600 tgaaaaccgt aagaatgtat attcgagcaa cgatattccg acctctatta tttatcgtat 6660 cgtggacgac aacctgccga agtttctgga aaacaaagcg aaatatgaat ctctgaaaga 6720 caaagcaccg gaagctatta actatgaaca gatcaagaaa gatctggcgg aagaactgac 6780 cttcgacatc gactataaaa cctccgaagt taaccagcgt gttttctcac tggacgaggt 6840 tttcgaaatc gctaatttca acaattacct gaatcaatct ggcatcacca aattcaacac 6900 cattattggt ggcaaatttg ttaacggcga aaacaccaag cgtaagggca tcaacgaata 6960 cattaacctc tatagccaac aaatcaacga caaaaccctg aaaaagtata aaatgtccgt 7020 tctgtttaaa cagattttat cggacaccga atctaaatcc ttcgtaattg ataaactgga 7080 agatgatagc gacgttgtca ccacgatgca gagcttttat gagcagattg cggcgttcaa 7140 aaccgtcgaa gagaaatcta ttaaagaaac tctgtccctg ctctttgacg acctcaaagc 7200 gcagaaacta gatctgtcta agatttactt taaaaacgac aaatctctga ccgatctcag 7260 tcaacaagtt ttcgatgact atagcgtgat cggcacggca gttttggaat acatcaccca 7320 acaaatcgcg ccgaaaaatc tggacaaccc gtccaagaag gaacaggaac tgattgcaaa 7380 gaaaacagaa aaagctaaat acctgagctt agaaactatc aaactggcac ttgaggaatt 7440 taataaacat cgtgatattg ataaacagtg tcgttttgag gaaattctgg cgaactttgc 7500 ggcaatcccg atgatcttcg acgaaattgc tcaaaacaaa gacaatctgg cgcagatctc 7560 tatcaagtac cagaatcagg gtaagaaaga tctgcttcaa gcatctgcgg aggacgatgt 7620 caaagcaatt aaagacttat tagatcagac gaataactta ttacacaagc tcaaaatctt 7680 ccacatcagc cagagcgagg acaaggcgaa cattctggat aaagatgaac acttctatct 7740 ggtgttcgaa gaatgttact tcgaactggc aaacatcgta cctctctaca ataaaatccg 7800 caactacatc acgcagaagc cttacagtga cgagaaattc aaactgaact tcgaaaacag 7860 cacgctggcg aacggctggg ataagaacaa agagccggac aacaccgcaa tcctgttcat 7920 caaagacgac aaatactatc tgggcgtaat gaacaagaag aacaacaaga tcttcgacga 7980 taaagcgatc aaagaaaaca agggtgaagg ctataagaaa atcgtgtaca agctcctgcc 8040 gggtgcgaac aaaatgttac cgaaagtgtt cttttccgcg aaaagcatca aattctacaa 8100 cccgtctgag gatattctgc gcatccgcaa tcatagcacg cacactaaaa acggtagccc 8160 gcagaaaggg tatgaaaaat tcgaatttaa tatagaggac tgccgtaaat tcatcgactt 8220 ctataaacag agcatttcca aacatccgga atggaaagac ttcggcttcc gtttctctga 8280 cactcagcgc tataatagca tcgacgagtt ctaccgcgaa gtggagaatc agggctataa 8340 actgaccttc gagaacatta gtgagtcgta catcgactcc gttgtgaatc agggtaaact 8400 gtacctgttt cagatctata ataaagactt tagcgcgtac agcaaaggcc gcccgaatct 8460 gcacaccctt tactggaaag cattatttga cgaacgtaac ctgcaagatg tggtgtataa 8520 actgaacggt gaggcggaac ttttctaccg taaacagagt atcccgaaga aaatcacgca 8580 tccggcaaaa gaagctattg ccaacaaaaa caaagacaac ccgaagaaag aaagtgtatt 8640 cgaatatgac ctgatcaaag ataaacgttt caccgaagat aagttctttt tccactgtcc 8700 gattaccatc aacttcaaat ctagcggtgc gaacaagttc aacgatgaaa ttaacttatt 8760 actgaaagag aaagctaatg acgtacacat cttatctatt gatcgcggtg aacgtcattt 8820 agcatactat acactggtag acggtaaagg taatattatt aaacaggata ctttcaatat 8880 tatcggtaat gaccgtatga aaaccaacta tcacgataag ctggcggcga tcgaaaaaga 8940 tcgtgattct gcgcgtaaag attggaagaa aattaacaat atcaaagaaa tgaaagaagg 9000 ctatctgagc caagtggtgc acgagatcgc aaaactggtg attgaatata acgctatcgt 9060 ggttttcgaa gatctgaact ttggttttaa acgtggtcgc ttcaaagtag aaaaacaggt 9120 gtaccaaaaa ctggaaaaaa tgctgattga aaaactgaac tatctggttt ttaaagacaa 9180 cgaatttgac aaaacgggtg gcgtactccg tgcctatcag cttaccgctc cgttcgaaac 9240 gtttaagaaa atgggtaaac aaacggggat tatctattat gtgccagccg gtttcacctc 9300 caagatttgt ccagttacgg gcttcgttaa ccagctttac ccgaaatacg agagcgttag 9360 caaatctcaa gaatttttca gcaaattcga caagatctgc tataatctgg ataaaggcta 9420 tttcgagttc agctttgatt acaaaaactt cggcgataaa gcggctaaag gtaagtggac 9480 tattgctagc tttggtagcc gtctgattaa ctttcgcaac tccgacaaaa accataattg 9540 ggacacgcgt gaagtgtatc cgaccaaaga actggaaaaa ttactgaaag actattccat 9600 cgaatatggt catggggagt gcattaaagc ggcgatttgc ggtgaatccg ataagaaatt 9660 tttcgccaaa ctgaccagcg tgcttaacac cattctccaa atgcgtaatt ctaaaacggg 9720 tacggagctt gactacctga tttctccggt agccgacgtt aacggcaact tcttcgattc 9780 tcgtcaagca ccgaaaaata tgccacaaga cgcggatgcc aacggtgcat accatatcgg 9840 ccttaaaggc ttaatgttat taggccgtat caagaataat caggagggca agaaattaaa 9900 tctggttatc aaaaacgaag aatacttcga gttcgttcag aatcgtaaca attaatgtat 9960 gcttaagcag atcggtaata aagacgaaca ataagacgct gaaaagcgtc ttttttcgtt 10020 ttggtcctgt tccggcgcga tagtgtgaac atgctataga cttctggtgc tacccgactg 10080 acaattaatc atccggctcg tataatgcta gcaatttcta ctgttgtaga tcattccgga 10140 acgttccagc gctgcaattt ctactgttgt agatctgatt tttcacatgt tacctttcaa 10200 tttctactgt tgtagatccg aaaacgtaaa gcttcagctg taatttctac tgttgtagat 10260 atcatatctg gcgttaatgg agtttcgtga cgaacaataa gtcctcccta acggggggca 10320 attttattg ataacaaaag taacttcgag cttgtctacc tcctagctcg taaattgcac 10380 gctgatagtc tcccaattgc gaaggaccaa aacgaaaaaa caccctttcg ggtgtctttt 10440 ctggaatttg gtacgcagta ctaggtatcg tgtaagtagc gaaggcccgt acgcgagata 10500 aactgctagg caaccgcgac tctacgactg gtgctcgatt taatttcgct gacgtaaaga 10560 aattatcggc agtgcgtcaa ctgccgtatc tttatcttaa ttaggtagtt ggacaagccc 10620 ttgaaagaaa tagcaagagc ctgcctctct attgaagtca cggcgaaagt cgggtagaaa 10680 tcaaagaaag cagaaattaa atcggagtaa tactaagttg ggataactcc gtaactgact 10740 acgcctttct ctagacttta cttgaccaga tacactgtct ttgacacgtt gaaggattag 10800 agcaatcaaa tccaagactg gctaagcacg aagcaactct tgagtgttaa aaagttactt 10860 cctgtattcg ggacgagggt actagaagat tgcagggact ccgacgttaa gtaaattaca 10920 aagtaataag tatcgttcag gatcacgtta ccgcaataag aagcgagaat aatataattt 10980 ccgaagtgct taccccagta gtgactattc ctataaccct tctgagtgtc cggaggcgga 11040 aatttgccac gaaagagaaa gtatttcccc gacaataata aaggggcgct cctcagcttt 11100 tccacttggt tgggtaagct aggcaactct gaaaggagtt tcggcgaagt gaagccgaca 11160 cctttgaatt gttttagggg cgttattcga gggcaatcgg agctaacttc aagactactt 11220 ctttgttgaa tactaaatag tgcaaaggtc gtgtttcctc aaggatactc cgctaacaat 11280 ataggattcc aatcagattc agcactggcg gtacgggtgt tgcggtgagg cgttcgggtt 11340 tacggctcga agctagcacg gtaggaagcc tgacaatcac caagcaaaag ggccgtcgaa 11400 ggcccacaag atacgaaagc tctcgaagcc ttatccttga ccgatccacc tatttaggca 11460 gttacgcaca aaagctaccc aataatccgt gacaggcaca atatcacgga acaaaaccga 11520 aaactctcgt acacggttag gttttcgcta ggaagaataa acctctatct tgattataag 11580 aaggctcccc aagcaccccc aaaaccgaaa tagcg 11615 <210> 11 <211> 11609 <212> DNA <213> artificial sequence <220> <223> payload p1900 plasmid <400> 11 tcccgcgtac ttaattccca ataagaaacg cgcccaagtc ctatcaggca aaattcagcc 60 ccttcccgtg ttagaacgag ggtaaaaata caagccgatt gaacaagggt tgggggcttc 120 aaatcgtcgt ttaccccact ttacaacgga gggtaagtag ttcaccctat agtacgaagc 180 agaactattt cgaggggcgt gcaataatcg aatcttctgc ggttgactta acacgctagg 240 gacgtgccct cgattcagtc gcaggtactc ctactcagac tgcctcacac ccagctagtc 300 actgagcgat aaaattgacc cgccctctaa ggtagcgagt acgtcccaaa gggctccgga 360 cagggctata taggagagtt tgatctcgcc ccgacaactg caaccctcaa ctcccttaga 420 taatattgtt agccgaagtt gcacgacccg ccgtccacgg actgctctta gggtgtggct 480 ccttaatctg acaacgtgca acccctatcg agggcgattg tttctgcgaa aggtgttgtc 540 ctaatagtcg cgacatttgg cccttgtagg tgtgaaacca cttagcttcg cgccgtagtc 600 ctaaaggccc acctattgac tttgtttcgg gtagcactag gaatcttaac aatttgaatt 660 tggacgtgga acgcgtacac cttgatcttc gaataattct agggatttgg aagtcctcta 720 cgttgacaca cctacaatgc tccaagtaaa tatacgaata acgcgggcct cgcggagccg 780 ttccgaatcg tcacgtgttc gtttactgtt aattggtggc aaataagcaa tatcgtagtc 840 cgtcaggccc agccctgtta tccacggcgt tatttgtcaa attgcgtaga actggattga 900 ctgcctgaca atacctaatt atcggtacga agtccccgaa tctgtccggc tatttcacta 960 atactttcca aacgccccgt atccaagaag aacgaattta tccacgctcc cgtctttggg 1020 acgaataccg ctacaagtgg acagaggatc ggtacgggcc tctaataaat ccaacactct 1080 acgccctctt caagagctag aagaacaggg tgcagttgga aagggaatta tttcgtaagg 1140 cgagccaata ccgtaattaa ttcggaagag ttaacacgat tggaagtagg aatagtttct 1200 aaccacggtt actaatccta ataacggaac gctgtctgat agattagtgt cagcgctcac 1260 taccaaagaa aaataaaaag acgctgaaaa gcgtcttttt atttttcggt ccagtgtaac 1320 tcaggcaaaa gcacgtaata ttcgtactca ccaaacgaaa ctcatccggc gcatcgcgct 1380 tcttcctccg taagcgtcac ccccattact taaagagtgc atgtgcatat tttgttatca 1440 ataaaaaagg ccgcgatttg cggccttatt gttcgtcttg ccggattaga tagctaccgg 1500 tgctttaata cccggatgcg gatcatagcc ttcgatttcg aagtcctcaa aacgataatc 1560 gaagatgctt tccggtttgc gtttgataat cagtttcggg agcgggcgtg gctcacggct 1620 taattgtaaa tgcgtctgat ccatgtgatt tgagtacagg tgagtatccc caccagtcca 1680 aacaaagtca ccaacttcca gatcacactg ctgtgccatc atatgaacta ataaggcgta 1740 ggaggcaatg ttaaacggta agcccagaaa cacgtcgcaa gaacgctggt acagttggca 1800 cgataactta ccatccgcaa catagaattg aaagaaggca tgacacggtg ctaaagccat 1860 tttgtctaat tccccccacgt tccatgcgga cacgataatc cggcgagagt ccggatcatt 1920 tttcagttgg ttaagaacgg tagtgatctg atcaatatgc cgaccatccg gcgtaggcca 1980 tgcacgccat tgcttaccat acactggccc taagtcaccg ttttcatctg cccactcatc 2040 ccagatggta acgttattct cgtgcaggta cgcaatgttc gtatcgcctt gcagaaacca 2100 taataactcg tgaataatag aacggaggtg gcaacgcttg gtagtgacca gcgggaaacc 2160 gtcttgcagg ttgaaacgca tctgatgacc aaagatagac agcgtaccag tgccagtacg 2220 atcattcttc tgagtgcctt cgtccagcac tttttgcatc agttccagat actgtttcat 2280 tttagcttcc ttagcttgcg aaatctcgat aactcaaaaa atagtagtga tcttatttca 2340 ttatggtgaa agttgtctta cgtgcaacat tttcgcaaaa agttggcgct ttatcaacac 2400 tgtccgaatg acaaatggtt acaattattg aacacccttc ggggtgtttt tttgtttctg 2460 gtttcccgag gccgaacttt tgttgcaatg gctgtctacc ctgtctacct gagtaaagaa 2520 aaatacattt aattcagtat attaacttgg gtagacagcc tttttttact gtctaccttc 2580 tgtctaccct ctctacctga ttttacctga atcagacagg gaggtagaca cggggtagac 2640 agtggataaa agcactctac cccactgaaa gcagtgccat tactggcatg gttgccagta 2700 aggttgataa ggtagacaag gggagggaca actcaaaact ttttaaacga gggggtaaaa 2760 cgcagatcaa aacgatctca agaagatcat cttattaatc agataaaata tttctagatt 2820 tcagtgcaat ttatctcttc aaatgtagca ccggcgcgcc gtgaccaatt attgaaggcc 2880 gctaacgcgg cctttttttg tttctggttt cccgaataga gcgacttctc cccaaaaagc 2940 ctcgctttca gcacctgtcg tttcctttct tttcagaggg tattttaaat aaaaacatta 3000 agttatgacg aagaagaacg gaaacgcctt aaaccggaaa attttcataa atagcgaaaa 3060 cccgcgaggt cgccgccccg taacctgtcg gatcaccgga aagaacctgt aaagtgataa 3120 tgattatcat ctacatatca caacgtgcgt aaagggtaag tatgaaggtc gtgtactcca 3180 tcgctaccaa attccagaaa acagacgctt tcgagcgtct tttttcgttt tggtcacgac 3240 gtacggtgga agattcgtta ccaattgaca gctagctcag tcctaggtat atacatacat 3300 gcttgtttgt ttgtaaacta ctgttttcat taaagaggag aaaggaagcc atgaccaaaa 3360 cgtttgatag cgagtttttt aacctgtaca gcctgcaaaa aaccgtgcgc tttgaattaa 3420 aaccagtggg cgaaaccgcg agctttgtgg aagattttaa aaacgaaggc ctgaaacgtg 3480 tggttagcga agatgaacgc cgtgcggtgg attatcagaa agtgaaagaa attattgatg 3540 attatcatcg cgattttatt gaagaaagtc tgaactattt tccggaacag gtgagcaaag 3600 atgcgctgga acaggcgttt catctgtatc agaaattaaa ggccgcgaaa gttgaagaaa 3660 gagaaaaagc gctgaaagaa tgggaagcac tgcaaaaaaa actgcgtgaa aaagtggtga 3720 aatgctttag cgatagcaat aaagcgcgtt tctcccgcat tgataaaaag gaactgatta 3780 aagaagatct gattaactgg ctggtcgcgc agaatcgcga agatgatatc ccgaccgtgg 3840 aaacctttaa caactttacc acgtatttta cgggcttcca tgaaaaccgt aaaaacattt 3900 atagcaaaga tgatcatgcg accgcgatta gctttcgcct gattcatgaa aacctgccga 3960 aattttttga taacgtgatt agctttaaca aactgaaaga aggttttccg gaactgaaat 4020 ttgataaagt gaaagaagat ttagaggtgg attatgatct gaaacatgcg tttgagattg 4080 aatattttgt taactttggg acccaggcgg gcatagatca gtataactat ctgttaggcg 4140 gtaaaaccct ggaagatggc accaaaaagc agggcatgaa tgaacagatt aacctgttta 4200 aacagcaaca aacgcgcgat aaagcgcgtc agattccgaa actgatcccg ctgtttaaac 4260 agattttaag cgaaaggacc gaaagtcaga gctttattcc gaaacagttt gaaagcgatc 4320 aggaattgtt tgatagcttg cagaaattac ataacaactg ccaggataaa tttaccgtgt 4380 tgcaacaagc gattctgggc ctggcggagg cggatctgaa aaaagtgttt attaaaacct 4440 ctgatctgaa cgcgctgtct aacaccattt ttggcaatta tagcgtgttt agcgatgcgc 4500 tgaatctgta taaagaaagt ctgaaaacca aaaaagcgca ggaagcgttt gaaaaactgc 4560 cagcgcatag cattcatgat ctgattcagt atctggaaca gtttaactcc agcttggatg 4620 cggaaaaaca gcaaagcacc gataccgtgc tgaactattt tatcaaaacg gatgaactgt 4680 attctcgctt tattaaaagc accagcgaag cctttaccca ggtgcaaccg ttgtttgaac 4740 tggaagcgct gtccagcaaa cgtcgcccgc cggaaagcga agatgagggc gcgaaaggcc 4800 aggaaggctt cgaacaaatc aaacgtatta aagcgtatct ggataccctg atggaagcgg 4860 tgcactttgc gaaaccgctg tatctggtga aaggtcgtaa aatgatcgaa ggcctcgata 4920 aagatcagag cttttacgaa gcgtttgaaa tggcgtatca ggaattagaa agcttaatca 4980 ttccgatcta taacaaagcg cgtagctatt tgtcgcgcaa accgtttaaa gcggataaat 5040 ttaaaattaa ctttgataac aacaccctgt taagcggttg ggacgcgaac aaagaaaccg 5100 ccaacgcgtc cattctgttt aaaaaagatg gcctgtatta tctgggtatt atgccgaagg 5160 gtaaaacctt tctctttgat tattttgtgt cgagcgaaga tagcgaaaaa ctgaaacagc 5220 gtcgccagaa aaccgccgaa gaagcgctgg cgcaggatgg cgaaagctat tttgaaaaaa 5280 ttcgttataa actgttaccg ggcgcgagca aaatgttacc gaaagtgttt tttagcaaca 5340 aaaacattgg cttttataac ccgagcgacg atattctgcg catccgcaac accgccagcc 5400 ataccaaaaa cggcaccccg cagaaaggcc atagcaaagt ggaatttaac ctgaacgatt 5460 gccataagat gattgatttt tttaaatcca gcattcagaa acatccggaa tggggatctt 5520 ttggctttac ctttagcgat accagcgatt ttgaagatat gagcgcgttt tatcgcgaag 5580 tggaaaatca gggttacgtg attagctttg ataaaatcaa agaaacctat atccagagtc 5640 aggtggaaca gggtaatctg tatctgtttc agatttataa caaagatttt agcccgtata 5700 gcaaaggcaa accaaacctg cacaccctgt attggaaagc gttatttgaa gaagccaacc 5760 tgaataacgt ggtggcgaaa ctgaacggtg aagcggaaat cttttttcgt cgtcatagca 5820 ttaaagcgag cgataaagtg gtgcatccgg caaaccaggc gattgataac aaaaatccgc 5880 ataccgaaaa aacgcagagc acctttgaat atgatctggt gaaagataaa cgctataccc 5940 aagataaatt tttttttcac gtgccgatca gcctcaactt taaagcgcag ggcgtgagca 6000 aatttaacga taaagtgaac ggcttcctga aaggcaaccc ggatgtcaac attattggta 6060 ttgatcgggg cgagcgccat ctgctttatt ttaccgtggt gaatcagaaa ggtgaaattc 6120 tcgttcagga aagcttaaac accctgatga gcgataaagg ccatgtgaac gattatcagc 6180 aaaaactgga taaaaaagaa caggagcgtg atgcggcacg taaatcttgg accacggtgg 6240 aaaacattaa agaattgaaa gaaggctatt taagccatgt ggtgcataaa ctggcgcacc 6300 tgatcattaa atataacgcg attgtgtgcc tggaggacct gaattttggc tttaaacgcg 6360 gtcgctttaa agtggaaaaa caggtttatc agaaatttga aaaagcgctg attgataaac 6420 tgaactatct ggtgtttaaa gaaaaagaat taggtgaagt ggggcattat ctgaccgcgt 6480 atcaactgac cgcgccgttc gaaagcttta aaaaactggg taaacagtct ggcattctgt 6540 tttacgtccc ggcggattat acctccaaaa tcgatccgac cacgggcttc gttaactttc 6600 tggatctgcg ctatcagagc gtggaaaaag cgaaacagct tctgtccgat tttaacgcga 6660 ttcgttttaa cagcgtgcag aactattttg aatttgaaat tgattataaa aaactgaccc 6720 cgaaacgtaa agtcggcacc caaagtaaat gggtatttg cacctatggc gatgtgcgct 6780 atcagaatcg tcgcaatcag aaaggtcatt gggaaaccga