Foot And Mouth Disease Virus Recombinant Vaccines And Uses Thereof

Abstract

The present invention encompasses FMDV vaccines or compositions. The vaccine or composition may be a vaccine or composition containing FMDV antigens. The invention also encompasses recombinant vectors encoding and expressing FMDV antigens, epitopes or immunogens which can be used to protect animals, in particular ovines, bovines, caprines, or porcines, against FMDV.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional application Ser. No. 61/313,164 filed Mar. 12, 2010 and U.S. provisional application Ser. No. 61/366,363 filed Jul. 21, 2010.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions for combating Foot and Mouth Disease Virus (FMDV) infection in animals. The present invention provides pharmaceutical compositions comprising an FMDV antigen, methods of vaccination against FMDV, and kits for use with such methods and compositions.

BACKGROUND OF THE INVENTION

[0003] Foot-and-mouth disease (FMD) is one of the most virulent and contagious diseases affecting farm animals. This disease is endemic in numerous countries in the world, especially in Africa, Asia and South America. In addition, epidemic outbreaks can occur periodically. The presence of this disease in a country may have very severe economic consequences resulting from loss of productivity, loss of weight and milk production in infected herds, and from trade embargoes imposed on these countries. The measures taken against this disease consist of strict application of import restrictions, hygiene controls and quarantine, slaughtering sick animals and vaccination programs using inactivated vaccines, either as a preventive measure at the national or regional level, or periodically when an epidemic outbreak occurs.

[0004] FMD is characterized by its short incubation period, its highly contagious nature, the formation of ulcers in the mouth and on the feet and sometimes, the death of young animals. FMD affects a number of animal species, in particular cattle, pigs, sheep and goats. The agent responsible for this disease is a ribonucleic acid (RNA) virus belonging to the Aphthovirus genus of the Picornaviridae family (Cooper et al., Intervirology, 1978, 10, 165-180). At present, at least seven types of foot-and-mouth disease virus (FMDV) are known: the European types (A, O and C), the African types (SAT1, SAT2 and SAT3) and an Asiatic type (Asia 1). Numerous sub-types have also been distinguished (Kleid et al. Science (1981), 214, 1125-1129).

[0005] FMDV is a naked icosahedral virus of about 25 nm in diameter, containing a single-stranded RNA molecule consisting of about 8500 nucleotides, with a positive polarity. This RNA molecule comprises a single open reading frame (ORF), encoding a single polyprotein containing, inter alia, the capsid precursor also known as protein P1 or P88. The protein P1 is myristylated at its amino-terminal end. During the maturation process, the protein P1 is cleaved by the protease 3C into three proteins known as VP0, VP1 and VP3 (or 1AB, 1D and 1C respectively; Belsham G. J., Progress in Biophysics and Molecular Biology, 1993, 60, 241-261). In the virion, the protein VP0 is then cleaved into two proteins, VP4 and VP2 (or 1A and 1B respectively). The mechanism for the conversion of the proteins VP0 into VP4 and VP2, and for the formation of mature virions is not known. The proteins VP1, VP2 and VP3 have a molecular weight of about 26,000 Da, while the protein VP4 is smaller at about 8,000 Da.

[0006] The simple combination of the capsid proteins forms the protomer or 5S molecule, which is the elementary constituent of the FMDV capsid. This protomer is then complexed into a pentamer to form the 12S molecule. The virion results from the encapsidation of a genomic RNA molecule by assembly of twelve 12S pentamers, thus constituting the 146S particles. The viral capsid may also be formed without the presence of an RNA molecule inside it (hereinafter "empty capsid"). The empty capsid is also designated as particle 70S. The formation of empty capsids may occur naturally during viral replication or may be produced artificially by chemical treatment.

[0007] Many hypotheses, research routes, and proposals have been developed in an attempt to design effective vaccines against FMD. Currently, the only vaccines on the market comprise inactivated virus. Concerns about safety of the FMDV vaccine exist, as outbreaks of FMD in Europe have been associated with shortcomings in vaccine manufacture (King, A. M. Q. et al, (1981) Nature 293: 479-480). The inactivated vaccines do not confer long-term immunity, thus requiring booster injections given every year, or more often in the event of epidemic outbreaks. In addition, there are risks linked to incomplete inactivation and/or to the escape of virus during the production of inactivated vaccines (King, A. M. Q., ibid). A goal in the art has been to construct conformationally correct immunogens lacking the infective FMDV genome to make effective and safe vaccines.

[0008] Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. Thus, a new role for poxviruses became important, that of a genetically engineered vector for the expression of foreign genes (Panicali and Paoletti, 1982; Paoletti et al., 1984). Genes encoding heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990). A highly attenuated strain of vaccines, designated MVA, has also been used as a vector for poxvirus-based vaccines. Use of MVA is described in U.S. Pat. No. 5,185,146.

[0009] Additional vaccine vector systems involve the use of avipox viruses, which are naturally host-restricted poxviruses. Both fowlpoxvirus (FPV; Taylor et al. 1988a, b) and canarypoxvirus (CPV; Taylor et al., 1991 & 1992) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry that has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982) and there are no reports in the literature of avipox virus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier against transmission of the virus to other species and makes the use of avipox virus based vaccine vectors in veterinary and human applications an attractive proposition.

[0010] Other attenuated poxvirus vectors have been prepared by genetic modifications of wild type strains of virus. The NYVAC vector, derived by deletion of specific virulence and host-range genes from the Copenhagen strain of vaccinia (Tartaglia et al., 1992) has proven useful as a recombinant vector in eliciting a protective immune response against an expressed foreign antigen. Another engineered poxvirus vector is ALVAC, derived from canarypox virus (see U.S. Pat. No. 5,756,103). ALVAC does not productively replicate in non-avian hosts, a characteristic thought to improve its safety profile (Taylor et al., 1991 & 1992). ALVAC was deposited under the terms of the Budapest Treaty with the American Type Culture Collection under accession number VR-2547. Yet another engineered poxvirus vector is TROVAC, derived from fowlpox virus (see U.S. Pat. No. 5,766,599).

[0011] Recombinant poxviruses can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587; 5,174,993; 5,494,807; and 5,505,941, the disclosures of which are incorporated herein by reference. It can thus be appreciated that provision of an FMDV recombinant poxvirus, and of compositions and products therefrom, particularly ALVAC or TROVAC-based FMDV recombinants and compositions and products therefrom, especially such recombinants containing the P1 genes and/or 3C protease gene of FMDV, and compositions and products therefrom, would be a highly desirable advance over the current state of technology.

[0012] Recently, plants have been investigated as a source for the production of therapeutic agents such as vaccines, antibodies, and biopharmaceuticals. However, the production of vaccines, antibodies, proteins, and biopharmaceuticals from plants is far from a remedial process, and there are numerous obstacles that are commonly associated with such vaccine production. Limitations to successfully producing plant vaccines include low yield of the bioproduct or expressed antigen (Chargelegue et al., Trends in Plant Science 2001, 6, 495-496), protein instability, inconsistencies in product quality (Schillberg et al., Vaccine 2005, 23, 1764-1769), and insufficient capacity to produce viral-like products of expected size and immunogenicity (Arntzen et al., Vaccine 2005, 23, 1753-1756). In order to address these problems, codon optimization, careful approaches to harvesting and purifying plant products, use of plant parts such as chloroplasts to increase uptake of the material, and improved subcellular targeting are all being considered as potential strategies (Koprowski, Vaccine 2005, 23, 1757-1763).