agaagtgaac gtgaccgaaa 6840 agctgaaagc gttatttgcg agcgatagca aaacgaccac ggttatcgat tatgccaacg 6900 acgacaacct gattgatgtg attttagaac aggataaagc gagctttttt aaagaattat 6960 tgtggttaact gaaactgacc atgaccctgc gccatagcaa aattaaaagc gaagatgatt 7020 ttattctgtc cccggtgaaa aatgaacagg gtgaatttta tgatagccgt aaagcgggcg 7080 aagtttggcc taaagatgcg gatgccaacg gcgcgtatca tatcgcgctg aaaggccttt 7140 ggaatttaca gcaaattaac cagtgggaaa aaggtaaaac cctgaattta gcgatcaaaa 7200 accaggatg gtttagcttt atccaggaaa aaccgtatca ggaatgatga aagcttatgc 7260 agatcggtaa taaagacgaa caataagacg ctgaaaagcg tcttttttcg ttttggtcct 7320 gttccggcgc gatagtgtga acatgctata gacttctggt gctacccgac tgacaattaa 7380 tcatccggct cgtataatgc tagcaatttc tactgttgta gatcattccg gaacgttcca 7440 gcgctgcaat ttctactgtt gtagatctga tttttcacat gttacctttc aatttctact 7500 gttgtagatc cgaaaacgta aagcttcagc tgtaatttct actgttgtag atatcatatc 7560 tggcgttaat ggagtttcgt gacgaacaat aagtcctccc taacgggggg caatttttat 7620 tgataacaaa agtaacttcg agcttgtcta cctcctagct cgtaaattgc acgctgatag 7680 tctcccaatt gcgaaggacc aaaacgaaaa aacacccttt cgggtgtctt ttctggaatt 7740 tggtacgcag tactaggtat cgtgtaagta gcgaaggccc gtacgcgaga taaactgcta 7800 ggcaaccgcg actctacgac tggtgctcga tttaatttcg ctgacgtaaa gaaattatcg 7860 gcagtgcgtc aactgccgta tctttatctt aattaggtag ttggacaagc ccttgaaaga 7920 aatagcaaga gcctgcctct ctattgaagt cacggcgaaa gtcgggtaga aatcaaagaa 7980 agcagaaatt aaatcggagt aatactaagt tggtataact ccgtaactga ctacgccttt 8040 ctctagactt tacttgacca gatacactgt ctttgacacg ttgaaggatt agagcaatca 8100 aatccaagac tggctaagca cgaagcaact cttgagtgtt aaaaagttac ttcctgtatt 8160 cgggacgagg gtactagaag attgcaggga ctccgacgtt aagtaaatta caaagtaata 8220 agtatcgttc aggatcacgt taccgcaata agaagcgaga ataatataat ttccgaagtg 8280 cttaccccag tagtgactat tcctataacc cttctgagtg tccggaggcg gaaatttgcc 8340 acgaaagaga aagtatttcc ccgacaataa taaaggggcg ctcctcagct tttccacttg 8400 gttgggtaag ctaggcaact ctgaaaggag tttcggcgaa gtgaagccga cacctttgaa 8460 ttgttttagg ggcgttattc gagggcaatc ggagctaact tcaagactac ttctttgttg 8520 aatactaaat agtgcaaagg tcgtgtttcc tcaaggatac tccgctaaca atataggatt 8580 ccaatcagat tcagcactgg cggtacgggt gttgcggtga ggcgttcggg tttacggctc 8640 gaagctagca cggtaggaag cctgacaatc accaagcaaa agggccgtcg aaggcccaca 8700 agatacgaaa gctctcgaag ccttatcctt gaccgatcca cctatttagg cagttacgca 8760 caaaagctac ccaataatcc gtgacaggca caatatcacg gaacaaaacc gaaaactctc 8820 gtacacggtt aggttttcgc taggaagaat aaacctctat cttgattata agaaggctcc 8880 ccaagcaccc ccaaaaccga aatagcggtt tgcaataagg gacaagttac gaggttagac 8940 acgcagaatt atccagcctt tagtctttag gaaggcaaag ctattgtacg cggtagccgt 9000 cgtagcaatt taccaactgt agaattattg gacacacgta acaagggctt acagttgaag 9060 tttaataagg tcacacgcaa aaccgctaag gaataatcgc accgttagcg aaagaatatt 9120 tcagagcggt tagtaaaggt tgagtaaagt gagattccaa agtgagcctt tataaaaagt 9180 aaagagctat aataaaaccg tcgatcggaa aacaatcgcc tgaaatctca agcacgttgc 9240 cctttctaac gtcgctaagg tttcgtaaac ccgtttgatt aggaagaaga ataagtaacc 9300 cgattaggtt tgagatcgcg ggttatcggt ttggattaaa agtggatacc agcggagtca 9360 acgccgacgc aaacgtacag tgatccaatc ctgttccacg gtcaagcaca atcagctagc 9420 aagatcttgg aatagagtcg ttgcaccgct ttgatttaca tgctctccat tgcacaacat 9480 tccggaagga ctggcttctc tgccatgatc ggataatgaa aaacatcagt atgccctgtc 9540 atttttcttt gggtgtcctc aaataattgc cctcacgtta tcgtatgtga cgcgctcatc 9600 tatgctcgaa gtattccttg ttctcccatc ttttaataga aagtctttaa tgaacgtgtc 9660 gttacgcagt gtatgaactc ttgttttata gggcagactt tggcgtggcc taagtgtgtt 9720 cgataagaag gcaaggacaa ctagctgacg cgctgtaata cggatattat ggcacggttg 9780 atacaaacgc tgatatcctg atttgctaat gtgcccaaca ctttagttga gtgccacgtt 9840 ccgactacaa gttgcttcaa gaggggaatt tggatttggc aatagccccc cgtttctacc 9900 tcaagaggcg acgagtatta accgcgccag ctttcggcac aagggccaaa gaagattcca 9960 atttcttatt cccgaataac ctccgaatcc ctgcgggaaa atcaccgacc gaatagccta 10020 gaagcaaggg ggaacagata ggtataatta gcttaagaga gtaccagccg tgacaacacc 10080 gtagtaacca caaacttacg ctggggcttc tttggcggat ttttacagat actaacaagg 10140 tgatttgaag taccttagtt gaggatttaa acgcgctatc cggtagtcta caaattggga 10200 aataccgttc aaagagggct agaattactt aaaagccttc acaccgcctg cgctatacgc 10260 gcccactctc ccgtttatcc gtccaagcgg aagcagggcg aacttccgct aagatattct 10320 tacgtgtaac gtagctaagt atcccaaata gctggcgtac gcgttgaaca ccgcctagag 10380 gatcgggagt cgccggacga gcgtgttatt ggggacttac gccagcgtag actacaacgc 10440 gcccagatta accctgcacg tattgccttg aataacgtac taatctctcc ggctctcgac 10500 aatctatcga gcgactcgat tatcaacggg tgtcttgcag ttctaatctc ttgcccccgc 10560 ccgtaatagc ctccaagtga ttcaagatag taaagggcaa gagcttattc ggcgttgaag 10620 gatagcggac tttcggtcaa ccacaattcc ccactcgaca aaaccagccg tgcgaagaac 10680 tctgaaagta caagcaaccc aagagggctg agcctaaact cagctaattc ctaagtgagc 10740 taaagactcg aagtgacagc tattaataaa tagagcggga acgtcgaacg gtcgtgaaag 10800 taatagtaca acgggtatta acttactgag gatattgctt gaagctgtac cgttttattg 10860 ggtgaacgaa taagatccag caattcagcc aaagaagcta ccaattttta gtttaagagt 10920 gtcacgtctg acctcgcggg tggatagccg aacgtagagc ttacgagcca gcggaaacag 10980 tagccgcagg ataagtaagg ggagtaagtg atcgaacgaa tcagaagtga caatatactt 11040 aggctggatc tcgtcccgtg aatcccaacc ctcaccaact acgagataag aggtaagcca 11100 gaaatcggca tggtggcgac caacgactgt tccccccctg taactaatcg ttccgtcaaa 11160 acctgactta cttcaaggcc aattccaagc gcaaacaata ccgtcctagt tcttcggtta 11220 agtttccgaa gtaggagtga gcctacctcc gtttgcgtct tgttaccact gacccagcta 11280 tttactttgt attgcctgca atcgaatttc tgaactctca gatagtgggg ataacgggaa 11340 agttcctata tttgcgaact aacttagccg tccacctcga agctacctac tcacacccac 11400 cccgcgcggg gtaaataagg cactaatccc agcttagagc ttgcgtagca cttagccaca 11460 agttaattaa cagttgtctg gtagtttggc ggtattagcg agatcctaga agcaaggcag 11520 agttagttct aacctaaagc cacaaataag acaggttgcc aaagcccgcc ggaaattaaa 11580 tcttgctcag ttcggtaacg gagtttccc 11609 <210> 12 <211> 1196 <212> PRT <213> artificial sequence <220> <223> chimeric STF (STF-V10-[Helix]) <400> 12 Met Ala Val Lys Ile Ser Gly Val Leu Lys Asp Gly Thr Gly Lys Pro 1 5 10 15 Val Gln Asn Cys Thr Ile Gln Leu Lys Ala Arg Arg Asn Ser Thr Thr 20 25 30 Val Val Val Asn Thr Val Gly Ser Glu Asn Pro Asp Glu Ala Gly Arg 35 40 45 Tyr Ser Met Asp Val Glu Tyr Gly Gln Tyr Ser Val Ile Leu Gln Val 50 55 60 Asp Gly Phe Pro Pro Ser His Ala Gly Thr Ile Thr Val Tyr Glu Asp 65 70 75 80 Ser Gln Pro Gly Thr Leu Asn Asp Phe Leu Cys Ala Met Thr Glu Asp 85 90 95 Asp Ala Arg Pro Glu Val Leu Arg Arg Leu Glu Leu Met Val Glu Glu 100 105 110 Val Ala Arg Asn Ala Ser Val Val Val Ala Gln Ser Thr Ala Asp Ala Lys 115 120 125 Lys Ser Ala Gly Asp Ala Ser Ala Ser Ala Ala Gln Val Ala Ala Leu 130 135 140 Val Thr Asp Ala Thr Asp Ser Ala Arg Ala Ala Ser Thr Ser Ala Gly 145 150 155 160 Gln Ala Ala Ser Ser Ala Gln Glu Ala Ser Ser Gly Ala Glu Ala Ala 165 170 175 Ser Ala Lys Ala Thr Glu Ala Glu Lys Ser Ala Ala Ala Ala Glu Ser 180 185 190 Ser Lys Asn Ala Ala Ala Thr Ser Ala Gly Ala Ala Lys Thr Ser Glu 195 200 205 Thr Asn Ala Ala Ala Ser Gln Gln Ser Ala Ala Thr Ser Ala Ser Thr 210 215 220 Ala Ala Thr Lys Ala Ser Glu Ala Ala Thr Ser Ala Arg Asp Ala Val 225 230 235 240 Ala Ser Lys Glu Ala Ala Lys Ser Ser Glu Thr Asn Ala Ser Ser Ser 245 250 255 Ala Gly Arg Ala Ala Ser Ser Ala Thr Ala Ala Glu Asn Ser Ala Arg 260 265 270 Ala Ala Lys Thr Ser Glu Thr Asn Ala Arg Ser Ser Glu Thr Ala Ala 275 280 285 Glu Arg Ser Ala Ser Ala Ala Ala Asp Ala Lys Thr Ala Ala Ala Gly 290 295 300 Ser Ala Ser Thr Ala Ser Thr Lys Ala Thr Glu Ala Ala Gly Ser Ala 305 310 315 320 Val Ser Ala Ser Gln Ser Lys Ser Ala Ala Glu Ala Ala Ala Ile Arg 325 330 335 Ala Lys Asn Ser Ala Lys Arg Ala Glu Asp Ile Ala Ser Ala Val Ala 340 345 350 Leu Glu Asp Ala Asp Thr Thr Arg Lys Gly Ile Val Gln Leu Ser Ser 355 360 365 Ala Thr Asn Ser Thr Ser Glu Thr Leu Ala Ala Thr Pro Lys Ala Val 370 375 380 Lys Val Val Met Asp Glu Thr Asn Arg Lys Ala Pro Leu Asp Ser Pro 385 390 395 400 Ala Leu Thr Gly Thr Pro Thr Ala Pro Thr Ala Leu Arg Gly Thr Asn 405 410 415 Asn Thr Gln Ile Ala Asn Thr Ala Phe Val Leu Ala Ala Ile Ala Asp 420 425 430 Val Ile Asp Ala Ser Pro Asp Ala Leu Asn Thr Leu Asn Glu Leu Ala 435 440 445 Ala Ala Leu Gly Asn Asp Pro Asp Phe Ala Thr Thr Met Thr Asn Ala 450 455 460 Leu Ala Gly Lys Gln Pro Lys Asn Ala Thr Leu Thr Ala Leu Ala Gly 465 470 475 480 Leu Ser Thr Ala Lys Asn Lys Leu Pro Tyr Phe Ala Glu Asn Asp Ala 485 490 495 Ala Ser Leu Thr Glu Leu Thr Gln Val Gly Arg Asp Ile Leu Ala Lys 500 505 510 Asn Ser Val Ala Asp Val Leu Glu Tyr Leu Gly Ala Gly Glu Asn Ser 515 520 525 Gly Ser Ala Thr Asp Val Met Ile Gln Leu Ala Ala Asn Asp Gly Phe 530 535 540 Lys Phe Ile Gly Gln Cys Pro Asp Ile Leu Thr Leu Arg Thr Ile Glu 545 550 555 560 Pro Glu Lys Asn Gly Gln Arg Ile Thr Leu Arg Gln His Thr Ile Gly 565 570 575 Thr Gly Leu Gly Gly Gly Val Phe Arg Ala Val Leu Asp Gly Thr Gly 580 585 590 Tyr Thr Asp Asp Asp Gly Val Val Ile Lys Thr Ala Gly Gly Ser Val 595 600 605 Trp Leu Arg Val Asn Ala Asp Lys Val Asn Pro Phe Met Phe Gly Ala 610 615 620 Thr Gly Val Ala Asp Asp Thr Ala Ala Leu Gln Lys Met Leu Glu Cys 625 630 635 640 Gly Arg Ala Ala Glu Leu Gly Thr Asn Val Trp Lys Ala Ser Asn Leu 645 650 655 Glu Leu Asn Asn Lys Ser Cys Ser Leu Ser Gly Ser Gly Leu His Val 660 665 670 Ser Arg Ile Glu Gln Ile Ser Gly Ala Thr Gly Ala Leu Leu Thr Ile 675 680 685 Thr Gln Asp Cys Ser Leu Ile Tyr Leu Ser Asp Cys Gly Leu Tyr Gly 690 695 700 Asp Gly Ile Thr Ala Gly Thr Ser Gly Val Thr Met Glu Thr Gly Asn 705 710 715 720 Pro Gly Gly Ala Pro Ser Tyr Pro Phe Asn Thr Ala Pro Asp Val Arg 725 730 735 Arg Asp Leu Tyr Ile Ser Asn Val His Ile Thr Gly Phe Asp Glu Leu 740 745 750 Gly Phe Asp Tyr Pro Glu Thr Asn Phe Ser Val Ser Thr His Gly Leu 755 760 765 Phe Ile Arg Asn Ile Lys Lys Thr Gly Ala Lys Ile Gly Thr Thr Asp 770 775 780 Phe Thr Trp Thr Asn Leu Gln Ile Asp Thr Cys Gly Gln Glu Cys Leu 785 790 795 800 Val Leu Asp Gly Ala Gly Asn Cys Arg Ile Ile Gly Ala Lys Leu Ile 805 810 815 Trp Ala Gly Ser Glu Asn Glu Thr Pro Tyr Ser Gly Leu Arg Ile Ser 820 825 830 Asn Ser Gln Asn Val Asn Met Thr Gly Val Glu Leu Gln Asp Cys Ala 835 840 845 Tyr Asp Gly Leu Tyr Ile Lys Asn Ser Thr Val Ala Ile Ser Gly Leu 850 855 860 Asn Thr Asn Arg Asn Ser Ala Ser Ser Asn Leu Ser Tyr His Asn Met 865 870 875 880 Val Phe Glu Asn Ser Ile Val Thr Val Asp Gly Tyr Val Cys Arg Asn 885 890 895 Tyr Ala Ala Thr Ser Leu Tyr Asp Leu Asn Ser Gln Ala Gly Asn Val 900 905 910 Arg Cys Ile Gly Ser Asp Ser Thr Val Leu Ile Asn Gly Ile Tyr Glu 915 920 925 Ser Glu Val Asn Ser Glu Arg Leu Met Gly Asp Asn Asn Leu Ile Gln 930 935 940 Pro Tyr Ser Gly Asp Leu Ile Ile Asn Gly Leu Lys Asn Tyr Tyr Thr 945 950 955 960 Tyr Thr Gly Ser Val Lys Asn Asn Ile Pro Thr Phe Asp Gly Val Val 965 970 975 Thr Thr Ala Thr Tyr Val Ser Ala Pro Ser Ile Leu Gly Gln Gly Asn 980 985 990 Met Leu Lys Leu Thr Gln Ser Asn Lys Asp Lys Leu Leu Phe Ser Asp 995 1000 1005 Lys Val Ser Arg His Gly Cys Thr Ile Gly Leu Val Leu Ile Pro Ser 1010 1015 1020 Phe Thr Gly Ala Thr Thr Met Thr Ala Phe Thr Leu Gly Ser Gly Tyr 1025 1030 1035 1040 Ser Pro Ser Gly Asn Ser Ala Val Met Gln Phe Ile Val Asn Ser Ser 1045 1050 1055 Gly Val Gln Thr Ile Ala Ile Leu Leu Ser Gly Asp Gly Ile Thr Gln 1060 1065 1070 Thr Leu Thr Ser Asp Leu Thr Thr Glu Gln Ala Leu Ala Ser Gly Gly 1075 1080 1085 Val Tyr His Phe Ala Met Gly Phe Ala Pro Gly Arg Leu Trp Trp Ser 1090 1095 1100 Ile Ile Asp Ile Asn Thr Gly Arg Arg Ile Arg Arg Ala Tyr Arg Gln 1105 1110 1115 1120 Pro Asp Leu His Ala Ala Phe Asn Ser Ile Phe Asn Ser Gly Thr Ser 1125 1130 1135 Ser Ile Thr Ala Phe Ser Gly Pro Leu Ala Gly Asp Ile Ala Cys Glu 1140 1145 1150 Gly Ala Gly Ser His Val Tyr Val Gly Gly Phe Ser Ser Glu Ser Asp 1155 1160 1165 Tyr Ala Ala Ser Arg Met Tyr Gly Leu Phe Thr Pro Val Asp Leu Asp 1170 1175 1180 Lys Gln Tyr Ser Phe Arg Thr Leu Asn Gly Asn Ile 1185 1190 1195 <210> 13 <211> 3588 <212> DNA <213> artificial sequence <220> <223> chimeric STF (STF-V10-[Helix]) <400> 13 atggcagtaa agatttcagg agtcctgaaa gacggcacag gaaaaccggt acagaactgc 60 accattcagc tgaaagccag acgtaacagc accacggtgg tggtgaacac ggtgggctca 120 gagaatccgg atgaagccgg gcgttacagc atggatgtgg agtacggtca gtacagtgtc 180 atcctgcagg ttgacggttt tccaccatcg cacgccggga ccatcaccgt gtatgaagat 240 tcacaaccgg ggacgctgaa tgattttctc tgtgccatga cggaggatga tgcccggccg 300 gaggtgctgc gtcgtcttga actgatggtg gaagaggtgg cgcgtaacgc gtccgtggtg 360 gcacagagta cggcagacgc gaagaaatca gccggcgatg ccagtgcatc agctgctcag 420 gtcgcggccc ttgtgactga tgcaactgac tcagcacgcg ccgccagcac gtccgccgga 480 caggctgcat cgtcagctca ggaagcgtcc tccggcgcag aagcggcatc agcaaaggcc 540 actgaagcgg aaaaaagtgc cgcagccgca gagtcctcaa aaaacgcggc ggccaccagt 600 gccggtgcgg cgaaaacgtc agaaacgaat gctgcagcgt cacaacaatc agccgccacg 660 tctgcctcca ccgcggccac gaaagcgtca gaggccgcca cttcagcacg agatgcggtg 720 gcctcaaaag aggcagcaaa atcatcagaa acgaacgcat catcaagtgc cggtcgtgca 780 gcttcctcgg caacggcggc agaaaattct gccagggcgg caaaaacgtc cgagacgaat 840 gccaggtcat ctgaaacagc agcggaacgg agcgcctctg ccgcggcaga cgcaaaaaca 900 gcggcggcgg ggaggtgcgtc aacggcatcc acgaaggcga cagaggctgc gggaagtgcg 960 gtatcagcat cgcagagcaa aagtgcggca gaagcggcgg caatacgtgc aaaaaattcg 1020 gcaaaacgtg cagaagatat agcttcagct gtcgcgcttg aggatgcgga cacaacgaga 1080 aaggggatag tgcagctcag cagtgcaacc aacagcacgt ctgaaacgct tgctgcaacg 1140 ccaaaggcgg ttaaggtggt aatggatgag actaatcgta aggcacctct ggacagtccg 1200 gcactgaccg gaacgccaac agcaccaacc gcgctcaggg gaacaaacaa tacccagatt 1260 gcgaacaccg cttttgtact ggccgcgatt gcagatgtta tcgacgcgtc acctgacgca 1320 ctgaatacgc tgaatgaact ggccgcagcg ctcgggaatg atccagattt tgctaccacc 1380 atgactaacg cgcttgcggg taaacaaccg aagaatgcga cactgacggc gctggcaggg 1440 ctttccacgg cgaaaaataa attaccgtat tttgcggaaa atgatgccgc cagcctgact 1500 gaactgactc aggttggcag ggatattctg gcaaaaaatt ccgttgcaga tgttcttgaa 1560 taccttgggg ccggtgagaa ttcggggagc gctacagacg ttatgattca gctggcggca 1620 aatgatggct tcaaattcat cggtcagtgc ccagacatct tgaccctgcg tactatcgag 1680 ccggaaaaaa acggtcagcg tatcacctta cgtcaacata cgattggcac tggcttaggc 1740 ggtggcgttt tccgtgcagt tctggacggc actggctata ccgatgacga cggtgtggtg 1800 atcaaaaccg ctgggggcag cgtttggctg cgtgtcaacg ctgacaaagt taacccgttc 1860 atgttcggtg caaccggagt agcggacgac accgccgccc tgcaaaaaat gctggaatgc 1920 ggtcgtgcgg cggaactggg gactaacgta tggaaagcaa gcaatctgga actgaacaac 1980 aaatcttgct ctctgtccgg cagtggcctg cacgtttctc gtattgaaca gatttccggt 2040 gcaaccggag cattgttaac catcacccaa gactgttcgc