[0013] Considering the susceptibility of animals (including humans, albeit rarely), to FMDV, a method of preventing FMDV infection and protecting animals is essential. Accordingly, there is a need for an effective vaccine against FMDV.

SUMMARY OF THE INVENTION

[0014] Compositions comprising an antigenic FMDV polypeptide and fragments and variants thereof are provided. The FMDV antigens and fragments and variants thereof possess immunogenic and protective properties. The FMDV antigens may be produced in a plant or algae.

[0015] The antigenic polypeptides and fragments and variants thereof can be formulated into vaccines and/or pharmaceutical compositions. Such vaccines can be used to vaccinate an animal and provide protection against at least one FMDV strain.

[0016] Methods of the invention include methods for making the antigenic polypeptides in duckweed plant. Methods also include methods of use including administering to an animal an effective amount of an antigenic polypeptide or fragment or variant thereof to produce a protective immunogenic response. After production in duckweed the antigenic polypeptide can be partially or substantially purified for use as a vaccine.

[0017] Kits comprising at least one antigenic polypeptide or fragment or variant thereof and instructions for use are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:

[0019] FIG. 1 depicts a table summarizing the DNA and Protein sequences, presented as a listing in the appendix of the instant application;

[0020] FIG. 2 represents the native and optimized FMDV sequence that was expressed in duckweed. The expressed polypeptide is cleaved into its individual proteins by the 3C protease that folds after translation, self-cleaves to release itself from the polypeptide, and finally perform internal cleavages within the P1 polypeptide.

[0021] FIG. 3 depicts the identity and placement of the duckweed-optimized FMDV antigens for the 4 "MerE" duckweed expression constructs;

[0022] FIG. 4 is a sequence alignment of FMDV optimized for expression in duckweed (SEQ ID NO:2) and FMDV optimized for expression in mammalian cells (SEQ ID NO:1). The sequence identity is indicated;

[0023] FIG. 5 depicts the pHM-1119-1 plasmid, which contains nucleic acid sequence encoding the mammalian optimized FMDV P1-3C (SEQ ID NO:1);

[0024] FIG. 6 depicts the pMerE01 plasmid, Strong Promoter+P1+2A/2B1 (A24)+3C (A12);

[0025] FIG. 7 depicts the pMerE02, Strong Promoter+P1;

[0026] FIG. 8 depicts the pMerE03 plasmid, Strong Promoter+P1+2A/2B1 (A24)+Weak Promoter 3C (A12);

[0027] FIG. 9 depicts the pMerE04 plasmid, Strong Promoter+P1+2A/2B1 (A24)+Weak Promoter 3C (A12) with optimized 5'UTR;

[0028] FIG. 10 depicts a representative RNA dot blot used to screen recombinant duckweed cell lines for expression of genes encoding FMDV antigens;

[0029] FIG. 11 depicts a representative quantitative PCR results for duckweed cell lines expressing FMDV constructs;

[0030] FIG. 12 depicts the Western blot for duckweed cell lines harboring and expressing MerE01 and MerE02;

[0031] FIG. 13 depicts electron micrographs of inventive FMDV virus like particles (VLP);

[0032] FIG. 14 depicts electron micrograph of clusters of FMDV VLP;

[0033] FIG. 15 depicts WB Analysis of MerE01 and MerE03 Crude Extract (5 ug TSP/lane) using Guinea Pig Serum. For MerE01, VP1 (VP3) band(s) observed, suggesting expression of both P1 and 3C and further processing of P1. For MerE03, neither P1 nor VP1 (VP3) were observed, suggesting expression of 3C and degradation of P1;

[0034] FIG. 16 presents an FMD viral particle schematic diagram;

[0035] FIG. 17 presents OD of decreasing concentrations of various extracts from duckweed expressing FMDV antigens as measured by ELISA;

[0036] FIG. 18 presents MerF01 plasmid map with feature summary table;

[0037] FIG. 19 presents MerF02 plasmid map with feature summary table;

[0038] FIG. 20 presents MerF03 plasmid map with feature summary table;

[0039] FIG. 21 presents MerF04 plasmid map with feature summary table;

[0040] FIG. 22 presents MerF05 plasmid map with feature summary table;

[0041] FIG. 23 presents MerF06 plasmid map with feature summary table.

DETAILED DESCRIPTION

[0042] Compositions comprising an FMDV polypeptide, antigen and fragments and variants thereof that elicit an immunogenic response in an animal are provided. The antigenic polypeptides or fragments or variants thereof are produced in a plant or algae. The antigenic polypeptides or fragments or variants may be formulated into vaccines or pharmaceutical compositions and used to elicit or stimulate a protective response in an animal. In one embodiment the polypeptide antigen is an FMDV P1 or 3C polypeptide or active fragment or variant thereof.

[0043] It is recognized that the antigenic polypeptides of the invention may be full length polypeptides or active fragments or variants thereof. By "active fragments" or "active variants" is intended that the fragments or variants retain the antigenic nature of the polypeptide. Thus, the present invention encompasses any FMDV polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in an animal. The FMDV polypeptide, antigen, epitope or immunogen may be any FMDV polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment or variant thereof, that elicits, induces or stimulates a response in an animal, such as an ovine, bovine, caprine or porcine.

[0044] Particular FMDV antigenic polypeptides include P1 and 3C. FMDV is a non-enveloped icosahedral virus of about 25 nm in diameter, containing a single-stranded RNA molecule consisting of about 8500 nucleotides, with a positive polarity. This RNA molecule comprises a single open reading frame (ORF), encoding a single polyprotein containing, inter alia, the capsid precursor also known as protein P1 or P88. The protein P1 is myristylated at its amino-terminal end. During the maturation process, the protein P1 is cleaved by the protease 3C into three proteins known as VP0, VP1 and VP3 (or 1AB, 1D and 1C respectively; Belsham G. J., Progress in Biophysics and Molecular Biology, 1993, 60, 241-261). In the virion, the protein VP0 is then cleaved into two proteins, VP4 and VP2 (or 1A and 1B respectively). The mechanism for the conversion of the proteins VP0 into VP4 and VP2, and for the formation of mature virions is not known. The proteins VP1, VP2 and VP3 have a molecular weight of about 26,000 Da, while the protein VP4 is smaller at about 8,000 Da. FMDV sequences are also described in U.S. Pat. No. 7,527,960 and U.S. Pat. No. 7,531,182, which documents are herein incorporated in their entirety.

[0045] The, simple combination of the capsid proteins forms the protomer or 5S molecule, which is the elementary constituent of the FMDV capsid. This protomer is then complexed into a pentamer to form the 12S molecule. The virion results from the encapsidation of a genomic RNA molecule by assembly of twelve 12S pentamers, thus constituting the 146S particles. The viral capsid may also be formed without the presence of an RNA molecule inside it (hereinafter "empty capsid"). The empty capsid is also designated as particle 70S. The formation of empty capsids may occur naturally during viral replication or may be produced artificially by chemical treatment.

[0046] The present invention relates to bovine, ovine, caprine, or porcine vaccines or compositions which may comprise an effective amount of a recombinant FMDV antigen and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.

[0047] In some embodiments, the vaccines further comprise adjuvants, such as the oil-in-water (O/W) emulsions described in U.S. Pat. No. 7,371,395.

[0048] In still other embodiments, the adjuvants include EMULSIGEN, Aluminum Hydroxide, Saponin, and CpG, or combinations thereof.

[0049] In some embodiments, the response in the animal is a protective immune response.