tgatttacct gtccgattgt 2100 ggcctgtacg gcgatggcat caccgcaggc acgagcggtg ttactatgga aacgggtaat 2160 ccgggtggcg ctccgtctta ccctttcaat accgctccgg acgttcgtcg tgacctgtac 2220 atctctaacg tgcacatcac gggcttcgac gagctgggtt ttgattatcc ggaaaccaat 2280 ttctctgttt cgacgcatgg cctcttcatc cgtaaca aaaaaacggg tgcaaagatt 2340 ggtactacgg acttcacttg gactaacctg caaattgata cttgcggtca ggaatgtctg 2400 gtgctggacg gtgcgggtaa ctgccgtatt attggtgcaa aactgatttg ggcaggtagc 2460 gaaaacgaaa cgccatactc tggcctgcgt attagcaact ctcaaaatgt aaatatgact 2520 ggcgtagagt tacaagactg cgcgtatgat ggtttataca tcaagaactc tacggttgca 2580 atttcaggct taaacaccaa tcgcaatagc gcatcctcta atctgtccta ccataacatg 2640 gtattcgaaa attctattgt aactgttgat ggttatgtgt gtcgtaacta cgcggcgact 2700 tcgctgtacg acctgaacag ccaagcaggc aacgtccgtt gcatcggtag cgacagcacc 2760 gttttaatca acggcatcta cgaaagcgaa gtcaatagcg agcgcctgat gggtgataac 2820 aacctgatcc agccgtatag tggtgatctg atcattaacg gcctgaaaaa ttactacacc 2880 tatactggta gcgtaaaaaa caacattccg accttcgacg gcgttgttac tacggcaacc 2940 tatgtgagcg caccgtctat tctgggtcag ggcaatatgc tcaaactgac ccagtctaat 3000 aaagacaaac tgttatttag cgataaagtt agccgtcatg gctgtaccat cggcttagtt 3060 ctgattccgt cctttacggg cgcgaccact atgacggcgt tcacgctggg tagcggttac 3120 tctccatccg gtaactccgc cgtgatgcag ttcattgtta acagttccgg tgtacaaacc 3180 attgcgattt tattatccgg cgacggtatt acccaaaccc tgaccagcga tctgaccacg 3240 gaacaagcac tggcgagcgg tggcgtgtat cattttgcaa tgggttttgc gccgggtcgt 3300 ttatggtgga gcattatcga tattaacacg ggcaggcgta ttcgtcgcgc ctaccgtcag 3360 ccggatctgc acgcggcgtt caactctatc ttcaactccg gcacgtcgtc tattaccgca 3420 tttagcgggc cactggcggg cgacattgct tgcgaaggtg caggtagcca tgtatacgtt 3480 ggcggttttt cgtcggaatc tgattacgcg gctagccgta tgtatggcct gttcactccg 3540 gtcgatctgg acaagcagta tagcttccgt accctgaacg gtaacatt 3588 <210> 14 <211> 1131 <212> PRT <213> artificial sequence <220> <223> chimeric gpJ (1A2) <400> 14 Met Gly Lys Gly Ser Ser Lys Gly His Thr Pro Arg Glu Ala Lys Asp 1 5 10 15 Asn Leu Lys Ser Thr Gln Leu Leu Ser Val Ile Asp Ala Ile Ser Glu 20 25 30 Gly Pro Ile Glu Gly Pro Val Asp Gly Leu Lys Ser Val Leu Leu Asn 35 40 45 Ser Thr Pro Val Leu Asp Thr Glu Gly Asn Thr Asn Ile Ser Gly Val 50 55 60 Thr Val Val Phe Arg Ala Gly Glu Gln Glu Gln Thr Pro Pro Glu Gly 65 70 75 80 Phe Glu Ser Ser Gly Ser Glu Thr Val Leu Gly Thr Glu Val Lys Tyr 85 90 95 Asp Thr Pro Ile Thr Arg Thr Ile Thr Ser Ala Asn Ile Asp Arg Leu 100 105 110 Arg Phe Thr Phe Gly Val Gln Ala Leu Val Glu Thr Thr Ser Lys Gly 115 120 125 Asp Arg Asn Pro Ser Glu Val Arg Leu Leu Val Gln Ile Gln Arg Asn 130 135 140 Gly Gly Trp Val Thr Glu Lys Asp Ile Thr Ile Lys Gly Lys Thr Thr 145 150 155 160 Ser Gln Tyr Leu Ala Ser Val Val Met Gly Asn Leu Pro Pro Arg Pro 165 170 175 Phe Asn Ile Arg Met Arg Arg Met Thr Pro Asp Ser Thr Thr Asp Gln 180 185 190 Leu Gln Asn Lys Thr Leu Trp Ser Ser Tyr Thr Glu Ile Ile Asp Val 195 200 205 Lys Gln Cys Tyr Pro Asn Thr Ala Leu Val Gly Val Gln Val Asp Ser 210 215 220 Glu Gln Phe Gly Ser Gln Gln Val Ser Arg Asn Tyr His Leu Arg Gly 225 230 235 240 Arg Ile Leu Gln Val Pro Ser Asn Tyr Asn Pro Gln Thr Arg Gln Tyr 245 250 255 Ser Gly Ile Trp Asp Gly Thr Phe Lys Pro Ala Tyr Ser Asn Asn Met 260 265 270 Ala Trp Cys Leu Trp Asp Met Leu Thr His Pro Arg Tyr Gly Met Gly 275 280 285 Lys Arg Leu Gly Ala Ala Asp Val Asp Lys Trp Ala Leu Tyr Val Ile 290 295 300 Gly Gln Tyr Cys Asp Gln Ser Val Pro Asp Gly Phe Gly Gly Thr Glu 305 310 315 320 Pro Arg Ile Thr Cys Asn Ala Tyr Leu Thr Thr Gln Arg Lys Ala Trp 325 330 335 Asp Val Leu Ser Asp Phe Cys Ser Ala Met Arg Cys Met Pro Val Trp 340 345 350 Asn Gly Gln Thr Leu Thr Phe Val Gln Asp Arg Pro Ser Asp Lys Thr 355 360 365 Trp Thr Tyr Asn Arg Ser Asn Val Val Met Pro Asp Asp Gly Ala Pro 370 375 380 Phe Arg Tyr Ser Phe Ser Ala Leu Lys Asp Arg His Asn Ala Val Glu 385 390 395 400 Val Asn Trp Ile Asp Pro Asn Asn Gly Trp Glu Thr Ala Thr Glu Leu 405 410 415 Val Glu Asp Thr Gln Ala Ile Ala Arg Tyr Gly Arg Asn Val Thr Lys 420 425 430 Met Asp Ala Phe Gly Cys Thr Ser Arg Gly Gln Ala His Arg Ala Gly 435 440 445 Leu Trp Leu Ile Lys Thr Glu Leu Leu Glu Thr Gln Thr Val Asp Phe 450 455 460 Ser Val Gly Ala Glu Gly Leu Arg His Val Pro Gly Asp Val Ile Glu 465 470 475 480 Ile Cys Asp Asp Asp Tyr Ala Gly Ile Ser Thr Gly Gly Arg Val Leu 485 490 495 Ala Val Asn Ser Gln Thr Arg Thr Leu Thr Leu Asp Arg Glu Ile Thr 500 505 510 Leu Pro Ser Ser Gly Thr Ala Leu Ile Ser Leu Val Asp Gly Ser Gly 515 520 525 Asn Pro Val Ser Val Glu Val Gln Ser Val Thr Asp Gly Val Lys Val 530 535 540 Lys Val Ser Arg Val Pro Asp Gly Val Ala Glu Tyr Ser Val Trp Glu 545 550 555 560 Leu Lys Leu Pro Thr Leu Arg Gln Arg Leu Phe Arg Cys Val Ser Ile 565 570 575 Arg Glu Asn Asp Asp Gly Thr Tyr Ala Ile Thr Ala Val Gln His Val 580 585 590 Pro Glu Lys Glu Ala Ile Val Asp Asn Gly Ala His Phe Asp Gly Glu 595 600 605 Gln Ser Gly Thr Val Asn Gly Val Thr Pro Pro Ala Val Gln His Leu 610 615 620 Thr Ala Glu Val Thr Ala Asp Ser Gly Glu Tyr Gln Val Leu Ala Arg 625 630 635 640 Trp Asp Thr Pro Lys Val Val Lys Gly Val Ser Phe Leu Leu Arg Leu 645 650 655 Thr Val Thr Ala Asp Asp Gly Ser Glu Arg Leu Val Ser Thr Ala Arg 660 665 670 Thr Thr Glu Thr Thr Tyr Arg Phe Thr Gln Leu Ala Leu Gly Asn Tyr 675 680 685 Arg Leu Thr Val Arg Ala Val Asn Ala Trp Gly Gln Gln Gly Asp Pro 690 695 700 Ala Ser Val Ser Phe Arg Ile Ala Ala Pro Ala Ala Pro Ser Arg Ile 705 710 715 720 Glu Leu Thr Pro Gly Tyr Phe Gln Ile Thr Ala Thr Pro His Leu Ala 725 730 735 Val Tyr Asp Pro Thr Val Gln Phe Glu Phe Trp Phe Ser Glu Lys Gln 740 745 750 Ile Ala Asp Ile Arg Gln Val Glu Thr Ser Thr Arg Tyr Leu Gly Thr 755 760 765 Ala Leu Tyr Trp Ile Ala Ala Ser Ile Asn Ile Lys Pro Gly His Asp 770 775 780 Tyr Tyr Phe Tyr Ile Arg Ser Val Asn Thr Val Gly Lys Ser Ala Phe 785 790 795 800 Val Glu Ala Val Gly Arg Ala Ser Asp Asp Ala Glu Gly Tyr Leu Asp 805 810 815 Phe Phe Lys Gly Lys Ile Thr Glu Ser His Leu Gly Lys Glu Leu Leu 820 825 830 Glu Lys Val Glu Leu Thr Glu Asp Asn Ala Ser Arg Leu Glu Glu Phe 835 840 845 Ser Lys Glu Trp Lys Asp Ala Ser Asp Lys Trp Asn Ala Met Trp Ala 850 855 860 Val Lys Ile Glu Gln Thr Lys Asp Gly Lys His Tyr Val Ala Gly Ile 865 870 875 880 Gly Leu Ser Met Glu Asp Thr Glu Glu Gly Lys Leu Ser Gln Phe Leu 885 890 895 Val Ala Ala Asn Arg Ile Ala Phe Ile Asp Pro Ala Asn Gly Asn Glu 900 905 910 Thr Pro Met Phe Val Ala Gln Gly Asn Gln Ile Phe Met Asn Asp Val 915 920 925 Phe Leu Lys Arg Leu Thr Ala Pro Thr Ile Thr Ser Gly Gly Asn Pro 930 935 940 Pro Ala Phe Ser Leu Thr Pro Asp Gly Lys Leu Thr Ala Lys Asn Ala 945 950 955 960 Asp Ile Ser Gly Asn Val Asn Ala Asn Ser Gly Thr Leu Asn Asn Val 965 970 975 Thr Ile Asn Glu Asn Cys Arg Val Leu Gly Lys Leu Ser Ala Asn Gln 980 985 990 Ile Glu Gly Asp Leu Val Lys Thr Val Gly Lys Ala Phe Pro Arg Asp 995 1000 1005 Ser Arg Ala Pro Glu Arg Trp Pro Ser Gly Thr Ile Thr Val Arg Val 1010 1015 1020 Tyr Asp Asp Gln Pro Phe Asp Arg Gln Ile Val Ile Pro Ala Val Ala 1025 1030 1035 1040 Phe Ser Gly Ala Lys His Glu Lys Glu His Thr Asp Ile Tyr Ser Ser 1045 1050 1055 Cys Arg Leu Ile Val Arg Lys Asn Gly Ala Glu Ile Tyr Asn Arg Thr 1060 1065 1070 Ala Leu Asp Asn Thr Leu Ile Tyr Ser Gly Val Ile Asp Met Pro Ala 1075 1080 1085 Gly His Gly His Met Thr Leu Glu Phe Ser Val Ser Ala Trp Leu Val 1090 1095 1100 Asn Asn Trp Tyr Pro Thr Ala Ser Ile Ser Asp Leu Leu Val Val Val 1105 1110 1115 1120 Met Lys Lys Ala Thr Ala Gly Ile Thr Ile Ser 1125 1130 <210> 15 <211> 3393 <212> DNA <213> artificial sequence <220> <223> chimeric gpJ (1A2) <400> 15 atgggtaaag gaagcagtaa ggggcatacc ccgcgcgaag cgaaggacaa cctgaagtcc 60 acgcagttgc tgagtgtgat cgatgccatc agcgaagggc cgattgaagg tccggtggat 120 ggcttaaaaa gcgtgctgct gaacagtacg ccggtgctgg acactgaggg gaataccaac 180 atatccggtg tcacggtggt gttccgggct ggtgagcagg agcagactcc gccggaggga 240 tttgaatcct ccggctccga gacggtgctg ggtacggaag tgaaatatga cacgccgatc 300 acccgcacca ttacgtctgc aaacatcgac cgtctgcgct ttaccttcgg tgtacaggca 360 ctggtggaaa ccacctcaaa gggtgacagg aatccgtcgg aagtccgcct gctggttcag 420 atacaacgta acggtggctg ggtgacggaa aaagacatca ccattaaggg caaaaccacc 480 tcgcagtatc tggcctcggt ggtgatgggt aacctgccgc cgcgcccgtt taatatccgg 540 atgcgcagga tgacgccgga cagcaccaca gaccagctgc agaacaaaac gctctggtcg 600 tcatacactg aaatcatcga tgtgaaacag tgctacccga acacggcact ggtcggcgtg 660 caggtggact cggagcagtt cggcagccag caggtgagcc gtaattatca tctgcgcggg 720 cgtattctgc aggtgccgtc gaactataac ccgcagacgc ggcaatacag cggtatctgg 780 gacggaacgt ttaaaccggc atacagcaac aacatggcct ggtgtctgtg ggatatgctg 840 acccatccgc gctacggcat ggggaaacgt cttggtgcgg cggatgtgga taaatgggcg 900 ctgtatgtca tcggccagta ctgcgaccag tcagtgccgg acggctttgg cggcacggag 960 ccgcgcatca cctgtaatgc gtacctgacc acacagcgta aggcgtggga tgtgctcagc 1020 gatttctgct cggcgatgcg ctgtatgccg gtatggaacg ggcagacgct gacgttcgtg 1080 caggaccgac cgtcggataa gacgtggacc tataaccgca gtaatgtggt gatgccggat 1140 gatggcgcgc cgttccgcta cagcttcagc gccctgaagg accgccataa tgccgttgag 1200 gtgaactgga ttgacccgaa caacggctgg gagacggcga cagagcttgt tgaagatacg 1260 caggccattg cccgttacgg tcgtaatgtt acgaagatgg atgcctttgg ctgtaccagc 1320 cgggggcagg cacaccgcgc cgggctgtgg ctgattaaaa cagaactgct ggaaacgcag 1380 accgtggatt tcagcgtcgg cgcagaaggg cttcgccatg taccgggcga tgttattgaa 1440 atctgcgatg atgactatgc cggtatcagc accggtggtc gtgtgctggc ggtgaacagc 1500 cagacccgga cgctgacgct cgaccgtgaa atcacgctgc catcctccgg taccgcgctg 1560 ataagcctgg ttgacggaag tggcaatccg gtcagcgtgg aggttcagtc cgtcaccgac 1620 ggcgtgaagg taaaagtgag ccgtgttcct gacggtgttg ctgaatacag cgtatggggag 1680 ctgaagctgc cgacgctgcg ccagcgactg ttccgctgcg tgagtatccg tgagaacgac 1740 gacggcacgt atgccatcac cgccgtgcag catgtgccgg aaaaagaggc catcgtggat 1800 aacggggcgc actttgacgg cgaacagagt ggcacggtga atggtgtcac gccgccagcg 1860 gtgcagcacc tgaccgcaga agtcactgca gacagcgggg aatatcaggt gctggcgcga 1920 tgggacacac cgaaggtggt gaagggcgtg agtttcctgc tccgtctgac cgtaacagcg 1980 gacgacggca gtgagcggct ggtcagcacg gcccggacga cggaaaccac ataccgcttc 2040 acgcaactgg cgctggggaa ctacaggctg acagtccggg cggtaaatgc gtgggggcag 2100 cagggcgatc cggcgtcggt atcgttccgg attgccgcac cggcagcacc gtcgaggatt 2160 gagctgacgc cgggctattt tcagataacc gccacgccgc atcttgccgt ttatgacccg 2220 acggtacagt ttgagttctg gttctcggaa aagcagattg cggatatcag acaggttgaa 2280 accagcacgc gttatcttgg tacggcgctg tactggatag ccgccagtat caatatcaaa 2340 ccgggccatg attattactt ttatatccgc agtgtgaaca ccgttggcaa atcggcattc 2400 gtggaggccg tcggtcgggc gagcgatgat gcggaaggtt acctggattt tttcaaaggc 2460 aagataaccg aatcccatct cggcaaggag ctgctggaaa aagtcgagct gacggaggat 2520 aacgccagca gactggagga gttttcgaaa gagtggaagg atgccagtga taagtggaat 2580 gccatgtggg ctgtcaaaat tgagcagacc aaagacggca aacattatgt cgcgggtatt 2640 ggcctcagca tggaggacac ggaggaaggc aaactgagcc agtttctggt tgccgccaat 2700 cgtatcgcat ttattgaccc ggcaaacggg aatgaaacgc cgatgtttgt ggcgcagggc 2760 aaccagatat tcatgaacga cgtgttcctg aagcgcctga cggcccccac cattaccagc 2820 ggcggcaatc ctccggcctt ttccctgaca ccggacggaa agctgaccgc taaaaatgcg 2880 gatatcagcg gtaacgtgaa tgcgaactcc gggacgctca acaacgtcac gattaacgag 2940 aactgtcggg ttctgggaaa attgtccgcg aaccagattg aaggcgatct cgttaaaaca 3000 gtgggcaaag ctttcccccg ggactcccgt gcaccggagc ggtggccatc aggaaccatt 3060 accgtcaggg tttatgacga tcagccgttt gaccggcaga ttgttattcc ggcggtggca 3120 ttcagcggcg ctaaacatga gaaagagcat actgatattt actcctcatg ccgtctgata 3180 gtgcggaaaa acggtgctga aatttataac cgtaccgcgc tggataatac gctgatttac 3240 agtggcgtta ttgatatgcc tgccggtcac ggtcacatga cactggagtt ttcggtgtca 3300 gcatggctgg taaataactg gtatcccaca gcaagtatca gcgatttgct ggttgtggtg 3360 atgaagaaag ccactgcagg catcacgatt agc 3393 <210> 16 <211> 2352 <212> DNA <213> artificial sequence <220> <223> p2.3 pri-ori p1319 <400> 16 gttgtccata tttgctacgt ttaaatcaaa actggtgaaa ctcacccacg gatttgcact 60 gacgaaaaac atattttcga taaacccttt agggaaatat gctaagtttt caccgtaaca 120 cgccacatct tgactatata tgtgtagaaa ctgccggaaa tcgtcatggt attctgacca 180 gagcgatgag aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 240 ccatatcacc agttcaccgt ctttcattgc catacgaaac tccggatgtg cattcatcag 300 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtttt 360 taaaaaggcc gtaatatcca gttgaacggt ttggttatag gtgcactgag caactgactg 420 gaatgcctca aaatgttctt tacgatgcca ttgacttata tcaactgtag tatatccagt 480 gatttttttc tccattttag attccttagc ttgcgaaatc tcgataactc aaaaaatagt 540 agtgatctta tttcattatg gtgaaagttg tcttacgtgc aacattttcg caaaaagttg 600 gcgctttatc aacactgtcc ctcctgttca gctactgacg gtactgcgga actgactaaa 660 gtagtgcgta acggcaaaag caccgccgga catcttttgt tgcaatggct gtctaccctg 720 tctacctgag taaagaaaaa tacatttaat tcagtacatt aacttgggta gacagccttt 780 ttttactgtc tacctactat ctaccctctc tacctgattt tacctgaatc agacaggggag 840 gtagatacgg ggtagatagt ggataaaagc actctacccc actgaaagcc gcgccattac 900 tggcatggtg gccagtaagg tagataaggt agacaagggg aggcacaact caaaactttt 960 taaacgaggg ggtaaaacgc agaccaaaac gatctcaaga agatcatctt attaatcaga 1020 taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagcacgt ttagccgaac 1080 gccccaaaaa gcctcgcttt cagcacctgc cgtttccttt cttttcagag ggtattttaa 1140 ataaaaacat taagttatga cgaagaagaa cggaaacgcc ttaaaccgga aaattttcat 1200 aaatagcgaa aacccgcgag gtcgccgccc cgtaaccagt cggatcaccg gaaagtacct 1260 gtaaagtgat aatgattatc atcttcatat cacaacgtgc gtaaagggac tagtggatac 1320 tagtattata cctagcactg agcacgggta gcaccagaag tctatagcat gtgcatacct 1380 ttggtcgaaa aaaaaagccc gcactgtcag gtgcgggctt ttttcagtgt ttccttgccg 1440 gattatttgt agagttcatc cataccgtgc gtgataccac tagcggtaac gaactctaat 1500 aacaccatgt ggtcgcgctt ttcgttcgga tccttagaca gtttagactg ggtggacagg 1560 tagtggttat ccggcagtaa aacaggagcg tctccaatcg gagtgttttg ttggtaatga 1620 tccgcaagct ggacgctacc atcttcaacg ttatgtcgaa ttttaaagtt agctttgata 1680 ccgttctttt gtttgtctgc ggtgatgtaa acgttatggg agttgaagtt atattccagt 1740 ttgtggccta aaatattgcc gtcctctttg aaatcaatgc ctttcagttc aatacgattc 1800 accagagtgt caccttcaaa tttaacctct gcacgggttt tatacgtgcc atcgtctttg 1860 aaagaaatgg tgcgctcctg tacataacct tccggcattg cagatttgaa gaaatcatgt 1920 tgcttcatgt ggtcagggta acgagaaaaa cactgaacac cataggtcag ggtagtcacc 1980 agagtaggcc acggtactgg taattttcca gtagtgcaga tgaatttcag ggtcagctta 2040 ccgttggttg catcgccttc accttcacca cgaacactga atttatgacc gttaacatcg 2100 ccgtccagtt caactaagat cggaacaaca ccagtaaata attcctcacc tttactcatg 2160 gcttcctttc tcctctttaa tgaaaactta cgccccgccc tgccactcat cgcagtattg 2220 