[0050] By "animal" it is intended mammals, birds, and the like. Animal or host includes mammals and human. The animal may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cattle), porcine (e.g., pig), caprine (e.g., goat), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), and fish. The term "animal" also includes an individual animal in all stages of development, including embryonic and fetal stages.

[0051] The term "plants" as used herein includes both dicotyledonous (dicot) plants and monocotyledonous (monocot) plant. Dicot plants include, but are not limited to, legumes such as pea, alfalfa and soybean, carrot, celery, tomato, potato, tobacco, pepper, oilseed rape, beet, cabbage, cauliflower, broccoli, lettuce, peanut, and the like. Monocot plants include, but are not limited to, cereals such as wheat, barley, sorghum and millet, rye, triticale, maize, rice or oats, sugarcane, members of the microalgae family, grasses, and the like. The term "plant" also include non-flowering plants including, but not limited to, ferns, horsetails, club mosses, mosses, liverworts, hornworts, algae, for example, red, brown, and green algae, gametophytes, and the like.

[0052] The term "algae" and "alga" as used herein includes any strain of algae capable of producing a polypeptide or fragment or variant thereof. The algae may be microalgae. The microalgae may be Thraustochytriaceae, for example, Schizochytrium, Thraustochytrium, Labyrinthuloides, and Japonochytrium.

[0053] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise.

[0054] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0055] The antigenic polypeptides of the invention are capable of protecting against FMDV. That is, they are capable of stimulating an immune response in an animal. By "antigen" or "immunogen" means a substance that induces a specific immune response in a host animal. The antigen may comprise a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. Alternately, the immunogen or antigen may comprise a toxin or antitoxin.

[0056] The term "immunogenic protein, polypeptide, or peptide" as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises or consists essentially of or consists of at least one epitope or antigenic determinant. An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By "immunogenic fragment" is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al., 1984; Geysen et al., 1986. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methods especially applicable to the proteins of T. parva are fully described in PCT/US2004/022605 incorporated herein by reference in its entirety.

[0057] As discussed the invention encompasses active fragments and variants of the antigenic polypeptide. Thus, the term "immunogenic protein, polypeptide, or peptide" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic--aspartate and glutamate; (2) basic--lysine, arginine, histidine; (3) non-polar--alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar--glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

[0058] The term "epitope" refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site". Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0059] An "immunological response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

[0060] Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al., 1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

[0061] Accordingly, a minimum structure of a polynucleotide expressing an epitope is that it comprises or consists essentially of or consists of nucleotides encoding an epitope or antigenic determinant of an FMDV polypeptide. A polynucleotide encoding a fragment of an FMDV polypeptide may comprise or consist essentially of or consist of a minimum of 15 nucleotides, about 30-45 nucleotides, about 45-75, or at least 57, 87 or 150 consecutive or contiguous nucleotides of the sequence encoding the polypeptide. Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer et al., 1998), Pepscan (Geysen et al., 1984; Geysen et al., 1985; Van der Zee R. et al., 1989; Geysen, 1990; Multipin.RTM. Peptide Synthesis Kits de Chiron) and algorithms (De Groot et al., 1999; PCT/US2004/022605) can be used in the practice of the invention.

[0062] The term "nucleic acid" and "polynucleotide" refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.

[0063] The term "gene" is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

[0064] The invention further comprises a complementary strand to a polynucleotide encoding an FMDV antigen, epitope or immunogen. The complementary strand can be polymeric and of any length, and can contain deoxyribonucleotides, ribonucleotides, and analogs in any combination.

[0065] The terms "protein", "peptide", "polypeptide" and "polypeptide fragment" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0066] An "isolated" biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.

[0067] The term "purified" as used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. That is the polypeptide is separated from cellular components. By "substantially purified" it is intended that such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed. Likewise, the polypeptide may be partially purified. By "partially purified" is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art.

[0068] As noted above, the antigenic polypeptides or fragments or variants thereof are FMDV antigenic polypeptides that are produced in duckweed. Fragments and variants of the disclosed polynucleotides and polypeptides encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the polynucleotide or a portion of the antigenic amino acid sequence encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence have immunogenic activity as noted elsewhere herein. Fragments of the polypeptide sequence retain the ability to induce a protective immune response in an animal.

[0069] "Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they the ability to elicit an immune response.

[0070] In one aspect, the present invention provides FMDV polypeptides from ovine, bovine, caprine, or porcine. In another aspect, the present invention provides a polypeptide having a sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29, and variant or fragment thereof.

[0071] Moreover, homologs of FMDV polypeptides from ovine, bovine, caprine, or porcine are intended to be within the scope of the present invention. As used herein, the term "homologs" includes orthologs, analogs and paralogs. The term "analogs" refers to two polynucleotides or polypeptides that have the same or similar function, but that have evolved separately in unrelated organisms. The term "orthologs" refers to two polynucleotides or polypeptides from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode polypeptides having the same or similar functions. The term "paralogs" refers to two polynucleotides or polypeptides that are related by duplication within a genome. Paralogs usually have different functions, but these functions may be related. Analogs, orthologs, and paralogs of a wild-type FMDV polypeptide can differ from the wild-type FMDV polypeptide by post-translational modifications, by amino acid sequence differences, or by both. In particular, homologs of the invention will generally exhibit at least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequence identity, with all or part of the wild-type FMDV or polynucleotide sequences, and will exhibit a similar function. Variants include allelic variants. The term "allelic variant" refers to a polynucleotide or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a protein and that exist within a natural population (e.g., a virus species or variety). Such natural allelic variations can typically result in 1-5% variance in a polynucleotide or a polypeptide. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different species, which can be readily carried out by using hybridization probes to identify the same gene genetic locus in those species. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity of gene of interest, are intended to be within the scope of the invention.

[0072] As used herein, the term "derivative" or "variant" refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that (1) the corresponding polypeptide has substantially equivalent function when compared to the wild type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide. These variants or derivatives include polypeptides having minor modifications of the FMDV polypeptide primary amino acid sequences that may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. The term "variant" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.

[0073] The term "conservative variation" denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, as described above.

[0074] The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code, e.g., optimized codon usage for a specific host. As used herein, "optimized" refers to a polynucleotide that is genetically engineered to increase its expression in a given species. To provide optimized polynucleotides coding for FMDV polypeptides, the DNA sequence of the FMDV protein gene can be modified to 1) comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T or G+C content in nucleotide base composition to that substantially found in said species; 3) form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of FMDV protein in said species can be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species. The term "frequency of preferred codon usage" refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the FMDV polypeptide encoded by the nucleotide sequence is functionally unchanged.

[0075] The sequence identity between two amino acid sequences may be established by the NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix, using the standard parameters (see, e.g., the BLAST or BLASTX algorithm available on the "National Center for Biotechnology Information" (NCBI, Bethesda, Md., USA) server, as well as in Altschul et al.; and thus, this document speaks of using the algorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term "blasts").

[0076] The "identity" with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and Lipman), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics.TM. Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.

[0077] The sequence identity or sequence similarity of two amino acid sequences, or the sequence identity between two nucleotide sequences can be determined using Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, Calif.).

[0078] The following documents provide algorithms for comparing the relative identity or homology of sequences, and additionally or alternatively with respect to the foregoing, the teachings in these references can be used for determining percent homology or identity: Needleman S B and Wunsch C D; Smith T F and Waterman M S; Smith T F, Waterman M S and Sadler J R; Feng D F and Dolittle R F; Higgins D G and Sharp P M; Thompson J D, Higgins D G and Gibson T J; and, Devereux J, Haeberlie P and Smithies O. And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.