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacttg 2280 gatcgccagt ggcattaaca ccttgtcgcc ttgcgtataa tattttccca tagtgaaaac 2340 gggggcgaag aa 2352 <210> 17 <211> 2778 <212> DNA <213> artificial sequence <220> <223> p2.8 p15a, p1220 <400> 17 gttgtccata tttgctacgt ttaaatcaaa actggtgaaa ctcacccacg gatttgcact 60 gacgaaaaac atattttcga taaacccttt agggaaatat gctaagtttt caccgtaaca 120 cgccacatct tgactatata tgtgtagaaa ctgccggaaa tcgtcatggt attctgacca 180 gagcgatgag aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 240 ccatatcacc agttcaccgt ctttcattgc catacgaaac tccggatgtg cattcatcag 300 gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtttt 360 taaaaaggcc gtaatatcca gttgaacggt ttggttatag gtgcactgag caactgactg 420 gaatgcctca aaatgttctt tacgatgcca ttgacttata tcaactgtag tatatccagt 480 gatttttttc tccattttag attccttagc ttgcgaaatc tcgataactc aaaaaatagt 540 agtgatctta tttcattatg gtgaaagttg tcttacgtgc aacattttcg caaaaagttg 600 gcgctttatc aacactgtcc ctcctgttca gctactgacg gtactgcgga actgactaaa 660 gtagtgcgta acggcaaaag caccgccgga catctgcgct agcggagtgt atactggctt 720 actatgttgg cactgatgag ggtgtaagtg aagtgcttca tgtggcagga gaaaaaaggc 780 tgcatcggtg cgtcagcaga atatgtgata caggatatat tccgcttcct cgctcactga 840 ctcgctacgc tcggtcgttc gactgtggcg agcggaaatg gcttacgaac ggggctgaga 900 tttcctggaa gatgccagga agatacttaa cagggaagtg agagggtcgc ggcaaagccg 960 tttttccata ggctccgccc ccctgacaag catcacgaaa tctgacgctc aaatcagtgg 1020 tggcgaaacc tgacaggact ataaagatac caggcgtttc cccctggcgg ctccctcgtg 1080 cgctctcctg ttcctgcctt tcggtttgcc ggtgtcattc ctctgttact gccgagtttg 1140 tctcattcca cgcctgacac tcagttccgg gtaggcagtt cgctccaagc tggactgtat 1200 gcacaaaccc cccgttcagt ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 1260 caacccggaa agacatgcaa aagcaccact ggcagcagcc actggtaatt gatttagagg 1320 agttagtctt gaagtcatgc gccggataag gctaaactga aaggacaagt tttggcgact 1380 gcgctcctcc aagccagtta cctcggtcca aagagttggt agctcagagg accttcgaaa 1440 aaccgccctg caaggcggtt ttttcgtttt cagagcaaga gattacgcgc agaccaaaac 1500 gatctcaagc caaaaagcct cgctttcagc acctgccgtt tcctttcttt tcagagggta 1560 ttttaaataa aaacattaag ttatgacgaa gaagaacgga aacgccttaa accggaaaat 1620 tttcataaat agcgaaaacc cgcgaggtcg ccgccccgta accagtcgga tcaccggaaa 1680 gtacctgtaa agtgataatg attatcatct tcatatcaca acgtgcgtaa agggactagt 1740 ggatactagt attataccta gcactgagca cgggtagcac cagaagtcta tagcatgtgc 1800 atacctttgg tcgaaaaaaa aagcccgcac tgtcaggtgc gggctttttt cagtgtttcc 1860 ttgccggatt atttgtagag ttcatccata ccgtgcgtga taccactagc ggtaacgaac 1920 tctaataaca ccatgtggtc gcgcttttcg ttcggatcct tagacagttt agactgggtg 1980 gacaggtagt ggttatccgg cagtaaaaca ggagcgtctc caatcggagt gttttgttgg 2040 taatgatccg caagctggac gctaccatct tcaacgttat gtcgaatttt aaagttagct 2100 ttgataccgt tcttttgttt gtctgcggtg atgtaaacgt tatgggagtt gaagttatat 2160 tccagtttgt ggcctaaaat attgccgtcc tctttgaaat caatgccttt cagttcaata 2220 cgattcacca gagtgtcacc ttcaaattta acctctgcac gggttttata cgtgccatcg 2280 tctttgaaag aaatggtgcg ctcctgtaca taaccttccg gcattgcaga tttgaagaaa 2340 tcatgttgct tcatgtggtc agggtaacga gaaaaacact gaacaccata ggtcagggta 2400 gtcaccagag taggccacgg tactggtaat tttccagtag tgcagatgaa tttcagggtc 2460 agcttaccgt tggttgcatc gccttcacct tcaccacgaa cactgaattt atgaccgtta 2520 acatcgccgt ccagttcaac taagatcgga acaacaccag taaataattc ctcaccttta 2580 ctcatggctt cctttctcct ctttaatgaa aacttacgcc ccgccctgcc actcatcgca 2640 gtattgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg 2700 aacttggatc gccagtggca ttaacacctt gtcgccttgc gtataatatt ttcccatagt 2760 gaaaacgggg gcgaagaa 2778 <210> 18 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 1 <400> 18 cttgattaaa cgattctggt gtaaaaa 27 <210> 19 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 2 <400> 19 cttgactaaa cgattcagtg gcaaaaa 27 <210> 20 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 3 <400> 20 cgtgattaaa cgatccccgtt ttcaaaa 27 <210> 21 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 4 <400> 21 cgtgattaaa cgatactgtt gcaaaaa 27 <210> 22 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 5 <400> 22 cgtgactaaa cgatccgggg ggcaaaa 27 <210> 23 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 6 <400> 23 cttgattaaa cgatccaggt ttaaaaa 27 <210> 24 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 7 <400> 24 cttgactaaa cgatactgtt tccaaaa 27 <210> 25 <211> 11789 <212> DNA <213> artificial sequence <220> <223> plasmid lacz6 pri-ori, p1322 <400> 25 ctaatctctt gcccccgccc gtaatagcct ccaagagatt gaagatagta aagggcaaga 60 gctgattcgg cgttgaagga tagcggactt tcggtcaacc acaattcccc actcgacaaa 120 accagccgtg cgaataactc tgaaagtaca agcaacccaa gagggctgag cctaaactca 180 gctaattcct aagtgagcta aagactcgaa gtgacagctc ttaataaata gagcgggaac 240 gtcgaacggt cgtgaaagta atagtacaac gggtattaac ttactgagga tattgcttga 300 agctgtaccg tttattggg tgaacgaata agatccagca attcagccaa agaagctacc 360 aatttttagt ttaagagtgt cacgtctgac ctcgcgggta gattgccgaa cgtagagctt 420 acgagccagc ggaaacagta gccgcaggat aagtaagggg agtaagtgat cgaacgaatc 480 agaagtgaca atatacttag gctggatctc gtcccgtgaa tcccaaccct caccaactac 540 gagataagag gtaagccaaa aatcgacttg gtggcgacca acgactgttc cccccctgta 600 actaatcgtt ccgtcaaaac ctgacttact tcaaggccaa ttccaagcgc aaacaatacc 660 gtcctagttc ttcggttaag tttccgaagt aggagtgagc ctacctccgt ttgcgtcttg 720 ttaccactga cccagctatt tactttgtat tgcctgcaat cgaatttctg aactctcaga 780 tagtggggat aacgggaaag ttcctatatt tgcgaactaa cttagccgtc cacctcgaag 840 ctacctactc acacccaccc cgcgcggggt aaataaggca ctaatcccag ctgagagctg 900 gcgtagcact tagccacaag ttaattaaca gttgtctggt agtttggcgg tattaggaag 960 atcctagaag caaggcagag ttagttctaa cctaaagcca caaataagac aggttgccaa 1020 agcccgccgg aaattaaatc ttgctcagtt cggtaacgga gtttccctcc cgcgtactta 1080 attcccaata agaaacgcgc ccaagtccta tcaggcaaaa ttcagcccct tcccgtgtta 1140 gaacgagggt aaaaatacaa gccgattgaa caagggttgg gggcttcaaa tcgtcgttta 1200 ccccacttta caacggagat taagtagttc accctatagt acgaagcaga actatttcga 1260 ggggcgtgca ataatcgaat cttctgcggt tgacttaaca cgctagggac gtgccctcga 1320 ttcaatcgaa ggtactccta ctcagactgc ctcacaccca gctagtcact gagcgataaa 1380 attgacccgc cctctaggga agcgagtacg tcccaaaggg ctccggacag ggctatatag 1440 gagagtttga tctcgccccg acaactgcaa ccctcaactc ccttagataa tattgttagc 1500 cgaagttgca cgacccgccg tccacggact gctcttaggg tgtggctcct taatctgaca 1560 acgtgcaacc cctatcgaag tcgattgttt ctgcgaaagg tgttgtccta atagtcccga 1620 aatttggccc ttgtaggtgt gaaaccactt agcttcgcgc cgtagtccta aaggcccacc 1680 tattgacttt gtttcgggta gcactaggaa tcttaacaat ttgaatttgg acgtggaacg 1740 cgtacacctt aatctccgaa taattctagg gatttggaag tcctctacgt tgacacacct 1800 acactgctcg aagtaaatat acgaataacg cgggcctcgc ggagccgttc cgaatcgtca 1860 cgtgttcgtt tactgttaat tggtggcaaa taagcaatat cgtagtccgt caggcccagc 1920 cctgttatcc acggcgttat ttgtcaaatt gcgtagaact ggattgactg cctgacaata 1980 cctaattatc ggtacgaagt ccccgaatct gtcgggctat ttcactaata ctttccaaac 2040 gccccgtatc caagaagaac gaatttatcc acgctcccgt ctttgggacg aataccgcta 2100 caagtggaca gaggatcggt acgggcctct aataaatcca acactctacg ccctcttcaa 2160 gagctagaag aacagggtgc agttggaaag ggaattattt cgtaaggcga gccaataccg 2220 taattaattc ggaagagtta acacgattgg aagtaggaat agtttctaac cacggttaact 2280 aatcctaata acggaacgct gtctgataga ttagtgtcag cgctcggtac caaagaaaaa 2340 taaaaagacg ctgaaaagcg tctttttatt tttcggtcca gtgtaactca ggcaaaagca 2400 cgtaatattc gtactttctt cctccgtaag cgtcacccac attccttaaa gagtgcatgt 2460 gcatattttg ttatcaataa aaaaggccgc gatttgcggc cttattgttc gtcttgccgg 2520 attacgcccc gccctgccac tcatcgcagt attgttgtaa ttcattaagc attctgccga 2580 catggaagcc atcacaaacg gcatgatgaa cttggatcgc cagtggcatt aacaccttgt 2640 cgccttgcgt ataatatttt cccatagtga aaacgggggc gaagaagttg tccatatttg 2700 ctacgtttaa atcaaaactg gtgaaactca cccacggatt ggcactgacg aaaaacatat 2760 tttcgataaa ccctttaggg aaatatgcta agttttcacc gtaacacgcc acatcttgac 2820 tatatatgtg tagaaactgc cggaaatcgt cgtggtattc tgaccagagc gatgaaaacg 2880 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct 2940 caccgtcttt cattgccata cgaaactccg gatgtgcatt catcaggcgg gcaagaatgt 3000 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtttttaaa aaggccgtaa 3060 tatccagctg aacggtttgg ttataggtgc actgagcaac tgactggaat gcctcaaaat 3120 gttctttacg atgccattga cttatatcaa ctgtagtata tccagtgatt tttttctcca 3180 ttttagcttc cttagcttgc gaaatctcga taactcaaaa aatagtagtg atcttatttc 3240 attatggtga aagttgtctt acgtgcaaca ttttcgcaaa aagttggcgc tttatcaaca 3300 ctgtcggaat gacaaatggt tccaattatt gaacaccctt cggggtgttt ttttgtttct 3360 ggtttcccga ggccggcctt ttgttgcaat ggctgtctac cctgtctacc tgagtaaaga 3420 aaaatacatt taattcagta cattaacttg ggtagacagc ctttttttac tgtctaccta 3480 ctatctaccc tctctacctg attttacctg aatcagacag ggaggtagat acggggtaga 3540 tagtggataa aagcactcta ccccactgaa agccgcgcca ttactggcat ggtggccagt 3600 aaggtagata aggtagacaa ggggaggcac aactcaaaac tttttaaacg agggggtaaa 3660 acgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat 3720 ttcagtgcaa tttatctctt caaatgtagc accggcgcgc cgtgaccaat tattgaaggc 3780 cgctaacgcg gccttttttt gtttctggta tcccgaatgg agcgacttct ccccaaaaag 3840 cctcgctttc agcacctgtc gtttcctttc ttttcagagg gtattttaaa taaaaacatt 3900 aagttatgac gaagaagaac ggaaacgcct taaaccggaa aattttcata aatagcgaaa 3960 acccgcgagg tcgccgcccc gtaacctgtc ggatcaccgg aaaggacccg taaagtgata 4020 atgattatca tctacatatc acaacgtgcg taaagggtaa gtatgaaggt cgtgtactcc 4080 atcgctacca aattccagaa aacagacgct ttcgagcgtc ttttttcgtt ttggtcacga 4140 cgtacggtgg aagattcgtt accaattgac agctagctca gtcctaggta tatacataca 4200 tgcttgtttg tttgtaaact actgttttca ttaaagagga gaaaggaagc catgtccatc 4260 tatcaggagt ttgttaacaa gtattccctg tctaaaaccc tgcgttttga actgatcccg 4320 cagggcaaaa ctttggaaaa cattaaagcg cgtggcctga ttctggatga cgaaaaacgt 4380 gcaaaggatt acaagaaagc taaacagatc atcgacaaat atcaccagtt ctttatcgaa 4440 gaaattctgt cgtcggtgtg catcagtgag gatctgttac agaattattc tgatgtatac 4500 tttaaactta aaaagtccga tgacgataat ctgcaaaaag atttcaagtc agccaaagat 4560 accatcaaga aacagatctc agaatatatt aaagatagcg aaaagttcaa aaacctgttt 4620 aaccaaaacc tcattgatgc taagaaaggc caagaatctg acctgatctt atggctgaaa 4680 cagagcaaag ataacggcat tgaactgttc aaagctaata gcgacatcac cgatattgat 4740 gaagcgctcg aaatcatcaa gtctttcaaa ggctggacga cgtatttcaa aggttttcat 4800 gaaaaccgta agaatgtata ttcgagcaac gatattccga cctctattat ttatcgtatc 4860 gtggacgaca acctgccgaa gtttctggaa aacaaagcga aatatgaatc tctgaaagac 4920 aaagcaccgg aagctattaa ctatgaacag atcaagaaag atctggcgga agaactgacc 4980 ttcgacatcg actataaaac ctccgaagtt aaccagcgtg ttttctcact ggacgaggtt 5040 ttcgaaatcg ctaatttcaa caattacctg aatcaatctg gcatcaccaa attcaacacc 5100 attattggtg gcaaatttgt taacggcgaa aacaccaagc gtaagggcat caacgaatac 5160 attaacctgt atagccaaca aatcaacgac aaaaccctga aaaagtataa aatgtccgtt 5220 ctgtttaaac agattttatc ggacaccgaa tctaaatcct tcgtaattga taaactggaa 5280 gatgatagcg acgttgtcac cacgatgcag agcttttatg agcagattgc ggcgttcaaa 5340 accgtggaag agaaatctat taaagaaact ctgtccctgc tctttgacga cctcaaagcg 5400 cagaaactag atctgtctaa gatttacttt aaaaacgaca aatctctgac cgatctcagt 5460 caacaagttt tcgatgacta tagcgtgatc ggcacggcag ttttggaata catcacccaa 5520 caaatcgcgc cgaaaaatct ggacaacccg tccaagaagg aacaggaact gattgcaaag 5580 aaaacagaaa aagctaaata cctgagctta gaaactatca aactggcact tgaggaattt 5640 aataaacatc gtgatattga taaacagtgt cgttttgagg aaattctggc gaactttgcg 5700 gcaatcccga tgatcttcga cgaaattgct caaaacaaag acaatctggc gcagatctct 5760 atcaagtacc agaatcaggg taagaaagat ctgcttcaag catctgcgga ggacgatgtg 5820 aaagcaatta aagacttatt agatcagacg aataacttat tacacaagct caaaatcttc 5880 cacatcagcc agagcgagga caaggcgaac attctggata aagatgaaca cttctatctg 5940 gtgttcgaag aatgttactt cgaactggca aacatcgtcc ctctctacaa taaaatccgc 6000 aactacatca cgcagaagcc ttactctgac gagaaattca aactgaactt cgaaaacagc 6060 acgctggcga acggctggga taagaacaaa gagccggaca acaccgcaat cctgttcatc 6120 aaagacgaca aatactatct gggcgtaatg aacaagaaga acaacaagat cttcgacgat 6180 aaagcgatca aagaaaacaa gggtgaaggc tataagaaaa tcgtgtacaa gctcctgccg 6240 ggtgcgaata aaatgttacc gaaagtgttc ttttccgcga aaagcatcaa attctacaac 6300 ccgtctgagg atattctgcg catccgcaat catagcacgc acactaaaaa cggtagcccg 6360 cagaaagggt atgaaaaatt cgaatttaat atagaggact gccgtaagtt catcgacttc 6420 tataaacaga gcatttccaa acatccggaa tggaaagact tcggcttccg tttctctgac 6480 actcagcgct ataatagcat cgacgagttc taccgcgaag tggagaatca gggctataaa 6540 ctgaccttcg agaacattag tgagtcgtac atcgactccg ttgtgaatca gggtaaactg 6600 tacctgtttc agatctataa taaagacttt agcgcgtaca gcaaaggccg tccgaatctg 6660 cacacccttt actggaaagc attatttgac gaacgtaacc tgcaagatgt ggtgtataaa 6720 ctgaacggtg aggcggaact tttctaccgt aaacagagta tcccgaagaa aatcacgcat 6780 ccggcaaaag aagctattgc caacaaaaac aaagacaacc cgaagaaaga atcagtattc 6840 gaatatgacc tgatcaaaga taaacgtttc accgaagata agttcttttt ccactgtccg 6900 attaccatca acttcaaatc tagcggtgcg aacaagttca acgatgaaat taacttatta 6960 ctgaaagaga aagctaatga cgtacacatc ttatctattg atcgcggtga acgtcattta 7020 gcatactata cactggtaga tggtaaaggt aatattatta aacaggatac tttcaatatt 7080 atcggtaatg accgtatgaa aaccaactat cacgataagc tggcggcgat cgaaaaagat 7140 cgtgattctg cgcgtaaaga ttggaagaaa attaacaata tcaaagaaat gaaagaaggc 7200 tatctgagcc aagtggtgca cgagatcgca aaactggtga ttgaatataa cgctatcgtg 7260 gttttcgaag atctgaactt tggttttaaa cgtggtcgct tcaaagtaga aaaacaggtg 7320 taccaaaaac tggaaaaaat gctgattgaa aaactgaact atctggtttt taaagacaac 7380 gaatttgaca aaacgggtgg cgtactccgt gcctatcagc tgaccgctcc gttcgaaacg 7440 ttcaagaaaa tgggtaaaca aacggggatt atctattatg tgccagctgg tttcacctcc 7500 aagatttgtc cagttacggg cttcgttaac cagctgtacc cgaaatacga gagcgttagc 7560 aaatctcaag aatttttcag caaattcgac aagatctgct ataatctgga taaaggctat 7620 ttcgagttca gcttcgatta caaaaacttc ggcgataaag cggctaaagg taagtggact 7680 attgctagct ttggtagccg tctgattaac tttcgcaact ccgacaaaaa ccataattgg 7740 gacacgcgtg aagtgtatcc gaccaaagaa ctggaaaaat tactgaaaga ctattccatc 7800 gaatatggtc atggggagtg cattaaagcg gcgatttgcg gtgaatccga taagaaattt 7860 ttcgccaaac tgaccagcgt gcttaacacc attctgcaaa tgcgtaattc taaaacgggt 7920 acggagctgg actacctgat ttctccggta gccgacgtta acggcaactt cttcgattct 7980 cgtcaagcac cgaaaaatat gccacaagac gcggatgcca acggtgcata ccatatcggc 8040 ttaaaaggct taatgttatt aggccgtatc aagaataatc aggagggcaa gaaattaaat 8100 ctggttatca aaaacgaaga atacttcgag ttcgttcaga atcgtaacaa ttaatgtatg 8160 cttaagcagc tcggtaccaa agacgaacaa taagacgctg aaaagcgtct tttttcgttt 8220 tggtcctgtt gcggcgcgat agtgtgaaca tgctatagac