[0079] Hybridization reactions can be performed under conditions of different "stringency." Conditions that increase stringency of a hybridization reaction are well known. See for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989).

[0080] The invention further encompasses the FMDV polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally to an enhancer.

[0081] A "vector" refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy, and may optionally be in the form of an expression cassette. As used herein, a vector needs not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0082] The term "recombinant" means a polynucleotide semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.

[0083] "Heterologous" means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

[0084] The present invention relates to ovine, bovine, caprine and porcine vaccines or pharmaceutical or immunological compositions which may comprise an effective amount of a recombinant FMDV antigens and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.

[0085] The subject matter described herein is directed in part, to compositions and methods related to the FMDV antigen prepared in a plant or alga expression system that was highly immunogenic and protected animals against challenge from homologous and heterologous FMDV strains.

Compositions

[0086] The present invention relates to an FMDV vaccine or composition which may comprise an effective amount of a recombinant FMDV antigen and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. In one embodiment, the recombinant FMDV antigen is expressed in a plant or alga.

[0087] In an embodiment, the subject matter disclosed herein is directed to a composition comprising an FMDV antigen produced by a duckweed expression system and plant material from duckweed, including the genus Lemna, and a pharmaceutical or veterinarily acceptable carrier, excipient or vehicle. In another embodiment, the subject matter disclosed herein is directed to an optionally aglycosylated protein produced by a duckweed expression system comprising an FMDV antigen.

[0088] In one embodiment, the recombinant FMDV antigen is expressed in algae. In yet another embodiment, the algae are selected from Schizochytrium. In one embodiment, the recombinant FMDV antigen may be expressed in a Schizochytrium protein expression system, as described, for example, in U.S. Pat. No. 7,001,772, US 2008/0022422, U.S. provisional App. No. 61/160,618, and US provisional applications concurrently filed on Dec. 28, 2009 by Martek BioScience Corp. (MD, USA).

[0089] In an embodiment, the subject matter disclosed herein is directed to a protein produced by a plant or alga expression system comprising an FMDV antigen and material from the plant or alga.

[0090] In an embodiment, the subject matter disclosed herein is directed to a vaccine or composition comprising an FMDV antigen produced by a duckweed expression system.

[0091] In an embodiment, the subject matter disclosed herein is directed to a vaccine or composition comprising an FMDV antigen produced by a duckweed expression system and plant material from duckweed.

[0092] In an embodiment, the subject matter disclosed herein is directed to a stably transformed plant or plant culture that expresses an FMDV antigen wherein the plant or plant culture is selected from duckweed.

[0093] The present invention encompasses any FMDV polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in an animal, such as an ovine, bovine, caprine or porcine. The FMDV polypeptide, antigen, epitope or immunogen may be any FMDV polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment thereof, that elicits, induces or stimulates a response in an animal, such as an ovine, bovine, caprine or porcine.

[0094] In an embodiment wherein the FMDV immunological composition or vaccine is a recombinant immunological composition or vaccine, the composition or vaccine comprising a recombinant vector and a pharmaceutical or veterinary acceptable excipient, carrier or vehicle; the recombinant vector is plant expression vector which may comprise a polynucleotide encoding a polypeptide, antigen, epitope or immunogen. The FMDV polypeptide, antigen, epitope or immunogen, may be VP1, VP2, VP3, VP4, VP5, NS1, VP7, NS2, VP6, NS3, NS3a, P1, VP0, 3C, or any fragment thereof.

[0095] In another embodiment, the FMDV antigen is P1, VP0, VP3, VP1, VP2, VP4, 2A, 2B, or 3C.

[0096] In an embodiment wherein the FMDV immunological composition or vaccine is a recombinant immunological composition or vaccine, the composition or vaccine comprising a recombinant vector and a pharmaceutical or veterinary acceptable excipient, carrier or vehicle; the recombinant vector is plant expression vector which may comprise a polynucleotide encoding an FMDV polypeptide, antigen, epitope or immunogen. The FMDV polypeptide, antigen, epitope or immunogen, may be an FMDV polypeptide VP1, VP2, VP3, VP4, 2A, 2B or 3C. In one embodiment, the nucleic acid molecule encoding one or more foot-and-mouth disease virus (FMDV) antigen(s) is a cDNA encoding FMDV P1 region and a cDNA encoding FMDV 3C protease of FMDV.

[0097] In one embodiment, the FMDV antigen may be a P1-3C polypeptide. In another embodiment, the FMDV antigen may be P1 alone, or P1-2A/2B1. In yet another embodiment, the FMDV antigen may be VP0-VP3. In another embodiment, the FMDV antigen may be VP4-VP2. In still another embodiment, the FMDV antigen may be 3C, or may be 3C with a 5'UTR optimized for expression in duckweed. In one embodiment, both P1-2A/2B1 and 3C polypeptides may be expressed in duckweed using a single construct and the expression may be regulated by one or more than one promoter sequences.

[0098] In another embodiment, the FMDV antigen may be FMDV O1 Manisa, O1 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia.

[0099] The present invention relates to an FMDV vaccine which may comprise an effective amount of a recombinant FMDV antigen and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. In one embodiment, the FMDV antigen may be FMDV P1, VP0, VP3, VP1, VP2, VP4, or 3C.

[0100] In another embodiment, the recombinant FMDV antigen is expressed in a plant or alga. In yet another embodiment, the plant is a duckweed plant, including a Lemna plant. In yet another embodiment, the plant is Lemna minor. In one embodiment, the recombinant FMDV antigen may be expressed in a proprietary Lemna minor protein expression system, advantageously Biolex's LEX System.sup.SM. In another embodiment, the algae are selected from Schizochytrium. In one embodiment, the recombinant FMDV antigen may be expressed in a Schizochytrium protein expression system, as described, for example, in U.S. Pat. No. 7,001,772, US 2008/0022422, US 2010/0233760 A1 by Martek BioScience Corp. (MD, USA).

[0101] In another embodiment, pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle may be a water-in-oil emulsion. In yet another embodiment, the water-in-oil emulsion may be an oil-in-water emulsion.

[0102] The invention further encompasses the FMDV polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally to an enhancer.

[0103] In one aspect, the present invention provides FMDV polypeptides, particularly ovine, bovine, caprine or porcine polypeptides having a sequence as set forth in SEQ ID NO:3 and variants or fragments thereof.

[0104] In another aspect, the present invention provides a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to an antigenic polypeptide of the invention, particularly to the polypeptides having a sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29.

[0105] In yet another aspect, the present invention provides fragments and variants of the FMDV polypeptides identified above (SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29) which may readily be prepared by one of skill in the art using well-known molecular biology techniques.

[0106] Variants are homologous polypeptides having an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29.

[0107] An immunogenic fragment of an FMDV polypeptide includes at least 8, 10, 15, or 20 consecutive amino acids, at least 21 amino acids, at least 23 amino acids, at least 25 amino acids, or at least 30 amino acids of an FMDV polypeptide having a sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29, or variants thereof. In another embodiment, a fragment of an FMDV polypeptide includes a specific antigenic epitope found on a full-length FMDV polypeptide.

[0108] In another aspect, the present invention provides a polynucleotide encoding an FMDV polypeptide, such as a polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29. In yet another aspect, the present invention provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29, or a conservative variant, an allelic variant, a homolog or an immunogenic fragment comprising at least eight or at least ten consecutive amino acids of one of these polypeptides, or a combination of these polypeptides.

[0109] In another aspect, the present invention provides a polynucleotide having a nucleotide sequence as set forth in SEQ ID NOs:1, 2, 4, 8, 9, 11, 13, 14, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30-35, or a variant thereof. In yet another aspect, the present invention provides a polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, 96%, 97%, 98% or 99% sequence identity to one of a polynucleotide having a sequence as set forth in SEQ ID NOs:1, 2, 4, 8, 9, 11, 13, 14, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30-35, or a variant thereof.

[0110] The polynucleotides of the invention may comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, 5'UTR, 3'UTR, transcription terminators, polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, homologous recombination, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.

[0111] Elements for the expression of an FMDV polypeptide, antigen, epitope or immunogen are advantageously present in an inventive vector. In minimum manner, this comprises, consists essentially of, or consists of an initiation codon (ATG), a stop codon and a promoter, and optionally also a polyadenylation sequence for certain vectors such as plasmid and certain viral vectors, e.g., viral vectors other than poxviruses. When the polynucleotide encodes a polyprotein fragment, e.g. an FMDV peptide, advantageously, in the vector, an ATG is placed at 5' of the reading frame and a stop codon is placed at 3'. Other elements for controlling expression may be present, such as enhancer sequences, stabilizing sequences, such as intron and signal sequences permitting the secretion of the protein.

[0112] The present invention also relates to preparations comprising vectors, such as expression vectors, e.g., therapeutic compositions. The preparations can comprise one or more vectors, e.g., expression vectors, such as in vivo expression vectors, comprising and expressing one or more FMDV polypeptides, antigens, epitopes or immunogens. In one embodiment, the vector contains and expresses a polynucleotide that comprises, consists essentially of, or consists of a polynucleotide coding for (and advantageously expressing) an FMDV antigen, epitope or immunogen, in a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle. Thus, according to an embodiment of the invention, the other vector or vectors in the preparation comprises, consists essentially of or consists of a polynucleotide that encodes, and under appropriate circumstances the vector expresses one or more other proteins of an FMDV polypeptide, antigen, epitope or immunogen, or a fragment thereof.

[0113] According to another embodiment, the vector or vectors in the preparation comprise, or consist essentially of, or consist of polynucleotide(s) encoding one or more proteins or fragment(s) thereof of an FMDV polypeptide, antigen, epitope or immunogen, the vector or vectors expressing the polynucleotide(s). In another embodiment, the preparation comprises one, two, or more vectors comprising polynucleotides encoding and expressing, advantageously in vivo, an FMDV polypeptide, antigen, fusion protein or an epitope thereof. The invention is also directed at mixtures of vectors that comprise polynucleotides encoding and expressing different FMDV polypeptides, antigens, epitopes or immunogens, e.g., an FMDV polypeptide, antigen, epitope or immunogen from different animal species such as, but not limited to, ovine, bovine, caprine or porcine

[0114] According to a yet further embodiment of the invention, the expression vector is a plasmid vector or a DNA plasmid vector, in particular an in vivo expression vector. In a specific, non-limiting example, the pVR1020 or 1012 plasmid (VICAL Inc.; Luke et al., 1997; Hartikka et al., 1996, see, e.g., U.S. Pat. Nos. 5,846,946 and 6,451,769) can be utilized as a vector for the insertion of a polynucleotide sequence. The pVR1020 plasmid is derived from pVR1012 and contains the human tPA signal sequence. In one embodiment the human tPA signal comprises from amino acid M(1) to amino acid S(23) in Genbank under the accession number HUMTPA14. In another specific, non-limiting example, the plasmid utilized as a vector for the insertion of a polynucleotide sequence can contain the signal peptide sequence of equine IGF1 from amino acid M(24) to amino acid A(48) in Genbank under the accession number U28070. Additional information on DNA plasmids which may be consulted or employed in the practice are found, for example, in U.S. Pat. Nos. 6,852,705; 6,818,628; 6,586,412; 6,576,243; 6,558,674; 6,464,984; 6,451,770; 6,376,473 and 6,221,362.

[0115] The term plasmid covers any DNA transcription unit comprising a polynucleotide according to the invention and the elements necessary for its in vivo expression in a cell or cells of the desired host or target; and, in this regard, it is noted that a supercoiled or non-supercoiled, circular plasmid, as well as a linear form, are intended to be within the scope of the invention.

[0116] Each plasmid comprises or contains or consists essentially of, in addition to the polynucleotide encoding an FMDV antigen, epitope or immunogen, optionally fused with a heterologous peptide sequence, variant, analog or fragment, operably linked to a promoter or under the control of a promoter or dependent upon a promoter. In general, it is advantageous to employ a strong promoter functional in eukaryotic cells. The strong promoter may be, but not limited to, the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or optionally having another origin such as the rat or guinea pig, the Super promoter (Ni, M. et al., Plant J. 7, 661-676, 1995.). The CMV-IE promoter can comprise the actual promoter part, which may or may not be associated with the enhancer part. Reference can be made to EP-A-260 148, EP-A-323 597, U.S. Pat. Nos. 5,168,062, 5,385,839, and 4,968,615, as well as to PCT Application No WO87/03905. The CMV-IE promoter is advantageously a human CMV-IE (Boshart et al., 1985) or murine CMV-IE.

[0117] In more general terms, the promoter has either a viral, a plant, or a cellular origin. A strong viral promoter other than CMV-IE that may be usefully employed in the practice of the invention is the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong cellular promoter that may be usefully employed in the practice of the invention is the promoter of a gene of the cytoskeleton, such as e.g. the desmin promoter (Kwissa et al., 2000), or the actin promoter (Miyazaki et al., 1989).

[0118] Any of constitutive, regulatable, or stimulus-dependent promoters may be used. For example, constitutive promoters may include the mannopine synthase promoter from Agrobacterium tumefaciens. Alternatively, it may be advantageous to use heat shock gene promoters, drought-inducible gene promoters, pathogen-inducible gene promoters, wound-inducible gene promoters, and light/dark-inducible gene promoters. It may be useful to use promoters that are controlled by plant growth regulators, such as abscissic acid, auxins, cytokinins, and gibberellic acid. Promoters may also be chosen that give tissue-specific expression (e.g., root, leaf, and floral-specific promoters).

[0119] The plasmids may comprise other expression control elements. It is particularly advantageous to incorporate stabilizing sequence(s), e.g., intron sequence(s), for example, maize alcohol dehydrogenase intron (Callis et al. Genes & Dev. 1(10):1183-1200, December 1987), the first intron of the hCMV-IE (PCT Application No. WO1989/01036), the intron II of the rabbit .beta.-globin gene (van Ooyen et al., 1979). In another embodiment, the plasmids may comprise 3' UTR. The 3' UTR may be, but not limited to, agrobacterium nopaline synthase (Nos) 3' UTR (Nopaline synthase: transcript mapping and DNA sequence. Depicker, A. et al. J. Mol. Appl. Genet., 1982; Bevan, N A R, 1984, 12(22): 8711-8721).

[0120] As to the polyadenylation signal (polyA) for the plasmids and viral vectors other than poxviruses, use can more be made of the poly(A) signal of the bovine growth hormone (bGH) gene (see U.S. Pat. No. 5,122,458), or the poly(A) signal of the rabbit .beta.-globin gene or the poly(A) signal of the SV40 virus.

[0121] A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.