ttctggtgct acccgactga 8280 caattaatca tccggctcgt ataatgctag caatttctac tgttgtagat cccgatgtac 8340 gcgcgcgtgg atgatcgaga cgaacaataa ggcctcccta acggggggcc ttttttattg 8400 ataacaaaag taacttcgag cttgtctacc tcctagcacc attattgcaa ttaataaaca 8460 actaacggac aattctacct aacagttttc atatatgacg agcagttaag tgatgagtaa 8520 aggtgaggaa ttatttactg gtgttgttcc gatcttagtt gaactggacg gcgatgttaa 8580 cggtcataaa ttcagtgttc gtggtgaagg tgaaggtgat gcaaccaacg gtaagctgac 8640 cctgaaattc atctgcacta ctggaaaatt accagtaccg tggcctactc tggtgactac 8700 cctgacctat ggtgttcagt gtttttctcg ttaccctgac cacatgaagc aacatgattt 8760 cttcaaatct gcaatgccgg aaggttatgt acaggagcgc accatttctt tcaaagacga 8820 tggcacgtat aaaacccgtg cagaggttaa atttgaaggt gacactctgg tgaatcgtat 8880 tgaactgaaa ggcattgatt tcaaagagga cggcaatatt ttaggccaca aactggaata 8940 taacttcaac tcccataacg tttacatcac cgcagacaaa cagaagaacg gtatcaaagc 9000 taacttcaaa attcgccata acgttgaaga tggtagcgta cagctggcgg atcattacca 9060 acagaacact ccgattggag atgctcctgt tttactgccg gataaccact acctgtccac 9120 ccagtctaaa ctgtcgaagg atccgaacga aaagcgcgac cacatggtgt tattagagtt 9180 cgttaccgct agtggtatca cgcacggtat ggatgaactc tacaaataag acgaacaata 9240 aggggagcgg gaaaccgctc ccctttttta ttgataacaa aagtaaattg cacgctgata 9300 gtctcccaat tgcgaaggac caaaacgaaa aaacaccctt tcgggtgtct tttctggaat 9360 ttggtaccga gtactaggta tcgtgtaagt agcgaaggcc cgtacgcgag ataaactgct 9420 aggcaaccgc gactctacga ctggtgctcg atttaatttc gctgacgtaa agaaattatc 9480 ggcagtgcgt caactgccgt atctttatct taattaggta gttggacaag cccttgaaag 9540 aaatagcaag agcctgcctc tctattgaag tcacggcgaa agtcgggtag aaatcaaaga 9600 aagcagaaat taaatcggag taacactaag gtgggataac tccgtaactg actacgcctt 9660 tctctagact ttacttgacc agatacactg tctttgacac gttgaaggat tagagcaatc 9720 aaatccaaga ctggctaagc acgaagcaac tcttgagtgt taaaaagtta tctcctgtat 9780 tcgggaagcg ggtactagaa gattgcaggg actccgacgt taagtaaatt acaaagtaat 9840 aagtatcgtt caggatcacg ttaccgcaat aagaagcgag aataatataa tttccgaagt 9900 gcttacccca gtagtgacta ttcctataac ccttctgagt gtccggaggc ggaaatttgc 9960 cacgaaagag aaagtatttc cccgacaata ataaaggggc gctcctcagc ttttccactt 10020 ggttgggtaa gctaggcaac tctgaaagga gtttcggcga attgaagccg acagctttga 10080 attgttttag gggcgttatt cgagggcaat cggagctaac ttcaagacta cttctttgtt 10140 gaatactaaa tagtgcaaag gtcgtgtttc ctcaaggata ctccgctaac aatataggat 10200 tccaatcaga ttcagcactg gcggtacggg tgttgcggtg aggcgttcgg gtttacggct 10260 cgaagctagc acggtaggaa gcctgacaat caccaagcaa aagggccgtc gaaggcccac 10320 aagatacgaa agctctcgaa gccttatcct tgaccgatcc acctatttag gcagttacgc 10380 acaaaagcta cccaataatc cgtgacaggc acaatatcac ggaacaaaac cgaaaactct 10440 cgtacacggt taggttttcg ctaggaagaa taaacctcta tcttgattat aagaaggctc 10500 cccaagcacc cccaaaaccg aaatagcggt ttgcaataag ggacaagtta cgagtgtaga 10560 cacgcagaat tatccagcct ttagtcttta ggaaggcaaa gctattgtac gcggtagccg 10620 tcgtagcaat ttaccaactg tagaattatt ggacacacgt aggaagggct tacagttgaa 10680 gtttaataag gtcacacgca aaaccgctaa ggaataatcg caccgttagc gaaagaatat 10740 ttcagagcgg ttagtaaagg ttgagtaaag tgagattcca aagtgagcct ttataaaaag 10800 taaagagcta taataaaacc gtcgagcaga aaacaatcgc ctgaaatctc aagcacgttg 10860 ccctttctaa cgtcgctaag gtttcgtaaa cccgtttgat taggaagaag aataagtaac 10920 ccgattaggt ttgagatcgc gggttatcgg tttggattaa aagtggatac cagcggagtc 10980 aacgccgacg caaacgtaca gtgatccaat cctgttgcac ggtcaagcac aatcagctcg 11040 caagatcttg gaatagtgtg cccaacagtt tagttgaggg ccacgttccg actacaagtt 11100 gcttcaagag gggaatttgg atttggcaat agccccccgt ttctacctca agaggcgacg 11160 agtattaacc gcgccagctg tcggcacaag ggccaaagaa gattccaatt tcttattccc 11220 gaataacctc cgaatccctg cgggaaaatc accgaccgaa tagcctagaa gcaaggggga 11280 acagataggt ataattagct taagagagta ccagccgtga caacagcgta gtaaccacaa 11340 acttacgctg gggcttcttt ggcggatttt tacagatact aacaaggtga tttgaagtac 11400 cttagttgag gatttaaacg cgctatccgg taatctccaa attgggaaat accgttcaaa 11460 gagggctaga attacttaaa agccttcaca ccgcctgcgc tatacgcgcc cactctcccg 11520 tttatccgtc caagcggaag cagggcgatc ctccgctaag atattcttac gtgtaacgta 11580 gctaagtatc ccaaatagct ggcgtacgcg ttgaacaccg cctagaggat cgtgactcgc 11640 cggacgagcg tgttattggg gacttacgcc agcgtagact acaacgcgcc cagattaacc 11700 ctgcacgtat tgccttgaat aacgtactaa tctctccggc tctcgacaat ctatcgagcg 11760 actcgattat caacgggtgt cttgcagtt 11789 <210> 26 <211> 27 <212> DNA <213> artificial sequence <220> <223> primase RBS 11 <400> 26 cttgattaaa cgattctgtg ttcaaaa 27 <210> 27 <211> 12299 <212> DNA <213> artificial sequence <220> <223> plasmid LacZ6 p15a, p780 <400> 27 ctaatctctt gcccccgccc gtaatagcct ccaagagatt gaagatagta aagggcaaga 60 gctgattcgg cgttgaagga tagcggactt tcggtcaacc acaattcccc actcgacaaa 120 accagccgtg cgaataactc tgaaagtaca agcaacccaa gagggctgag cctaaactca 180 gctaattcct aagtgagcta aagactcgaa gtgacagctc ttaataaata gagcgggaac 240 gtcgaacggt cgtgaaagta atagtacaac gggtattaac ttactgagga tattgcttga 300 agctgtaccg tttattggg tgaacgaata agatccagca attcagccaa agaagctacc 360 aatttttagt ttaagagtgt cacgtctgac ctcgcgggta gattgccgaa cgtagagctt 420 acgagccagc ggaaacagta gccgcaggat aagtaagggg agtaagtgat cgaacgaatc 480 agaagtgaca atatacttag gctggatctc gtcccgtgaa tcccaaccct caccaactac 540 gagataagag gtaagccaaa aatcgacttg gtggcgacca acgactgttc cccccctgta 600 actaatcgtt ccgtcaaaac ctgacttact tcaaggccaa ttccaagcgc aaacaatacc 660 gtcctagttc ttcggttaag tttccgaagt aggagtgagc ctacctccgt ttgcgtcttg 720 ttaccactga cccagctatt tactttgtat tgcctgcaat cgaatttctg aactctcaga 780 tagtggggat aacgggaaag ttcctatatt tgcgaactaa cttagccgtc cacctcgaag 840 ctacctactc acacccaccc cgcgcggggt aaataaggca ctaatcccag ctgagagctg 900 gcgtagcact tagccacaag ttaattaaca gttgtctggt agtttggcgg tattaggaag 960 atcctagaag caaggcagag ttagttctaa cctaaagcca caaataagac aggttgccaa 1020 agcccgccgg aaattaaatc ttgctcagtt cggtaacgga gtttccctcc cgcgtactta 1080 attcccaata agaaacgcgc ccaagtccta tcaggcaaaa ttcagcccct tcccgtgtta 1140 gaacgagggt aaaaatacaa gccgattgaa caagggttgg gggcttcaaa tcgtcgttta 1200 ccccacttta caacggagat taagtagttc accctatagt acgaagcaga actatttcga 1260 ggggcgtgca ataatcgaat cttctgcggt tgacttaaca cgctagggac gtgccctcga 1320 ttcaatcgaa ggtactccta ctcagactgc ctcacaccca gctagtcact gagcgataaa 1380 attgacccgc cctctaggga agcgagtacg tcccaaaggg ctccggacag ggctatatag 1440 gagagtttga tctcgccccg acaactgcaa ccctcaactc ccttagataa tattgttagc 1500 cgaagttgca cgacccgccg tccacggact gctcttaggg tgtggctcct taatctgaca 1560 acgtgcaacc cctatcgaag tcgattgttt ctgcgaaagg tgttgtccta atagtcccga 1620 aatttggccc ttgtaggtgt gaaaccactt agcttcgcgc cgtagtccta aaggcccacc 1680 tattgacttt gtttcgggta gcactaggaa tcttaacaat ttgaatttgg acgtggaacg 1740 cgtacacctt aatctccgaa taattctagg gatttggaag tcctctacgt tgacacacct 1800 acactgctcg aagtaaatat acgaataacg cgggcctcgc ggagccgttc cgaatcgtca 1860 cgtgttcgtt tactgttaat tggtggcaaa taagcaatat cgtagtccgt caggcccagc 1920 cctgttatcc acggcgttat ttgtcaaatt gcgtagaact ggattgactg cctgacaata 1980 cctaattatc ggtacgaagt ccccgaatct gtcgggctat ttcactaata ctttccaaac 2040 gccccgtatc caagaagaac gaatttatcc acgctcccgt ctttgggacg aataccgcta 2100 caagtggaca gaggatcggt acgggcctct aataaatcca acactctacg ccctcttcaa 2160 gagctagaag aacagggtgc agttggaaag ggaattattt cgtaaggcga gccaataccg 2220 taattaattc ggaagagtta acacgattgg aagtaggaat agtttctaac cacggttaact 2280 aatcctaata acggaacgct gtctgataga ttagtgtcag cgctcggtac caaagaaaaa 2340 taaaaagacg ctgaaaagcg tctttttatt tttcggtcca gtgtaactca ggcaaaagca 2400 cgtaatattc gtactttctt cctccgtaag cgtcacccac attccttaaa gagtgcatgt 2460 gcatattttg ttatcaataa aaaaggccgc gatttgcggc cttattgttc gtcttgccgg 2520 attacgcccc gccctgccac tcatcgcagt attgttgtaa ttcattaagc attctgccga 2580 catggaagcc atcacaaacg gcatgatgaa cttggatcgc cagtggcatt aacaccttgt 2640 cgccttgcgt ataatatttt cccatagtga aaacgggggc gaagaagttg tccatatttg 2700 ctacgtttaa atcaaaactg gtgaaactca cccacggatt ggcactgacg aaaaacatat 2760 tttcgataaa ccctttaggg aaatatgcta agttttcacc gtaacacgcc acatcttgac 2820 tatatatgtg tagaaactgc cggaaatcgt cgtggtattc tgaccagagc gatgaaaacg 2880 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct 2940 caccgtcttt cattgccata cgaaactccg gatgtgcatt catcaggcgg gcaagaatgt 3000 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtttttaaa aaggccgtaa 3060 tatccagctg aacggtttgg ttataggtgc actgagcaac tgactggaat gcctcaaaat 3120 gttctttacg atgccattga cttatatcaa ctgtagtata tccagtgatt tttttctcca 3180 ttttagcttc cttagcttgc gaaatctcga taactcaaaa aatagtagtg atcttatttc 3240 attatggtga aagttgtctt acgtgcaaca ttttcgcaaa aagttggcgc tttatcaaca 3300 ctgtcggaat gacaaatggt tccaattatt gaacaccctt cggggtgttt ttttgtttct 3360 ggtttcccga ggccggcctg cgctagcgga gtgtatactg gcttactatg ttggcactga 3420 tgagggtgta agtgaagtgc ttcatgtggc aggagaaaaa aggctgcatc ggtgcgtcag 3480 cagaatatgt gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc 3540 gttcgactgt ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc 3600 aggaagatac ttaacaggga agtgagaggg tcgcggcaaa gccgtttttc cataggctcc 3660 gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga aacctgacag 3720 gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg 3780 cctttcggtt tgccggtgtc attcctctgt tacggccgag tttgtctcat tccacgcctg 3840 acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt 3900 cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat 3960 gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc 4020 atgcgccgga taaggctaaa ctgaaaggac aagttttggc gactgcgctc ctccaagcca 4080 gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc 4140 ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca 4200 tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc 4260 accggcgcgc cgtgaccaat tattgaaggc cgctaacgcg gccttttttt gtttctggta 4320 tcccgaatgg agcgacttct ccccaaaaag cctcgctttc agcacctgtc gtttcctttc 4380 ttttcagagg gtattttaaa taaaaacatt aagttatgac gaagaagaac ggaaacgcct 4440 taaaccggaa aattttcata aatagcgaaa acccgcgagg tcgccgcccc gtaacctgtc 4500 ggatcaccgg aaaggacccg taaagtgata atgattatca tctacatatc acaacgtgcg 4560 taaagggtaa gtatgaaggt cgtgtactcc atcgctacca aattccagaa aacagacgct 4620 ttcgagcgtc ttttttcgtt ttggtcacga cgtacggtgg aagattcgtt accaattgac 4680 agctagctca gtcctaggta tatacataca tgcttgtttg tttgtaaact actgttttca 4740 ttaaagagga gaaaggaagc catgtccatc tatcaggagt ttgttaacaa gtattccctg 4800 tctaaaaccc tgcgttttga actgatcccg cagggcaaaa ctttggaaaa cattaaagcg 4860 cgtggcctga ttctggatga cgaaaaacgt gcaaaggatt acaagaaagc taaacagatc 4920 atcgacaaat atcaccagtt ctttatcgaa gaaattctgt cgtcggtgtg catcagtgag 4980 gatctgttac agaattattc tgatgtatac tttaaactta aaaagtccga tgacgataat 5040 ctgcaaaaag atttcaagtc agccaaagat accatcaaga aacagatctc agaatatatt 5100 aaagatagcg aaaagttcaa aaacctgttt aaccaaaacc tcattgatgc taagaaaggc 5160 caagaatctg acctgatctt atggctgaaa cagagcaaag ataacggcat tgaactgttc 5220 aaagctaata gcgacatcac cgatattgat gaagcgctcg aaatcatcaa gtctttcaaa 5280 ggctggacga cgtatttcaa aggttttcat gaaaaccgta agaatgtata ttcgagcaac 5340 gatattccga cctctattat ttatcgtatc gtggacgaca acctgccgaa gtttctggaa 5400 aacaaagcga aatatgaatc tctgaaagac aaagcaccgg aagctattaa ctatgaacag 5460 atcaagaaag atctggcgga agaactgacc ttcgacatcg actataaaac ctccgaagtt 5520 aaccagcgtg ttttctcact ggacgaggtt ttcgaaatcg ctaatttcaa caattacctg 5580 aatcaatctg gcatcaccaa attcaacacc attattggtg gcaaatttgt taacggcgaa 5640 aacaccaagc gtaagggcat caacgaatac attaacctgt atagccaaca aatcaacgac 5700 aaaaccctga aaaagtataa aatgtccgtt ctgtttaaac agattttatc ggacaccgaa 5760 tctaaatcct tcgtaattga taaactggaa gatgatagcg acgttgtcac cacgatgcag 5820 agcttttatg agcagattgc ggcgttcaaa accgtggaag agaaatctat taaagaaact 5880 ctgtccctgc tctttgacga cctcaaagcg cagaaactag atctgtctaa gatttacttt 5940 aaaaacgaca aatctctgac cgatctcagt caacaagttt tcgatgacta tagcgtgatc 6000 ggcacggcag ttttggaata catcacccaa caaatcgcgc cgaaaaatct ggacaacccg 6060 tccaagaagg aacaggaact gattgcaaag aaaacagaaa aagctaaata cctgagctta 6120 gaaactatca aactggcact tgaggaattt aataaacatc gtgatattga taaacagtgt 6180 cgttttgagg aaattctggc gaactttgcg gcaatcccga tgatcttcga cgaaattgct 6240 caaaacaaag acaatctggc gcagatctct atcaagtacc agaatcaggg taagaaagat 6300 ctgcttcaag catctgcgga ggacgatgg aaagcaatta aagacttatt agatcagacg 6360 aataacttat tacacaagct caaaatcttc cacatcagcc agagcgagga caaggcgaac 6420 attctggata aagatgaaca cttctatctg gtgttcgaag aatgttactt cgaactggca 6480 aacatcgtcc ctctctacaa taaaatccgc aactacatca cgcagaagcc ttactctgac 6540 gagaaattca aactgaactt cgaaaacagc acgctggcga acggctggga taagaacaaa 6600 gagccggaca acaccgcaat cctgttcatc aaagacgaca aatactatct gggcgtaatg 6660 aacaagaaga acaacaagat cttcgacgat aaagcgatca aagaaaacaa gggtgaaggc 6720 tataagaaaa tcgtgtacaa gctcctgccg ggtgcgaata aaatgttacc gaaagtgttc 6780 ttttccgcga aaagcatcaa attctacaac ccgtctgagg atattctgcg catccgcaat 6840 catagcacgc acactaaaaa cggtagcccg cagaaagggt atgaaaaatt cgaatttaat 6900 atagaggact gccgtaagtt catcgacttc tataaacaga gcatttccaa acatccggaa 6960 tggaaagact tcggcttccg tttctctgac actcagcgct ataatagcat cgacgagttc 7020 taccgcgaag tggagaatca gggctataaa ctgaccttcg agaacattag tgagtcgtac 7080 atcgactccg ttgtgaatca gggtaaactg tacctgtttc agatctataa taaagacttt 7140 agcgcgtaca gcaaaggccg tccgaatctg cacacccttt actggaaagc attatttgac 7200 gaacgtaacc tgcaagatgt ggtgtataaa ctgaacggtg aggcggaact tttctaccgt 7260 aaacagagta tcccgaagaa aatcacgcat ccggcaaaag aagctattgc caacaaaaac 7320 aaagacaacc cgaagaaaga atcagtattc gaatatgacc tgatcaaaga taaacgtttc 7380 accgaagata agttcttttt ccactgtccg attaccatca acttcaaatc tagcggtgcg 7440 aacaagttca acgatgaaat taacttatta ctgaaagaga aagctaatga cgtacacatc 7500 ttatctattg atcgcggtga acgtcattta gcatactata cactggtaga tggtaaaggt 7560 aatattatta aacaggatac tttcaatatt atcggtaatg accgtatgaa aaccaactat 7620 cacgataagc tggcggcgat cgaaaaagat cgtgattctg cgcgtaaaga ttggaagaaa 7680 attaacaata tcaaagaaat gaaagaaggc tatctgagcc aagtggtgca cgagatcgca 7740 aaactggtga ttgaatataa cgctatcgtg gttttcgaag atctgaactt tggttttaaa 7800 cgtggtcgct tcaaagtaga aaaacaggtg taccaaaaac tggaaaaaat gctgattgaa 7860 aaactgaact atctggtttt taaagacaac gaatttgaca aaacgggtgg cgtactccgt 7920 gcctatcagc tgaccgctcc gttcgaaacg ttcaagaaaa tgggtaaaca aacggggatt 7980 atctattatg tgccagctgg tttcacctcc aagatttgtc cagttacggg cttcgttaac 8040 cagctgtacc cgaaatacga gagcgttagc aaatctcaag aatttttcag caaattcgac 8100 aagatctgct ataatctgga taaaggctat ttcgagttca gcttcgatta caaaaacttc 8160 ggcgataaag cggctaaagg taagtggact attgctagct ttggtagccg tctgattaac 8220 tttcgcaact ccgacaaaaa ccataattgg gacacgcgtg aagtgtatcc gaccaaagaa 8280 ctggaaaaat tactgaaaga ctattccatc gaatatggtc atggggagtg cattaaagcg 8340 gcgatttgcg gtgaatccga taagaaattt ttcgccaaac tgaccagcgt gcttaacacc 8400 attctgcaaa tgcgtaattc taaaacgggt acggagctgg actacctgat ttctccggta 8460 gccgacgtta acggcaactt cttcgattct cgtcaagcac cgaaaaatat gccacaagac 8520 gcggatgcca acggtgcata ccatatcggc ttaaaaggct taatgttatt aggccgtatc 8580 aagaataatc aggagggcaa gaaattaaat ctggttatca aaaacgaaga atacttcgag 8640 ttcgttcaga atcgtaacaa ttaatgtatg cttaagcagc tcggtaccaa agacgaacaa 8700 taagacgctg aaaagcgtct tttttcgttt tggtcctgtt gcggcgcgat agtgtgaaca 8760 tgctatagac ttctggtgct acccgactga caattaatca tccggctcgt ataatgctag 8820 caatttctac tgttgtagat cccgatgtac gcgcgcgtgg atgatcgaga cgaacaataa 8880 ggcctcccta acggggggcc ttttttattg ataacaaaag taacttcgag cttgtctacc 8940 tcctagcacc attattgcaa ttaataaaca actaacggac aattctacct aacagttttc 9000 atatatgacg agcagttaag tgatgagtaa aggtgaggaa ttatttactg gtgttgttcc 9060 gatcttagtt gaactggacg gcgatgttaa cggtcataaa ttcagtgttc gtggtgaagg 9120 tgaaggtgat gcaaccaacg gtaagctgac cctgaaattc atctgcacta ctggaaaatt 9180 accagtaccg tggcctactc tggtgactac cctgacctat ggtgttcagt gtttttctcg 9240 ttaccctgac cacatgaagc aacatgattt cttcaaatct gcaatgccgg aaggttatgt 9300 acaggagcgc accatttctt tcaaagacga tggcacgtat aaaacccgtg cagaggttaa 9360 atttgaaggt gacactctgg tgaatcgtat tgaactgaaa ggcattgatt tcaaagagga 9420 cggcaatatt ttaggccaca aactggaata taacttcaac tcccataacg tttacatcac 9480 cgcagacaaa cagaagaacg gtatcaaagc taacttcaaa attcgccata acgttgaaga 9540 tggtagcgta cagctggcgg atcattacca acagaacact ccgattggag atgctcctgt 9600 tttactgccg gataaccact acctgtccac ccagtctaaa ctgtcgaagg atccgaacga 9660 aaagcgcgac cacatggtgt tattagagtt cgttaccgct agtggtatca cgcacggtat 9720 ggatgaactc tacaaataag acgaacaata aggggagcgg gaaaccgctc ccctttttta 9780 ttgataacaa aagtaaattg cacgctgata gtctcccaat tgcgaaggac caaaacgaaa 9840 aaacaccctt tcgggtgtct tttctggaat ttggtaccga gtactaggta tcgtgtaagt 9900 agcgaaggcc cgtacgcgag ataaactgct aggcaaccgc gactctacga ctggtgctcg 9960 atttaatttc gctgacgtaa agaaattatc ggcagtgcgt caactgccgt atctttatct 10020 taattaggta gttggacaag cccttgaaag aaatagcaag agcctgcctc tctattgaag 10080 tcacggcgaa agtcgggtag aaatcaaaga aagcagaaat taaatcggag taacactaag 10140 gtgggataac tccgtaactg actacgcctt tctctagact ttacttgacc agatacactg 10200 tctttgacac gttgaaggat tagagcaatc aaatccaaga ctggctaagc acgaagcaac 10260 tcttgagtgt taaaaagtta tctcctgtat tcgggaagcg ggtactagaa gattgcaggg 10320 actccgacgt taagtaaatt acaaagtaat aagtatcgtt caggatcacg ttaccgcaat 10380 aagaagcgag aataatataa tttccgaagt gcttacccca gtagtgacta ttcctataac 10440 ccttctgagt gtccggaggc ggaaatttgc cacgaaagag aaagtatttc cccgacaata 10500 ataaaggggc gctcctcagc ttttccactt ggttgggtaa gctaggcaac tctgaaagga 10560 gtttcggcga attgaagccg acagctttga attgttttag gggcgttatt cgagggcaat 10620 cggagctaac ttcaagacta cttctttgtt gaatactaaa tagtgcaaag gtcgtgtttc 10680 ctcaaggata ctccgctaac aatataggat tccaatcaga ttcagcactg gcggtacggg 10740 tgttgcggtg aggcgttcgg gtttacggct cgaagctagc acggtaggaa gcctgacaat 10800 caccaagcaa aagggccgtc gaaggcccac aagatacgaa agctctcgaa gccttatcct 10860 tgaccgatcc acctatttag gcagttacgc acaaaagcta cccaataatc cgtgacaggc 10920 acaatatcac ggaacaaaac cgaaaactct cgtacacggt taggttttcg ctaggaagaa 10980 taaacctcta tcttgattat aagaaggctc cccaagcacc cccaaaaccg aaatagcggt 11040 ttgcaataag ggacaagtta cgagtgtaga cacgcagaat tatccagcct ttagtcttta 11100 ggaaggcaaa gctattgtac gcggtagccg tcgtagcaat taccaactg tagaattatt 11160 ggacacacgt aggaagggct tacagttgaa gtttaataag gtcacacgca aaaccgctaa 11220 ggaataatcg caccgttagc gaaagaatat ttcagagcgg ttagtaaagg ttgagtaaag 11280 tgagattcca aagtgagcct ttataaaaag taaagagcta taataaaacc gtcgagcaga 11340 aaacaatcgc ctgaaatctc aagcacgttg ccctttctaa cgtcgctaag gtttcgtaaa 11400 cccgtttgat taggaagaag aataagtaac ccgattaggt ttgagatcgc gggttatcgg 11460 tttggattaa aagtggatac cagcggagtc aacgccgacg caaacgtaca gtgatccaat 11520 cctgttgcac ggtcaagcac aatcagctcg caagatcttg gaatagtgg cccaacagtt 11580 tagttgaggg ccacgttccg actacaagtt gcttcaagag gggaatttgg atttggcaat 11640 agccccccgt ttctacctca agaggcgacg agtattaacc gcgccagctg tcggcacaag 11700 ggccaaagaa gattccaatt tcttattccc gaataacctc cgaatccctg cgggaaaatc 11760 accgaccgaa tagcctagaa gcaaggggga acagataggt ataattagct taagagagta 11820 ccagccgtga caacagcgta gtaaccacaa acttacgctg gggcttcttt ggcggatttt 11880 tacagatact aacaaggtga tttgaagtac cttagttgag gatttaaacg cgctatccgg 11940 taatctccaa attgggaaat accgttcaaa gagggctaga attacttaaa agccttcaca 12000 ccgcctgcgc tatacgcgcc cactctcccg tttatccgtc caagcggaag cagggcgatc 12060 ctccgctaag atattcttac gtgtaacgta gctaagtatc ccaaatagct ggcgtacgcg 12120 ttgaacaccg cctagaggat cgtgactcgc cggacgagcg tgttattggg gacttacgcc 12180 agcgtagact acaacgcgcc cagattaacc ctgcacgtat tgccttgaat aacgtactaa 12240 tctctccggc tctcgacaat ctatcgagcg actcgattat caacgggtgt cttgcagtt 12299 <210> 28 <211> 11782 <212> DNA <213> artificial sequence <220> <223> plasmid LacZ6 pri-ori deltaGAAABCC, p1326 <400> 28 ctaatctctt gcccccgccc gtaatagcct ccaagagatt gaagatagta aagggcaaga 60 gctgattcgg cgttgaagga tagcggactt tcggtcaacc acaattcccc actcgacaaa 120 accagccgtg cgaataactc tgaaagtaca agcaacccaa gagggctgag cctaaactca 180 gctaattcct aagtgagcta aagactcgaa gtgacagctc ttaataaata gagcgggaac 240 gtcgaacggt cgtgaaagta atagtacaac gggtattaac ttactgagga tattgcttga 300 agctgtaccg tttattggg tgaacgaata agatccagca attcagccaa agaagctacc 360 aatttttagt ttaagagtgt cacgtctgac ctcgcgggta gattgccgaa cgtagagctt 420 acgagccagc ggaaacagta gccgcaggat aagtaagggg agtaagtgat cgaacgaatc 480 agaagtgaca atatacttag gctggatctc gtcccgtgaa tcccaaccct caccaactac 540 gagataagag gtaagccaaa aatcgacttg gtggcgacca acgactgttc cccccctgta 600 actaatcgtt ccgtcaaaac ctgacttact tcaaggccaa ttccaagcgc aaacaatacc 660 gtcctagttc ttcggttaag tttccgaagt aggagtgagc ctacctccgt ttgcgtcttg 720 ttaccactga cccagctatt tactttgtat tgcctgcaat cgaatttctg aactctcaga 780 tagtggggat aacgggaaag ttcctatatt tgcgaactaa cttagccgtc cacctcgaag 840 ctacctactc acacccaccc cgcgcggggt aaataaggca ctaatcccag ctgagagctg 900 gcgtagcact tagccacaag ttaattaaca gttgtctggt agtttggcgg tattaggaag 960 atcctagaag caaggcagag ttagttctaa cctaaagcca caaataagac aggttgccaa 1020 agcccgccgg aaattaaatc ttgctcagtt cggtaacgga gtttccctcc cgcgtactta 1080 attcccaata agaaacgcgc ccaagtccta tcaggcaaaa ttcagcccct tcccgtgtta 1140 gaacgagggt aaaaatacaa gccgattgaa caagggttgg gggcttcaaa tcgtcgttta 1200 ccccacttta caacggagat taagtagttc accctatagt acgaagcaga actatttcga 1260 ggggcgtgca ataatcgaat cttctgcggt tgacttaaca cgctagggac gtgccctcga 1320 ttcaatcgaa ggtactccta ctcagactgc ctcacaccca gctagtcact gagcgataaa 1380 attgacccgc cctctaggga agcgagtacg tcccaaaggg ctccggacag ggctatatag 1440 gagagtttga tctcgccccg acaactgcaa ccctcaactc ccttagataa tattgttagc 1500 cgaagttgca cgacccgccg tccacggact gctcttaggg tgtggctcct taatctgaca 1560 acgtgcaacc cctatcgaag tcgattgttt ctgcgaaagg tgttgtccta atagtcccga 1620 aatttggccc ttgtaggtgt gaaaccactt agcttcgcgc cgtagtccta aaggcccacc 1680 tattgacttt gtttcgggta gcactaggaa tcttaacaat ttgaatttgg acgtggaacg 1740 cgtacacctt aatctccgaa taattctagg gatttggaag tcctctacgt tgacacacct 1800 acactgctcg aagtaaatat acgaataacg cgggcctcgc ggagccgttc cgaatcgtca 1860 cgtgttcgtt tactgttaat tggtggcaaa taagcaatat cgtagtccgt caggcccagc 1920 cctgttatcc acggcgttat ttgtcaaatt gcgtagaact ggattgactg cctgacaata 1980 cctaattatc ggtacgaagt ccccgaatct gtcgggctat ttcactaata ctttccaaac 2040 gccccgtatc caagaagaac gaatttatcc acgctcccgt ctttgggacg aataccgcta 2100 caagtggaca gaggatcggt acgggcctct aataaatcca acactctacg ccctcttcaa 2160 gagctagaag aacagggtgc agttggaaag ggaattattt cgtaaggcga gccaataccg 2220 taattaattc ggaagagtta acacgattgg aagtaggaat agtttctaac cacggttaact 2280 aatcctaata acggaacgct gtctgataga ttagtgtcag cgctcggtac caaagaaaaa 2340 taaaaagacg ctgaaaagcg tctttttatt tttcggtcca gtgtaactca ggcaaaagca 2400 cgtaatattc gtactttctt cctccgtaag cgtcacccac attccttaaa gagtgcatgt 2460 gcatattttg ttatcaataa aaaaggccgc gatttgcggc cttattgttc gtcttgccgg 2520 attacgcccc gccctgccac tcatcgcagt attgttgtaa ttcattaagc attctgccga 2580 catggaagcc atcacaaacg gcatgatgaa cttggatcgc cagtggcatt aacaccttgt 2640 cgccttgcgt ataatatttt cccatagtga aaacgggggc gaagaagttg tccatatttg 2700 ctacgtttaa atcaaaactg gtgaaactca cccacggatt ggcactgacg aaaaacatat 2760 tttcgataaa ccctttaggg aaatatgcta agttttcacc gtaacacgcc acatcttgac 2820 tatatatgtg tagaaactgc cggaaatcgt cgtggtattc tgaccagagc gatgaaaacg 2880 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct 2940 caccgtcttt cattgccata cgaaactccg gatgtgcatt catcaggcgg gcaagaatgt 3000 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtttttaaa aaggccgtaa 3060 tatccagctg aacggtttgg ttataggtgc actgagcaac tgactggaat gcctcaaaat 3120 gttctttacg atgccattga cttatatcaa ctgtagtata tccagtgatt tttttctcca 3180 ttttagcttc cttagcttgc gaaatctcga taactcaaaa aatagtagtg atcttatttc 3240 attatggtga aagttgtctt acgtgcaaca ttttcgcaaa aagttggcgc tttatcaaca 3300 ctgtcggaat gacaaatggt tccaattatt gaacaccctt cggggtgttt ttttgtttcc 3360 cgaggccggc cttttgttgc aatggctgtc taccctgtct acctgagtaa agaaaaatac 3420 atttaattca gtacattaac ttgggtagac agcctttttt tactgtctac ctactatcta 3480 ccctctctac ctgattttac ctgaatcaga cagggaggta gatacggggt agatagtgga 3540 taaaagcact ctaccccact gaaagcagcg ccattactgg catggtggcc agtaaggtag 3600 ataaggtaga caaggggagg cacaactcaa aactttttaa acgagggggt aaaacgcaga 3660 ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta gatttcagtg 3720 caatttatct cttcaaatgt agcaccggcg cgccgtgacc aattattgaa ggccgctaac 3780 gcggcctttt tttgtttctg gtatcccgaa tggagcgact tctccccaaa aagcctcgct 3840 ttcagcacct gtcgtttcct ttcttttcag agggtatttt aaataaaaac attaagttat 3900 gacgaagaag aacggaaacg ccttaaaccg gaaaattttc ataaatagcg aaaacccgcg 3960 aggtcgccgc cccgtaacct gtcggatcac cggaaaggac ccgtaaagtg ataatgatta 4020 tcatctacat atcacaacgt gcgtaaaggg taagtatgaa ggtcgtgtac tccatcgcta 4080 ccaaattcca gaaaacagac gctttcgagc gtcttttttc gttttggtca cgacgtacgg 4140 tggaagattc gttaccaatt gacagctagc tcagtcctag gtatatacat acatgcttgt 4200 ttgtttgtaa actactgttt tcattaaaga ggagaaagga agccatgtcc atctatcagg 4260 agtttgttaa caagtattcc ctgtctaaaa ccctgcgttt tgaactgatc ccgcagggca 4320 aaactttgga aaacattaaa gcgcgtggcc tgattctgga tgacgaaaaa cgtgcaaagg 4380 attacaagaa agctaaacag atcatcgaca aatatcacca gttctttatc gaagaaattc 4440 tgtcgtcggt gtgcatcagt gaggatctgt tacagaatta ttctgatgta tactttaaac 4500 ttaaaaagtc cgatgacgat aatctgcaaa aagatttcaa gtcagccaaa gataccatca 4560 agaaacagat ctcagaatat attaaagata gcgaaaagtt caaaaacctg tttaaccaaa 4620 acctcattga tgctaagaaa ggccaagaat ctgacctgat cttatggctg aaacagagca 4680 aagataacgg cattgaactg ttcaaagcta atagcgacat caccgatatt gatgaagcgc 4740 tcgaaatcat caagtctttc aaaggctgga cgacgtattt caaaggtttt catgaaaacc 4800 gtaagaatgt atattcgagc aacgatattc cgacctctat tatttatcgt atcgtggacg 4860 acaacctgcc gaagtttctg gaaaacaaag cgaaatatga atctctgaaa gacaaagcac 4920 cggaagctat taactatgaa cagatcaaga aagatctggc ggaagaactg accttcgaca 4980 tcgactataa aacctccgaa gttaaccagc gtgttttctc actggacgag gttttcgaaa 5040 tcgctaattt caacaattac ctgaatcaat ctggcatcac caaattcaac accattattg 5100 gtggcaaatt tgttaacggc gaaaacacca agcgtaaggg catcaacgaa tacattaacc 5160 tgtatagcca acaaatcaac gacaaaaccc tgaaaaagta taaaatgtcc gttctgttta 5220 aacagatttt atcggacacc gaatctaaat ccttcgtaat tgataaactg gaagatgata 5280 gcgacgttgt caccacgatg cagagctttt atgagcagat tgcggcgttc aaaaccgtgg 5340 aagagaaatc tattaaagaa actctgtccc tgctctttga cgacctcaaa gcgcagaaac 5400 tagatctgtc taagatttac tttaaaaacg acaaatctct gaccgatctc agtcaacaag 5460 ttttcgatga ctatagcgtg atcggcacgg cagttttgga atacatcacc caacaaatcg 5520 cgccgaaaaa tctggacaac ccgtccaaga aggaacagga actgattgca aagaaaacag 5580 aaaaagctaa atacctgagc ttagaaacta tcaaactggc acttgaggaa tttaataaac 5640 atcgtgatat tgataaacag tgtcgttttg aggaaattct ggcgaacttt gcggcaatcc 5700 cgatgatctt cgacgaaatt gctcaaaaca aagacaatct ggcgcagatc tctatcaagt 5760 accagaatca gggtaagaaa gatctgcttc aagcatctgc ggaggacgat gtgaaagcaa 5820 ttaaagactt attagatcag acgaataact tattacacaa gctcaaaatc ttccacatca 5880 gccagagcga ggacaaggcg aacattctgg ataaagatga acacttctat ctggtgttcg 5940 aagaatgtta cttcgaactg gcaaacatcg tccctctcta caataaaatc cgcaactaca 6000 tcacgcagaa gccttactct gacgagaaat tcaaactgaa cttcgaaaac agcacgctgg 6060 cgaacggctg ggataagaac aaagagccgg acaacaccgc aatcctgttc atcaaagacg 6120 acaaatacta tctgggcgta atgaacaaga agaacaacaa gatcttcgac gataaagcga 6180 tcaaagaaaa caagggtgaa ggctataaga aaatcgtgta caagctcctg ccgggtgcga 6240 ataaaatgtt accgaaagtg ttcttttccg cgaaaagcat caaattctac aacccgtctg 6300 aggatattct gcgcatccgc aatcatagca cgcacactaa aaacggtagc ccgcagaaag 6360 ggtatgaaaa attcgaattt aatatagagg actgccgtaa gttcatcgac ttctataaac 6420 agagcatttc caaacatccg gaatggaaag acttcggctt ccgtttctct gacactcagc 6480 gctataatag catcgacgag ttctaccgcg aagtggagaa tcagggctat aaactgacct 6540 tcgagaacat tagtgagtcg tacatcgact ccgttgtgaa tcagggtaaa ctgtacctgt 6600 ttcagatcta taataaagac tttagcgcgt acagcaaagg ccgtccgaat ctgcacaccc 6660 tttactggaa agcattattt gacgaacgta acctgcaaga tgtggtgtat aaactgaacg 6720 gtgaggcgga acttttctac cgtaaacaga gtatcccgaa gaaaatcacg catccggcaa 6780 aagaagctat tgccaacaaa aacaaagaca acccgaagaa agaatcagta ttcgaatatg 6840 acctgatcaa agataaacgt ttcaccgaag ataagttctt tttccactgt ccgattacca 6900 tcaacttcaa atctagcggt gcgaacaagt tcaacgatga aattaactta ttactgaaag 6960 agaaagctaa tgacgtacac atcttatcta ttgatcgcgg tgaacgtcat ttagcatact 7020 atacactggt agatggtaaa ggtaatatta ttaaacagga tactttcaat attatcggta 7080 atgaccgtat gaaaaccaac tatcacgata agctggcggc gatcgaaaaa gatcgtgatt 7140 ctgcgcgtaa agattggaag aaaattaaca atatcaaaga aatgaaagaa ggctatctga 7200 gccaagtggt gcacgagatc gcaaaactgg tgattgaata taacgctatc gtggttttcg 7260 aagatctgaa ctttggtttt aaacgtggtc gcttcaaagt agaaaaacag gtgtaccaaa 7320 aactggaaaa aatgctgatt gaaaaactga actatctggt ttttaaagac aacgaatttg 7380 acaaaacggg tggcgtactc cgtgcctatc agctgaccgc tccgttcgaa acgttcaaga 7440 aaatgggtaa acaaacgggg attatctatt atgtgccagc tggtttcacc tccaagattt 7500 gtccagttac gggcttcgtt aaccagctgt acccgaaata cgagagcgtt agcaaatctc 7560 aagaattttt cagcaaattc gacaagatct gctataatct ggataaaggc tatttcgagt 7620 tcagcttcga ttacaaaaac ttcggcgata aagcggctaa aggtaagtgg actattgcta 7680 gctttggtag ccgtctgatt aactttcgca actccgacaa aaaccataat tgggacacgc 7740 gtgaagtgta tccgaccaaa gaactggaaa aattactgaa