[0122] In one embodiment, the recombinant FMDV antigen is expressed in a transgenic plant or alga. In another embodiment, the transgenic plant is a Lemna plant. In yet another embodiment, the transgenic plant is Lemna minor (duckweed). In yet another embodiment, the recombinant FMDV antigen may be expressed in the Lemna minor (duckweed) protein expression system, the Biolex's LEX System.sup.SM. Details of the Lemna minor (duckweed) protein expression system may be found, for example, in U.S. Pat. Nos. 6,815,184, 7,022,309, 7,160,717, 7,176,024, 6,040,498, and 7,161,064, the disclosures of which are incorporated by reference in their entireties. In yet another embodiment, the transgenic alga is Schizochytrium. Details of the algal protein expression system may be found, for example, in U.S. Pat. No. 7,001,772, US 2008/0022422, U.S. provisional application 61/160,618, the disclosures of which are incorporated by reference in their entireties. The FMDV antigen in the embodiments may be any polypeptide disclosed herein, or a polypeptide encoded by any polynucleotide disclosed herein.

Methods for Expressing FMDV Polypeptides in Duckweed or Microalga

[0123] Thus, in some embodiments of the invention, antigenic FMDV polypeptides, or fragments or variants thereof, are expressed in duckweed or microalga. These methods comprise the use of expression cassettes that are introduced into a duckweed plant or microalga using any suitable transformation method known in the art. Polynucleotides within these expression cassettes can be modified for enhanced expression of the antigenic FMDV polypeptide, or fragment or variant thereof, in duckweed or microalga, as follows.

[0124] Cassettes for Duckweed or Microalga Expression of Antigenic FMDV Polypeptides

[0125] Transgenic duckweed or microalga expressing an antigenic FMDV polypeptide, or fragment or variant thereof, is obtained by transformation of duckweed or microalga with an expression cassette comprising a polynucleotide encoding the antigenic FMDV polypeptide, or fragment or variant thereof. In this manner, a polynucleotide encoding the antigenic FMDV polypeptide of interest, or fragment or variant thereof, is constructed within an expression cassette and introduced into a duckweed plant or microalga culture by any suitable transformation method known in the art.

[0126] In some embodiments, the duckweed plant or microalga that is transformed with an expression cassette comprising polynucleotide encoding the antigenic FMDV polypeptide of interest, or fragment or variant thereof, has also been transformed with an expression cassette that provides for expression of another heterologous polypeptide of interest, for example, another antigenic FMDV polypeptide, fragment, or variant thereof. The expression cassette providing for expression of another heterologous polypeptide of interest can be provided on the same polynucleotide (for example, on the same transformation vector) for introduction into a duckweed plant or microalga, or on a different polynucleotide (for example, on different transformation vectors) for introduction into the duckweed plant or microalga at the same time or at different times, by the same or by different methods of introduction, for example, by the same or different transformation methods.

[0127] The expression cassettes for use in transformation of duckweed or microalga comprise expression control elements that at least comprise a transcriptional initiation region (e.g., a promoter) operably linked to the polynucleotide of interest, i.e., a polynucleotide encoding an antigenic FMDV polypeptide, fragment, or variant thereof "Operably linked" as used herein in reference to nucleotide sequences refers to multiple nucleotide sequences that are placed in a functional relationship with each other. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. Such an expression cassette is provided with a plurality of restriction sites for insertion of the polynucleotide or polynucleotides of interest (e.g., one polynucleotide of interest, two polynucleotides of interest, etc.) to be under the transcriptional regulation of the promoter and other expression control elements. In particular embodiments of the invention, the polynucleotide to be transferred contains two or more expression cassettes, each of which contains at least one polynucleotide of interest.

[0128] By "expression control element" is intended a regulatory region of DNA, usually comprising a TATA box, capable of directing RNA polymerase II, or in some embodiments, RNA polymerase III, to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. An expression control element may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, which influence (e.g., enhance) the transcription initiation rate. Furthermore, an expression control element may additionally comprise sequences generally positioned downstream or 3' to the TATA box, which influence (e.g., enhance) the transcription initiation rate.

[0129] The transcriptional initiation region (e.g., a promoter) may be native or homologous or foreign or heterologous to the duckweed or microalga host, or could be the natural sequence or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type duckweed or microalga host into which the transcriptional initiation region is introduced. By "functional promoter" is intended the promoter, when operably linked to a sequence encoding an antigenic FMDV polypeptide of interest, or fragment or variant thereof, is capable of driving expression (i.e., transcription and translation) of the encoded polypeptide, fragment, or variant. The promoters can be selected based on the desired outcome. Thus the expression cassettes of the invention can comprise constitutive, inducible, tissue-preferred, or other promoters for expression in duckweed.

[0130] Any suitable promoter known in the art can be employed in the expression cassettes according to the present invention, including bacterial, yeast, fungal, insect, mammalian, and plant promoters. For example, plant promoters, including duckweed or microalga promoters, may be used. Exemplary promoters include, but are not limited to, the Cauliflower Mosaic Virus 35S promoter, the opine synthetase promoters (e.g., nos, mas, ocs, etc.), the ubiquitin promoter, the actin promoter, the ribulose bisphosphate (RubP) carboxylase small subunit promoter, and the alcohol dehydrogenase promoter. The duckweed RubP carboxylase small subunit promoter is known in the art (Silverthorne et al. (1990) Plant Mol. Biol. 15:49). Other promoters from viruses that infect plants or microalgae are also suitable, including, but not limited to, promoters isolated from Dasheen mosaic virus, Chlorella virus (e.g., the Chlorella virus adenine methyltransferase promoter; Mitra et al. (1994) Plant Mol. Biol. 26:85), tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus, tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus, sugarcane baciliform badnavirus and the like.

[0131] Expression control elements, including promoters, can be chosen to give a desired level of regulation. For example, in some instances, it may be advantageous to use a promoter that confers constitutive expression (e.g., the mannopine synthase promoter from Agrobacterium tumefaciens). Alternatively, in other situations, it may be advantageous to use promoters that are activated in response to specific environmental stimuli (e.g., heat shock gene promoters, drought-inducible gene promoters, pathogen-inducible gene promoters, wound-inducible gene promoters, and light/dark-inducible gene promoters) or plant growth regulators (e.g., promoters from genes induced by abscissic acid, auxins, cytokinins, and gibberellic acid). As a further alternative, promoters can be chosen that give tissue-specific expression (e.g., root, leaf, and floral-specific promoters).

[0132] The overall strength of a given promoter can be influenced by the combination and spatial organization of cis-acting nucleotide sequences such as upstream activating sequences. For example, activating nucleotide sequences derived from the Agrobacterium tumefaciens octopine synthase gene can enhance transcription from the Agrobacterium tumefaciens mannopine synthase promoter (see U.S. Pat. No. 5,955,646 to Gelvin et al.). In the present invention, the expression cassette can contain activating nucleotide sequences inserted upstream of the promoter sequence to enhance the expression of the antigenic FMDV polypeptide of interest, or fragment or variant thereof. In one embodiment, the expression cassette includes three upstream activating sequences derived from the Agrobacterium tumefaciens octopine synthase gene operably linked to a promoter derived from an Agrobacterium tumefaciens mannopine synthase gene (see U.S. Pat. No. 5,955,646, herein incorporated by reference).