agactattcc atcgaatatg 7800 gtcatgggga gtgcattaaa gcggcgattt gcggtgaatc cgataagaaa tttttcgcca 7860 aactgaccag cgtgcttaac accattctgc aaatgcgtaa ttctaaaacg ggtacggagc 7920 tggactacct gatttctccg gtagccgacg ttaacggcaa cttcttcgat tctcgtcaag 7980 caccgaaaaa tatgccacaa gacgcggatg ccaacggtgc ataccatatc ggcttaaaag 8040 gcttaatgtt attaggccgt atcaagaata atcaggaggg caagaaatta aatctggtta 8100 tcaaaaacga agaatacttc gagttcgttc agaatcgtaa caattaatgt atgcttaagc 8160 agctcggtac caaagacgaa caataagacg ctgaaaagcg tcttttttcg ttttggtcct 8220 gttgcggcgc gatagtgtga acatgctata gacttctggt gctacccgac tgacaattaa 8280 tcatccggct cgtataatgc tagcaatttc tactgttgta gatcccgatg tacgcgcgcg 8340 tggatgatcg agacgaacaa taaggcctcc ctaacggggg gcctttttta ttgataacaa 8400 aagtaacttc gagcttgtct acctcctagc accattattg caattaataa acaactaacg 8460 gacaattcta cctaacagtt ttcatatatg acgagcagtt aagtgatgag taaaggtgag 8520 gaattattta ctggtgttgt tccgatctta gttgaactgg acggcgatgt taacggtcat 8580 aaattcagtg ttcgtggtga aggtgaaggt gatgcaacca acggtaagct gaccctgaaa 8640 ttcatctgca ctactggaaa attaccagta ccgtggccta ctctggtgac taccctgacc 8700 tatggtgttc agtgtttttc tcgttaccct gaccacatga agcaacatga tttcttcaaa 8760 tctgcaatgc cggaaggtta tgtacaggag cgcaccattt ctttcaaaga cgatggcacg 8820 tataaaaccc gtgcagaggt taaatttgaa ggtgacactc tggtgaatcg tattgaactg 8880 aaaggcattg atttcaaaga ggacggcaat attttaggcc acaaactgga atataacttc 8940 aactcccata acgtttacat caccgcagac aaacagaaga acggtatcaa agctaacttc 9000 aaaattcgcc ataacgttga agatggtagc gtacagctgg cggatcatta ccaacagaac 9060 actccgattg gagatgctcc tgttttactg ccggataacc actacctgtc cacccagtct 9120 aaactgtcga aggatccgaa cgaaaagcgc gaccacatgg tgttattaga gttcgttacc 9180 gctagtggta tcacgcacgg tatggatgaa ctctacaaat aagacgaaca ataaggggag 9240 cgggaaaccg ctcccctttt ttattgataa caaaagtaaa ttgcacgctg atagtctccc 9300 aattgcgaag gaccaaaacg aaaaaacacc ctttcgggtg tcttttctgg aatttggtac 9360 cgagtactag gtatcgtgta agtagcgaag gcccgtacgc gagataaact gctaggcaac 9420 cgcgactcta cgactggtgc tcgatttaat ttcgctgacg taaagaaatt atcggcagtg 9480 cgtcaactgc cgtatcttta tcttaattag gtagttggac aagcccttga aagaaatagc 9540 aagagcctgc ctctctattg aagtcacggc gaaagtcggg tagaaatcaa agaaagcaga 9600 aattaaatcg gagtaacact aaggtgggat aactccgtaa ctgactacgc ctttctctag 9660 actttacttg accagataca ctgtctttga cacgttgaag gattagagca atcaaatcca 9720 agactggcta agcacgaagc aactcttgag tgttaaaaag ttatctcctg tattcgggaa 9780 gcgggtacta gaagattgca gggactccga cgttaagtaa attacaaagt aataagtatc 9840 gttcaggatc acgttaccgc aataagaagc gagaataata taatttccga agtgcttacc 9900 ccagtagtga ctattcctat aacccttctg agtgtccgga ggcggaaatt tgccacgaaa 9960 gagaaagtat ttccccgaca ataataaagg ggcgctcctc agcttttcca cttggttggg 10020 taagctaggc aactctgaaa ggaggtttcgg cgaattgaag ccgacagctt tgaattgttt 10080 tagggggcgtt attcgagggc aatcggagct aacttcaaga ctacttcttt gttgaatact 10140 aaatagtgca aaggtcgtgt ttcctcaagg atactccgct aacaatatag gattccaatc 10200 agattcagca ctggcggtac gggtgttgcg gtgaggcgtt cgggtttacg gctcgaagct 10260 agcacggtag gaagcctgac aatcaccaag caaaagggcc gtcgaaggcc cacaagatac 10320 gaaagctctc gaagccttat ccttgaccga tccacctatt taggcagtta cgcacaaaag 10380 ctacccaata atccgtgaca ggcacaatat cacggaacaa aaccgaaaac tctcgtacac 10440 ggttaggttt tcgctaggaa gaataaacct ctatcttgat tataagaagg ctccccaagc 10500 acccccaaaa ccgaaatagc ggtttgcaat aagggacaag ttacgagtgt agacacgcag 10560 aattatccag cctttagtct ttaggaaggc aaagctattg tacgcggtag ccgtcgtagc 10620 aatttaccaa ctgtagaatt attggacaca cgtaggaagg gcttacagtt gaagtttaat 10680 aaggtcacac gcaaaaccgc taaggaataa tcgcaccgtt agcgaaagaa tatttcagag 10740 cggttagtaa aggttgagta aagtgagatt ccaaagtgag cctttataaa aagtaaagag 10800 ctataataaa accgtcgagc agaaaacaat cgcctgaaat ctcaagcacg ttgccctttc 10860 taacgtcgct aaggtttcgt aaacccgttt gattaggaag aagaataagt aacccgatta 10920 ggtttgagat cgcgggttat cggtttggat taaaagtgga taccagcgga gtcaacgccg 10980 acgcaaacgt acagtgatcc aatcctgttg cacggtcaag cacaatcagc tcgcaagatc 11040 ttggaatagt gtgcccaaca gtttagttga gggccacgtt ccgactacaa gttgcttcaa 11100 gaggggaatt tggatttggc aatagccccc cgtttctacc tcaagaggcg acgagtatta 11160 accgcgccag ctgtcggcac aagggccaaa gaagattcca atttcttatt cccgaataac 11220 ctccgaatcc ctgcgggaaa atcaccgacc gaatagccta gaagcaaggg ggaacagata 11280 ggtataatta gcttaagaga gtaccagccg tgacaacagc gtagtaacca caaacttacg 11340 ctggggcttc tttggcggat ttttacagat actaacaagg tgatttgaag taccttagtt 11400 gaggatttaa acgcgctatc cggtaatctc caaattggga aataccgttc aaagagggct 11460 agaattactt aaaagccttc acaccgcctg cgctatacgc gcccactctc ccgtttatcc 11520 gtccaagcgg aagcagggcg atcctccgct aagatattct tacgtgtaac gtagctaagt 11580 atcccaaata gctggcgtac gcgttgaaca ccgcctagag gatcgtgact cgccggacga 11640 gcgtgttatt ggggacttac gccagcgtag actacaacgc gcccagatta accctgcacg 11700 tattgccttg aataacgtac taatctctcc ggctctcgac aatctatcga gcgactcgat 11760 tatcaacggg tgtcttgcag tt 11782 <210> 29 <211> 11915 <212> DNA <213> artificial sequence <220> <223> plasmid 4stx pri-ori deltagaaabcc, p1327 <400> 29 ctaatctctt gcccccgccc gtaatagcct ccaagagatt gaagatagta aagggcaaga 60 gctgattcgg cgttgaagga tagcggactt tcggtcaacc acaattcccc actcgacaaa 120 accagccgtg cgaataactc tgaaagtaca agcaacccaa gagggctgag cctaaactca 180 gctaattcct aagtgagcta aagactcgaa gtgacagctc ttaataaata gagcgggaac 240 gtcgaacggt cgtgaaagta atagtacaac gggtattaac ttactgagga tattgcttga 300 agctgtaccg tttattggg tgaacgaata agatccagca attcagccaa agaagctacc 360 aatttttagt ttaagagtgt cacgtctgac ctcgcgggta gattgccgaa cgtagagctt 420 acgagccagc ggaaacagta gccgcaggat aagtaagggg agtaagtgat cgaacgaatc 480 agaagtgaca atatacttag gctggatctc gtcccgtgaa tcccaaccct caccaactac 540 gagataagag gtaagccaaa aatcgacttg gtggcgacca acgactgttc cccccctgta 600 actaatcgtt ccgtcaaaac ctgacttact tcaaggccaa ttccaagcgc aaacaatacc 660 gtcctagttc ttcggttaag tttccgaagt aggagtgagc ctacctccgt ttgcgtcttg 720 ttaccactga cccagctatt tactttgtat tgcctgcaat cgaatttctg aactctcaga 780 tagtggggat aacgggaaag ttcctatatt tgcgaactaa cttagccgtc cacctcgaag 840 ctacctactc acacccaccc cgcgcggggt aaataaggca ctaatcccag ctgagagctg 900 gcgtagcact tagccacaag ttaattaaca gttgtctggt agtttggcgg tattaggaag 960 atcctagaag caaggcagag ttagttctaa cctaaagcca caaataagac aggttgccaa 1020 agcccgccgg aaattaaatc ttgctcagtt cggtaacgga gtttccctcc cgcgtactta 1080 attcccaata agaaacgcgc ccaagtccta tcaggcaaaa ttcagcccct tcccgtgtta 1140 gaacgagggt aaaaatacaa gccgattgaa caagggttgg gggcttcaaa tcgtcgttta 1200 ccccacttta caacggagat taagtagttc accctatagt acgaagcaga actatttcga 1260 ggggcgtgca ataatcgaat cttctgcggt tgacttaaca cgctagggac gtgccctcga 1320 ttcaatcgaa ggtactccta ctcagactgc ctcacaccca gctagtcact gagcgataaa 1380 attgacccgc cctctaggga agcgagtacg tcccaaaggg ctccggacag ggctatatag 1440 gagagtttga tctcgccccg acaactgcaa ccctcaactc ccttagataa tattgttagc 1500 cgaagttgca cgacccgccg tccacggact gctcttaggg tgtggctcct taatctgaca 1560 acgtgcaacc cctatcgaag tcgattgttt ctgcgaaagg tgttgtccta atagtcccga 1620 aatttggccc ttgtaggtgt gaaaccactt agcttcgcgc cgtagtccta aaggcccacc 1680 tattgacttt gtttcgggta gcactaggaa tcttaacaat ttgaatttgg acgtggaacg 1740 cgtacacctt aatctccgaa taattctagg gatttggaag tcctctacgt tgacacacct 1800 acactgctcg aagtaaatat acgaataacg cgggcctcgc ggagccgttc cgaatcgtca 1860 cgtgttcgtt tactgttaat tggtggcaaa taagcaatat cgtagtccgt caggcccagc 1920 cctgttatcc acggcgttat ttgtcaaatt gcgtagaact ggattgactg cctgacaata 1980 cctaattatc ggtacgaagt ccccgaatct gtcgggctat ttcactaata ctttccaaac 2040 gccccgtatc caagaagaac gaatttatcc acgctcccgt ctttgggacg aataccgcta 2100 caagtggaca gaggatcggt acgggcctct aataaatcca acactctacg ccctcttcaa 2160 gagctagaag aacagggtgc agttggaaag ggaattattt cgtaaggcga gccaataccg 2220 taattaattc ggaagagtta acacgattgg aagtaggaat agtttctaac cacggttaact 2280 aatcctaata acggaacgct gtctgataga ttagtgtcag cgctcggtac caaagaaaaa 2340 taaaaagacg ctgaaaagcg tctttttatt tttcggtcca gtgtaactca ggcaaaagca 2400 cgtaatattc gtactttctt cctccgtaag cgtcacccac attccttaaa gagtgcatgt 2460 gcatattttg ttatcaataa aaaaggccgc gatttgcggc cttattgttc gtcttgccgg 2520 attacgcccc gccctgccac tcatcgcagt attgttgtaa ttcattaagc attctgccga 2580 catggaagcc atcacaaacg gcatgatgaa cttggatcgc cagtggcatt aacaccttgt 2640 cgccttgcgt ataatatttt cccatagtga aaacgggggc gaagaagttg tccatatttg 2700 ctacgtttaa atcaaaactg gtgaaactca cccacggatt ggcactgacg aaaaacatat 2760 tttcgataaa ccctttaggg aaatatgcta agttttcacc gtaacacgcc acatcttgac 2820 tatatatgtg tagaaactgc cggaaatcgt cgtggtattc tgaccagagc gatgaaaacg 2880 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct 2940 caccgtcttt cattgccata cgaaactccg gatgtgcatt catcaggcgg gcaagaatgt 3000 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtttttaaa aaggccgtaa 3060 tatccagctg aacggtttgg ttataggtgc actgagcaac tgactggaat gcctcaaaat 3120 gttctttacg atgccattga cttatatcaa ctgtagtata tccagtgatt tttttctcca 3180 ttttagcttc cttagcttgc gaaatctcga taactcaaaa aatagtagtg atcttatttc 3240 attatggtga aagttgtctt acgtgcaaca ttttcgcaaa aagttggcgc tttatcaaca 3300 ctgtcggaat gacaaatggt tccaattatt gaacaccctt cggggtgttt ttttgtttct 3360 ggtttcccga ggccggcctt tttgcaaggc tgtctaccct gtctacctga gtaaagaaaa 3420 atacatttaa ttcagtacat taacttgggt agacagcctt tttttactgt ctacctacta 3480 tctaccctct ctacctgatt ttacctgaat cagacaggga ggtagatacg gggtagatag 3540 tggataaaag cactctaccc cactgaaagc agcgccatta ctggcatggt ggccagtaag 3600 gtagataagg tagacaaggg gaggcacaac tcaaaacttt ttaaacgagg gggtaaaacg 3660 cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc 3720 agtgcaattt atctcttcaa atgtagcacc ggcgcgccgt gaccaattat tgaaggccgc 3780 taacgcggcc tttttttgtt tctggtatcc cgaatggagc gacttctccc caaaaagcct 3840 cgctttcagc acctgtcgtt tcctttcttt tcagagggta ttttaaataa aaacattaag 3900 ttatgacgaa gaagaacgga aacgccttaa accggaaaat tttcataaat agcgaaaacc 3960 cgcgaggtcg ccgccccgta acctgtcgga tcaccggaaa ggacccgtaa agtgataatg 4020 attatcatct acatatcaca acgtgcgtaa agggtaagta tgaaggtcgt gtactccatc 4080 gctaccaaat tccagaaaac agacgctttc gagcgtcttt tttcgttttg gtcacgacgt 4140 acggtggaag attcgttacc aattgacagc tagctcagtc ctaggtatat acatacatgc 4200 ttgtttgttt gtaaactact gttttcatta aagaggagaa aggaagccat gtccatctat 4260 caggagtttg ttaacaagta ttccctgtct aaaaccctgc gttttgaact gatcccgcag 4320 ggcaaaactt tggaaaacat taaagcgcgt ggcctgattc tggatgacga aaaacgtgca 4380 aaggattaca agaaagctaa acagatcatc gacaaatatc accagttctt tatcgaagaa 4440 attctgtcgt cggtgtgcat cagtgaggat ctgttacaga attattctga tgtatacttt 4500 aaacttaaaa agtccgatga cgataatctg caaaaagatt tcaagtcagc caaagatacc 4560 atcaagaaac agatctcaga atatattaaa gatagcgaaa agttcaaaaa cctgtttaac 4620 caaaacctca ttgatgctaa gaaaggccaa gaatctgacc tgatcttatg gctgaaacag 4680 agcaaagata acggcattga actgttcaaa gctaatagcg acatcaccga tattgatgaa 4740 gcgctcgaaa tcatcaagtc tttcaaaggc tggacgacgt atttcaaagg ttttcatgaa 4800 aaccgtaaga atgtatattc gagcaacgat attccgacct ctattattta tcgtatcgtg 4860 gacgacaacc tgccgaagtt tctggaaaac aaagcgaaat atgaatctct gaaagacaaa 4920 gcaccggaag ctattaacta tgaacagatc aagaaagatc tggcggaaga actgaccttc 4980 gacatcgact ataaaacctc cgaagttaac cagcgtgttt tctcactgga cgaggttttc 5040 gaaatcgcta atttcaacaa ttacctgaat caatctggca tcaccaaatt caacaccatt 5100 attggtggca aatttgttaa cggcgaaaac accaagcgta agggcatcaa cgaatacatt 5160 aacctgtata gccaacaaat caacgacaaa accctgaaaa agtataaaat gtccgttctg 5220 tttaaacaga ttttatcgga caccgaatct aaatccttcg taattgataa actggaagat 5280 gatagcgacg ttgtcaccac gatgcagagc ttttatgagc agattgcggc gttcaaaacc 5340 gtggaagaga aatctattaa agaaactctg tccctgctct ttgacgacct caaagcgcag 5400 aaactagatc tgtctaagat ttactttaaa aacgacaaat ctctgaccga tctcagtcaa 5460 caagttttcg atgactatag cgtgatcggc acggcagttt tggaatacat cacccaacaa 5520 atcgcgccga aaaatctgga caacccgtcc aagaaggaac aggaactgat tgcaaagaaa 5580 acagaaaaag ctaaatacct gagcttagaa actatcaaac tggcacttga ggaatttaat 5640 aaacatcgtg atattgataa acagtgtcgt tttgaggaaa ttctggcgaa ctttgcggca 5700 atcccgatga tcttcgacga aattgctcaa aacaaagaca atctggcgca gatctctatc 5760 aagtaccaga atcagggtaa gaaagatctg cttcaagcat ctgcggagga cgatgtgaaa 5820 gcaattaaag acttattaga tcagacgaat aacttattac acaagctcaa aatcttccac 5880 atcagccaga gcgaggacaa ggcgaacatt ctggataaag atgaacactt ctatctggtg 5940 ttcgaagaat gttacttcga actggcaaac atcgtccctc tctacaataa aatccgcaac 6000 tacatcacgc agaagcctta ctctgacgag aaattcaaac tgaacttcga aaacagcacg 6060 ctggcgaacg gctgggataa gaacaaagag ccggacaaca ccgcaatcct gttcatcaaa 6120 gacgacaaat actatctggg cgtaatgaac aagaagaaca acaagatctt cgacgataaa 6180 gcgatcaaag aaaacaaggg tgaaggctat aagaaaatcg tgtacaagct cctgccgggt 6240 gcgaataaaa tgttaccgaa agtgttcttt tccgcgaaaa gcatcaaatt ctacaacccg 6300 tctgaggata ttctgcgcat ccgcaatcat agcacgcaca ctaaaaacgg tagcccgcag 6360 aaagggtatg aaaaattcga atttaatata gaggactgcc gtaagttcat cgacttctat 6420 aaacagagca tttccaaaca tccggaatgg aaagacttcg gcttccgttt ctctgacact 6480 cagcgctata atagcatcga cgagttctac cgcgaagtgg agaatcaggg ctataaactg 6540 accttcgaga acattagtga gtcgtacatc gactccgttg tgaatcaggg taaactgtac 6600 ctgtttcaga tctataataa agactttagc gcgtacagca aaggccgtcc gaatctgcac 6660 accctttaact ggaaagcatt atttgacgaa cgtaacctgc aagatgtggt gtataaactg 6720 aacggtgagg cggaactttt ctaccgtaaa cagagtatcc cgaagaaaat cacgcatccg 6780 gcaaaagaag ctattgccaa caaaaacaaa gacaacccga agaaagaatc agtattcgaa 6840 tatgacctga tcaaagataa acgtttcacc gaagataagt tctttttcca ctgtccgatt 6900 accatcaact tcaaatctag cggtgcgaac aagttcaacg atgaaattaa cctattactg 6960 aaagagaaag ctaatgacgt acacatctta tctattgatc gcggtgaacg tcatttagca 7020 tactatacac tggtagatgg taaaggtaat attattaaac aggatacttt caatattatc 7080 ggtaatgacc gtatgaaaac caactatcac gataagctgg cggcgatcga aaaagatcgt 7140 gattctgcgc gtaaagatg gaagaaaatt aacaatatca aagaaatgaa agaaggctat 7200 ctgagccaag tggtgcacga gatcgcaaaa ctggtgattg aatataacgc tatcgtggtt 7260 ttcgaagatc tgaactttgg ttttaaacgt ggtcgcttca aagtagaaaa acaggtgtac 7320 caaaaactgg aaaaaatgct gattgaaaaa ctgaactatc tggtttttaa agacaacgaa 7380 tttgacaaaa cgggtggcgt actccgtgcc tatcagctga ccgctccgtt cgaaacgttc 7440 aagaaaatgg gtaaacaaac ggggattatc tattatgtgc cagctggttt cacctccaag 7500 atttgtccag ttacgggctt cgttaaccag ctgtacccga aatacgagag cgttagcaaa 7560 tctcaagaat ttttcagcaa attcgacaag atctgctata atctggataa aggctatttc 7620 gagttcagct tcgattacaa aaacttcggc gataaagcgg ctaaaggtaa gtggactatt 7680 gctagctttg gtagccgtct gattaacttt cgcaactccg acaaaaacca taattgggac 7740 acgcgtgaag tgtatccgac caaagaactg gaaaaattac tgaaagacta ttccatcgaa 7800 tatggtcatg gggaggtgcat taaagcggcg