[0133] The expression cassette thus includes in the 5'-3' direction of transcription, an expression control element comprising a transcriptional and translational initiation region, a polynucleotide of encoding an antigenic FMDV polypeptide of interest (or fragment or variant thereof), and a transcriptional and translational termination region functional in plants. Any suitable termination sequence known in the art may be used in accordance with the present invention. The termination region may be native with the transcriptional initiation region, may be native with the coding sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthetase and nopaline synthetase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141; Proudfoot (1991) Cell 64:671; Sanfacon et al. (1991) Genes Dev. 5:141; Mogen et al. (1990) Plant Cell 2:1261; Munroe et al. (1990) Gene 91:151; Ballas et al. (1989) Nucleic Acids Res. 17:7891; and Joshi et al. (1987) Nucleic Acids Res. 15:9627. Additional exemplary termination sequences are the pea RubP carboxylase small subunit termination sequence and the Cauliflower Mosaic Virus 35S termination sequence.

[0134] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed duckweed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds. Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. See DeBlock et al. (1987) EMBO J. 6:2513; DeBlock et al., (1989) Plant Physiol. 91:691; Fromm et al. (1990) BioTechnology 8:833; Gordon-Kamm et al. (1990) Plant Cell 2:603. For example, resistance to glyphosate or sulfonylurea herbicides has been obtained using genes coding for the mutant target enzymes, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS). Resistance to glufosinate ammonium, boromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterial genes encoding phosphinothricin acetyltransferase, a nitrilase, or a 2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respective herbicides.

[0135] For purposes of the present invention, selectable marker genes include, but are not limited to, genes encoding neomycin phosphotransferase II (Fraley et al. (1986) CRC Critical Reviews in Plant Science 4:1); cyanamide hydratase (Maier-Greiner et al. (1991) Proc. Natl. Acad. Sci. USA 88:4250); aspartate kinase; dihydrodipicolinate synthase (Perl et al. (1993) BioTechnology 11:715); bar gene (Toki et al. (1992) Plant Physiol. 100:1503; Meagher et al. (1996) Crop Sci. 36:1367); tryptophan decarboxylase (Goddijn et al. (1993) Plant Mol. Biol. 22:907); neomycin phosphotransferase (NEO; Southern et al. (1982) J. Mol. Appl. Gen. 1:327); hygromycin phosphotransferase (HPT or HYG; Shimizu et al. (1986) Mol. Cell. Biol. 6:1074); dihydrofolate reductase (DHFR; Kwok et al. (1986) Proc. Natl. Acad. Sci. USA 83:4552); phosphinothricin acetyltransferase (DeBlock et al. (1987) EMBO J. 6:2513); 2,2-dichloropropionic acid dehalogenase (Buchanan-Wollatron et al. (1989) J. Cell. Biochem. 13D:330); acetohydroxyacid synthase (U.S. Pat. No. 4,761,373 to Anderson et al.; Haughn et al. (1988) Mol. Gen. Genet. 221:266); 5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al. (1985) Nature 317:741); haloarylnitrilase (WO 87/04181 to Stalker et al.); acetyl-coenzyme A carboxylase (Parker et al. (1990) Plant Physiol. 92:1220); dihydropteroate synthase (sulI; Guerineau et al. (1990) Plant Mol. Biol. 15:127); and 32 kDa photosystem II polypeptide (psbA; Hirschberg et al. (1983) Science 222:1346 (1983).

[0136] Also included are genes encoding resistance to: gentamycin (e.g., aacC1, Wohlleben et al. (1989) Mol. Gen. Genet. 217:202-208); chloramphenicol (Herrera-Estrella et al. (1983) EMBO J. 2:987); methotrexate (Herrera-Estrella et al. (1983) Nature 303:209; Meijer et al. (1991) Plant Mol. Biol. 16:807); hygromycin (Waldron et al. (1985) Plant Mol. Biol. 5:103; Zhijian et al. (1995) Plant Science 108:219; Meijer et al. (1991) Plant Mol. Bio. 16:807); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210:86); spectinomycin (Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131); bleomycin (Hille et al. (1986) Plant Mol. Biol. 7:171); sulfonamide (Guerineau et al. (1990) Plant Mol. Bio. 15:127); bromoxynil (Stalker et al. (1988) Science 242:419); 2,4-D (Streber et al. (1989) BioTechnology 7:811); phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513); spectinomycin (Bretagne-Sagnard and Chupeau, Transgenic Research 5:131).

[0137] The bar gene confers herbicide resistance to glufosinate-type herbicides, such as phosphinothricin (PPT) or bialaphos, and the like. As noted above, other selectable markers that could be used in the vector constructs include, but are not limited to, the pat gene, also for bialaphos and phosphinothricin resistance, the ALS gene for imidazolinone resistance, the HPH or HYG gene for hygromycin resistance, the EPSP synthase gene for glyphosate resistance, the Hm1 gene for resistance to the Hc-toxin, and other selective agents used routinely and known to one of ordinary skill in the art. See Yarranton (1992) Curr. Opin. Biotech. 3:506; Chistopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314; Yao et al. (1992) Cell 71:63; Reznikoff (1992) Mol. Microbiol. 6:2419; Barkley et al. (1980) The Operon 177-220; Hu et al. (1987) Cell 48:555; Brown et al. (1987) Cell 49:603; Figge et al. (1988) Cell 52:713; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA 86:5400; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549; Deuschle et al. (1990) Science 248:480; Labow et al. (1990) Mol. Cell. Biol. 10:3343; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072; Wyborski et al. (1991) Nuc. Acids Res. 19:4647; Hillenand-Wissman (1989) Topics in Mol. And Struc. Biol. 10:143; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591; Kleinschnidt et al. (1988) Biochemistry 27:1094; Gatz et al. (1992) Plant J. 2:397; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913; Hlavka et al. (1985) Handbook of Experimental Pharmacology 78; and Gill et al. (1988) Nature 334:721. Such disclosures are herein incorporated by reference.

[0138] The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.

[0139] Modification of Nucleotide Sequences for Enhanced Expression in a Plant or Microalga Host

[0140] Where the antigenic FMDV polypeptide or fragment or variant thereof is expressed within duckweed or microalga, the expressed polynucleotide sequence encoding the FMDV polypeptide or fragment or variant thereof can be modified to enhance its expression in duckweed or microalga, respectively. One such modification is the synthesis of the polynucleotide using plant-preferred codons, particularly duckweed-preferred codons, or using microalga-preferred codons, such as Schizochytrium-preferred codons. Methods are available in the art for synthesizing nucleotide sequences with plant-preferred codons. See, e.g., U.S. Pat. Nos. 5,380,831 and 5,436,391; EP 0 359 472; EP 0 385 962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 15:3324; Iannacome et al. (1997) Plant Mol. Biol. 34:485; and Murray et al. (1989) Nucleic Acids. Res. 17:477, herein incorporated by reference. Synthesis can be accomplished using any method known to one of skill in the art. The preferred codons may be determined from the codons of highest frequency in the proteins expressed in duckweed or microalga. For example, the frequency of codon usage for Lemna minor is found in Table 1, the frequency of codon usage for Schizochytrium is found in Table 2.