atttgcggtg aatccgataa gaaatttttc 7860 gccaaactga ccagcgtgct taacaccatt ctgcaaatgc gtaattctaa aacgggtacg 7920 gagctggact acctgatttc tccggtagcc gacgttaacg gcaacttctt cgattctcgt 7980 caagcaccga aaaatatgcc acaagacgcg gatgccaacg gtgcatacca tatcggctta 8040 aaaggcttaa tgttattagg ccgtatcaag aataatcagg agggcaagaa attaaatctg 8100 gttatcaaaa acgaagaata cttcgagttc gttcagaatc gtaacaatta atgtatgctt 8160 aagcagctcg gtaccaaaga cgaacaataa gacgctgaaa agcgtctttt ttcgttttgg 8220 tcctgttgcg gcgcgatagt gtgaacatgc tatagacttc tggtgctacc cgactgacaa 8280 ttaatcatcc ggctcgtata atgctagcaa tttctactgt tgtagatcat tccggaacgt 8340 tccagcgctg caatttctac tgttgtagat ctgatttttc acatgttacc tttcaatttc 8400 tactgttgta gatccgaaaa cgtaaagctt cagctgtaat ttctactgtt gtagatatca 8460 tatctggcgt taatggagtt tcgagacgaa caataaggcc tccctaacgg ggggcctttt 8520 ttattgataa caaaagtaac ttcgagcttg tctacctcct agcaccatta ttgcaattaa 8580 taaacaacta acggacaatt ctacctaaca gttttcatat atgacgagca gttaagtgat 8640 gagtaaaggt gaggaattat ttactggtgt tgttccgatc ttagttgaac tggacggcga 8700 tgttaacggt cataaattca gtgttcgtgg tgaaggtgaa ggtgatgcaa ccaacggtaa 8760 gctgaccctg aaattcatct gcactactgg aaaattacca gtaccgtggc ctactctggt 8820 gactaccctg acctatggtg ttcagtgttt ttctcgttac cctgaccaca tgaagcaaca 8880 tgatttcttc aaatctgcaa tgccggaagg ttatgtacag gagcgcacca tttctttcaa 8940 agacgatggc acgtataaaa cccgtgcaga ggttaaattt gaaggtgaca ctctggtgaa 9000 tcgtattgaa ctgaaaggca ttgatttcaa agaggacggc aatattttag gccacaaact 9060 ggaatataac ttcaactccc ataacgttta catcaccgca gacaaacaga agaacggtat 9120 caaagctaac ttcaaaattc gccataacgt tgaagatggt agcgtacagc tggcggatca 9180 ttaccaacag aacactccga ttggagatgc tcctgtttta ctgccggata accactacct 9240 gtccacccag tctaaactgt cgaaggatcc gaacgaaaag cgcgaccaca tggtgttatt 9300 agagttcgtt accgctagtg gtatcacgca cggtatggat gaactctaca aataagacga 9360 acaataaggg gagcgggaaa ccgctcccct ttttattga taacaaaagt aaattgcacg 9420 ctgatagtct cccaattgcg aaggaccaaa acgaaaaaac accctttcgg gtgtcttttc 9480 tggaatttgg taccgagtac taggtatcgt gtaagtagcg aaggcccgta cgcgagataa 9540 actgctaggc aaccgcgact ctacgactgg tgctcgattt aatttcgctg acgtaaagaa 9600 attatcggca gtgcgtcaac tgccgtatct ttatcttaat taggtagttg gacaagccct 9660 tgaaagaaat agcaagagcc tgcctctcta ttgaagtcac ggcgaaagtc gggtagaaat 9720 caaagaaagc agaaattaaa tcggagtaac actaaggtgg gataactccg taactgacta 9780 cgcctttctc tagactttac ttgaccagat acactgtctt tgacacgttg aaggattaga 9840 gcaatcaaat ccaagactgg ctaagcacga agcaactctt gagtgttaaa aagttatctc 9900 ctgtattcgg gaagcgggta ctagaagatt gcagggactc cgacgttaag taaattacaa 9960 agtaataagt atcgttcagg atcacgttac cgcaataaga agcgagaata atataatttc 10020 cgaagtgctt accccagtag tgactattcc tataaccctt ctgagtgtcc ggaggcggaa 10080 atttgccacg aaagagaaag tatttccccg acaataataa aggggcgctc ctcagctttt 10140 ccacttggtt gggtaagcta ggcaactctg aaaggagttt cggcgaattg aagccgacag 10200 ctttgaattg ttttaggggc gttattcgag ggcaatcgga gctaacttca agactacttc 10260 tttgttgaat actaaatagt gcaaaggtcg tgtttcctca aggatactcc gctaacaata 10320 taggattcca atcagattca gcactggcgg tacgggtgtt gcggtgaggc gttcgggttt 10380 acggctcgaa gctagcacgg taggaagcct gacaatcacc aagcaaaagg gccgtcgaag 10440 gcccacaaga tacgaaagct ctcgaagcct tatccttgac cgatccacct atttaggcag 10500 ttacgcacaa aagctaccca ataatccgtg acaggcacaa tatcacggaa caaaaccgaa 10560 aactctcgta cacggttagg ttttcgctag gaagaataaa cctctatctt gattataaga 10620 aggctcccca agcaccccca aaaccgaaat agcggtttgc aataagggac aagttacgag 10680 tgtagacacg cagaattatc cagcctttag tctttaggaa ggcaaagcta ttgtacgcgg 10740 tagccgtcgt agcaatttac caactgtaga attattggac acacgtagga agggcttaca 10800 gttgaagttt aataaggtca cacgcaaaac cgctaaggaa taatcgcacc gttagcgaaa 10860 gaatatttca gagcggttag taaaggttga gtaaagtgag attccaaagt gagcctttat 10920 aaaaagtaaa gagctataat aaaaccgtcg agcagaaaac aatcgcctga aatctcaagc 10980 acgttgccct ttctaacgtc gctaaggttt cgtaaacccg tttgattagg aagaagaata 11040 agtaacccga ttaggtttga gatcgcgggt tatcggtttg gattaaaagt ggataccagc 11100 ggagtcaacg ccgacgcaaa cgtacagtga tccaatcctg ttgcacggtc aagcacaatc 11160 agctcgcaag atcttggaat agtgtgccca acagtttagt tgagggccac gttccgacta 11220 caagttgctt caagagggga atttggattt ggcaatagcc ccccgtttct acctcaagag 11280 gcgacgagta ttaaccgcgc cagctgtcgg cacaagggcc aaagaagatt ccaatttctt 11340 attcccgaat aacctccgaa tccctgcggg aaaatcaccg accgaatagc ctagaagcaa 11400 gggggaacag ataggtataa ttagcttaag agagtaccag ccgtgacaac agcgtagtaa 11460 ccacaaactt acgctggggc ttctttggcg gatttttaca gatactaaca aggtgatttg 11520 aagtacctta gttgaggatt taaacgcgct atccggtaat ctccaaattg ggaaataccg 11580 ttcaaagagg gctagaatta cttaaaagcc ttcacaccgc ctgcgctata cgcgcccact 11640 ctcccgttta tccgtccaag cggaagcagg gcgatcctcc gctaagatat tcttacgtgt 11700 aacgtagcta agtatcccaa atagctggcg tacgcgttga acaccgccta gaggatcgtg 11760 actcgccgga cgagcgtgtt attggggact tacgccagcg tagactacaa cgcgcccaga 11820 ttaaccctgc acgtattgcc ttgaataacg tactaatctc tccggctctc gacaatctat 11880 cgagcgactc gattatcaac gggtgtcttg cagtt 11915 <210> 30 <211> 861 <212> DNA <213> artificial sequence <220> <223> beta-lactamase gene <400> 30 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagacg ctgaggatca gttgggagcc 120 cgtgtgggtt acatcgagct ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtgttgatg ccggacaaga gcaacttggt cgccgtatac actattctca gaatgacttg 300 gttgagtact caccagttac cgaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac acggcagcca acttacttct gacaacgatc 420 ggagggccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg agccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cccgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tctcgtcaac aattaataga ctggatggag gcggataaag ttgcaggccc acttctgcgt 660 tcggcccttc cggctggctg gtttatgct gataaatctg gagcaggcga gcgtggatct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gatagtgca 840 tcactgatta agcattggta a 861

Claims (30)

A method for regulating the microbiome of a host organism in vivo, by delivering a nucleic acid of interest to a targeted recipient bacterial cell of the microbiome, wherein the nucleic acid of interest exerts a desired effect on the targeted recipient bacterial cell. create,
The method comprises the steps of administering a nucleic acid vector containing the target nucleic acid to the organism,
wherein the vector further comprises an origin of conditional replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell, wherein the vector is free of an antibiotic resistance marker;
thereby delivering said nucleic acid of interest to a targeted recipient bacterial cell,
wherein upon delivery to the targeted recipient bacterial cell, the nucleic acid of interest produces the desired effect on the targeted recipient bacterial cell even though the vector does not replicate in the targeted recipient bacterial cell. The method of claim 1 , wherein the nucleic acid of interest is expressed in the targeted recipient bacterium, thereby producing the desired effect. The method according to claim 1 or 2, wherein the regulation of the microbiome is regulation of microbiome function or microbiome composition. 4. The method according to any one of claims 1 to 3, wherein the predetermined effect is selected from the group consisting of killing the recipient bacterial cell, causing the recipient bacterial cell to stop producing the molecule of interest, and causing the recipient bacterial cell to produce the molecule of interest. A control method selected from the group consisting of 5. The method according to any one of claims 1 to 4, wherein the predetermined effect causes a recipient bacterial cell to produce a molecule of interest and the molecule of interest is a host regulatory molecule. 6. The method of claim 5, wherein the host regulatory molecule is selected from the group consisting of non-coding nucleic acids, coding nucleic acids, proteins, lipids, saccharides, LPS, metabolites, and small molecules. 7. The method of claim 5 or 6, wherein the host regulatory molecule is selected from the group consisting of host endogenous molecules, host exogenous molecules naturally expressed by other organisms, and synthetic compounds. 8. The method according to any one of claims 5 to 7, wherein the host regulatory molecule is selected from the group consisting of secreted molecules, intracellular molecules and membrane-displayed molecules. The method according to any one of claims 5 to 8, wherein the molecule of interest is a gene encoding the host regulatory molecule, several genes encoding a protein complex that is a host regulatory molecule, a metabolic pathway leading to the production of the host regulatory molecule A method of regulation, which is encoded by a nucleic acid selected from the group consisting of a gene or group of genes encoding the enzyme(s) of, a coding nucleic acid that is a host regulatory molecule, and a non-coding nucleic acid that is a host regulatory molecule. 5. The method according to any one of claims 1 to 4, wherein the predetermined effect is causing the recipient bacterial cell to stop producing the predetermined molecule, which molecule is a toxin, virulence factor, virulence protein, virulence factor, antibiotic resistance The method of regulation, which is selected from the group consisting of proteins encoded by genes, remodeling genes or proteins encoded by regulatory genes. 11. The method of claim 10, wherein the nucleic acid of interest is a gene or group of genes encoding one or more exogenous enzyme(s) causing genetic modification. The method according to claim 11, wherein the target nucleic acid is a gene encoding a base-editor or a prime-editor. 5. The method according to any one of claims 1 to 4, wherein the predetermined effect is the killing of a recipient bacterial cell, and the nucleic acid of interest is a gene encoding a nuclease. 14. The method of any one of claims 1 to 13, wherein the conditional origin of replication is an origin of replication whose replication is dependent on the presence of a given protein, peptide, nucleic acid, RNA, molecule or any combination thereof. 15. The method of claim 14, wherein the conditional origin of replication is active in the donor bacterial cell because the donor bacterial cell expresses the predetermined protein, peptide, nucleic acid, RNA, molecule or any combination thereof. 16. The method of claim 15, wherein said protein, peptide, RNA, molecule or any combination thereof is expressed in trans in said donor bacterial cell. 16. The method according to claim 14 or 15, wherein the conditional origin of replication is an origin of replication derived from a phage-derived chromosome island (PICI). 18. The method according to claim 17, wherein the conditional origin of replication is active in the donor bacterial cell because the donor bacterial cell expresses a rep protein, in particular a primase-helicase. 19. The method according to claim 17 or 18, wherein the conditional origin of replication is derived from an origin of replication from PICI of Escherichia coli strain CFT073. 20. The method of any one of claims 1 to 19, wherein the vector does not contain any restriction sites of restriction enzymes frequently encoded in the targeted recipient bacterial cell. 21. The method according to any one of claims 1 to 20, for treating a disease in said host subject. 21. The method according to any one of claims 1 to 20, for cosmetic treatment of said host subject. A nucleic acid vector for use in the in vivo delivery of a nucleic acid of interest to a targeted recipient bacterial cell, wherein the nucleic acid of interest produces a desired effect on the targeted recipient bacterial cell;
The vector is
- said nucleic acid of interest, and
- a conditional origin of replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell
including,
The vector is a nucleic acid vector without an antibiotic resistance marker. 24. The nucleic acid vector according to claim 23, wherein the conditional origin of replication is primase ori from PICI of Escherichia coli strain CFT073 or a derivative thereof. 24. The nucleic acid vector of claim 23, wherein said conditional origin of replication comprises or consists of the sequence SEQ ID NO: 6 or SEQ ID NO: 7. A bacterial delivery vehicle for use in the in vivo delivery of a nucleic acid of interest to a targeted recipient bacterial cell, wherein the bacterial delivery vehicle comprises a vector according to any one of claims 23 to 25 . A donor cell line comprising a vector according to any one of claims 23 to 25 or producing a bacterial delivery vehicle according to claim 26, said donor cell line according to any one of claims 23 to 25 A donor cell line stably containing a vector according to and capable of replicating said vector. 28. The method of claim 27, wherein the conditional origin of replication of the vector is an origin of replication whose replication is dependent on the presence of a given protein, peptide, nucleic acid, RNA, molecule or any combination thereof, and wherein the donor cell line has said protein, peptide, nucleic acid , RNA, molecule or any combination thereof. 28. The donor cell line of claim 27, wherein the protein, peptide, nucleic acid, RNA, molecule or any combination thereof is expressed in trans. A method for modulating a microbiome from an environment ex vivo, by collecting targeted recipient bacterial cells from the environment and delivering a target nucleic acid to the targeted recipient bacterial cells of the microbiome, the target nucleic acid comprising: produce a certain effect on targeted recipient bacterial cells;
The method is a step of contacting a nucleic acid vector containing the target nucleic acid with the microbiome,
wherein the vector further comprises an origin of conditional replication that is inactive in the targeted recipient bacterial cell but active in the donor bacterial cell, wherein the vector is free of an antibiotic resistance marker;
thereby delivering said nucleic acid of interest to a targeted recipient bacterial cell,
wherein, upon delivery to the targeted recipient bacterial cell, the nucleic acid of interest produces the desired effect on the targeted recipient bacterial cell even though the vector does not replicate in the targeted recipient bacterial cell.