TABLE-US-00001 TABLE 1 Lemna minor [gbpln]: 4 CDS's (1597 codons) fields: [triplet] [frequency: per thousand] ([number]) UUU 17.5(28) UCU 13.8(22) UAU 8.8(14) UGU 5.0(8) UUC 36.3(58) UCC 17.5(28) UAC 15.7(25) UGC 14.4(23) UUA 5.6(9) UCA 14.4(23) UAA 0.0(0) UGA 1.9(3) UUG 13.8(22) UCG 13.8(22) UAG 0.6(1) UGG 16.3(26) CUU 15.7(25) CCU 11.9(19) CAU 6.9(11) CGU 4.4(7) CUC 25.7(41) CCC 15.7(25) CAC 16.9(27) CGC 18.2(29) CUA 5.0(8) CCA 11.3(18) CAA 10.0(16) CGA 6.3(10) CUG 21.3(34) CCG 14.4(23) CAG 22.5(36) CGG 10.6(17) AUU 18.8(30) ACU 9.4(15) AAU 13.8(22) AGU 10.0(16) AUC 19.4(31) ACC 17.5(28) AAC 21.9(35) AGC 15.0(24) AUA 1.9(3) ACA 5.0(8) AAA 15.7(25) AGA 20.7(33) AUG 20.7(33) ACG 10.0(16) AAG 35.7(57) AGG 17.5(28) GUU 15.0(24) GCU 25.0(40) GAU 20.0(32) GGU 8.1(13) GUC 25.0(40) GCC 22.5(36) GAC 26.3(42) GGC 21.9(35) GUA 6.3(10) GCA 14.4(23) GAA 26.3(42) GGA 16.9(27) GUG 30.7(49) GCG 18.2(29) GAG 40.1(64) GGG 18.2(29)

TABLE-US-00002 TABLE 2 Schizochytrium sp. ATCC_20888 [gbpln]: 3 CDS's (6473 codons) fields: [triplet] [frequency: per thousand] ([number]) UUU 12.2(79) UCU 7.0(45) UAU 1.1(7) UGU 0.8(5) UUC 19.9(129) UCC 23.8(154) UAC 21.5(139) UGC 15.3(99) UUA 0.0(0) UCA 0.5(3) UAA 0.5(3) UGA 0.0(0) UUG 0.6(4) UCG 18.8(122) UAG 0.0(0) UGG 8.3(54) CUU 12.7(82) CCU 11.7(76) CAU 2.3(15) CGU 7.1(46) CUC 61.2(396) CCC 23.8(154) CAC 12.8(83) CGC 42.9(278) CUA 0.0(0) CCA 1.5(10) CAA 2.3(15) CGA 0.3(2) CUG 7.4(48) CCG 16.2(105) CAG 27.7(179) CGG 0.8(5) AUU 13.9(90) ACU 9.1(59) AAU 1.9(12) AGU 1.5(10) AUC 33.5(217) ACC 29.2(189) AAC 32.4(210) AGC 15.6(101) AUA 0.0(0) ACA 1.5(10) AAA 2.2(14) AGA 0.2(1) AUG 27.8(180) ACG 9.6(62) AAG 54.5(353) AGG 0.0(0) GUU 8.3(54) GCU 24.4(158) GAU 13.4(87) GGU 13.0(84) GUC 53.0(343) GCC 86.0(557) GAC 45.0(291) GGC 54.5(353) GUA 0.2(1) GCA 4.0(26) GAA 7.3(47) GGA 3.9(25) GUG 14.4(93) GCG 15.9(103) GAG 62.3(403) GGG 0.5(3)

[0141] For purposes of the present invention, "duckweed-preferred codons" refers to codons that have a frequency of codon usage in duckweed of greater than 17%. "Lemna-preferred codons" as used herein refers to codons that have a frequency of codon usage in the genus Lemna of greater than 17%. "Lemna minor-preferred codons" as used herein refers to codons that have a frequency of codon usage in Lemna minor of greater than 17% where the frequency of codon usage in Lemna minor is obtained from the Codon Usage Database (GenBank Release 160.0, Jun. 15, 2007). "Microalgae-preferred codons" refers to codons that have a frequency of codon usage in microalgae of greater than 17%. "microalgae-preferred codons" as used herein refers to codons that have a frequency of codon usage in the family Thraustochytriaceae of greater than 17%. "Schizochytrium-preferred codons" as used herein refers to codons that have a frequency of codon usage in schizochytrium of greater than 17% where the frequency of codon usage in schizochytrium is obtained from the Codon Usage Database.

[0142] It is further recognized that all or any part of the polynucleotide encoding the antigenic FMDV polypeptide of interest, or fragment or variant thereof, may be optimized or synthetic. In other words, fully optimized or partially optimized sequences may also be used. For example, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons may be duckweed-preferred or microalgae-preferred codons. In one embodiment, between 90 and 96% of the codons are duckweed-preferred or microalgae-preferred codons. The coding sequence of a polynucleotide sequence encoding an antigenic FMDV polypeptide of interest, or fragment or variant thereof, may comprise codons used with a frequency of at least 17% in Lemna gibba or at least 17% in Lemna minor. In another such embodiment, the expression cassette comprises SEQ ID NO:9, which contains Lemna minor-preferred codons encoding the P1 polypeptide set forth in SEQ ID NO:10. In a related embodiment, the FMDV polypeptide is a P1-3C polypeptide, for example, the P1-3C polypeptide as set forth in SEQ ID NO:3, and the expression cassette comprises an optimized coding sequence for this P1-3C polypeptide, where the coding sequence comprises duckweed-preferred codons, for example, Lemna minor-preferred or Lemna gibba-preferred codons. In one such embodiment, the expression cassette comprises SEQ ID NO:2, which contains Lemna minor-preferred codons encoding the FMDV polypeptide as set forth in SEQ ID NO:3.

[0143] Other modifications can also be made to the polynucleotide encoding the antigenic FMDV polypeptide of interest, or fragment or variant thereof, to enhance its expression in duckweed or microalga. These modifications include, but are not limited to, elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for duckweed, as calculated by reference to known genes expressed in this plant. When possible, the polynucleotide encoding the heterologous polypeptide of interest may be modified to avoid predicted hairpin secondary mRNA structures.

[0144] There are known differences between the optimal translation initiation context nucleotide sequences for translation initiation codons in animals, plants and algae. "Translation initiation context nucleotide sequence" as used herein refers to the identity of the three nucleotides directly 5' of the translation initiation codon. "Translation initiation codon" refers to the codon that initiates the translation of the mRNA transcribed from the nucleotide sequence of interest. The composition of these translation initiation context nucleotide sequences can influence the efficiency of translation initiation. See, for example, Lukaszewicz et al. (2000) Plant Science 154:89-98; and Joshi et al. (1997); Plant Mol. Biol. 35:993-1001. In the present invention, the translation initiation context nucleotide sequence for the translation initiation codon of the polynucleotide encoding the antigenic FMDV polypeptide of interest, or fragment or variant thereof, may be modified to enhance expression in duckweed. In one embodiment, the nucleotide sequence is modified such that the three nucleotides directly upstream of the translation initiation codon are "ACC." In a second embodiment, these nucleotides are "ACA."

[0145] Expression of an antigenic FMDV polypeptide in duckweed or alga can also be enhanced by the use of 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include, but are not limited to, picornavirus leaders, e.g., EMCV leader (Encephalomyocarditis 5' noncoding region; Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci USA 86:6126); potyvirus leaders, e.g., TEV leader (Tobacco Etch Virus; Allison et al. (1986) Virology 154:9); human immunoglobulin heavy-chain binding protein (BiP; Macajak and Sarnow (1991) Nature 353:90); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke (1987) Nature 325:622); tobacco mosaic virus leader (TMV; Gallie (1989) Molecular Biology of RNA, 23:56); potato etch virus leader (Tomashevskaya et al. (1993) J. Gen. Virol. 74:2717-2724); Fed-1 5' untranslated region (Dickey (1992) EMBO J. 11:2311-2317); RbcS 5' untranslated region (Silverthorne et al. (1990) J. Plant. Mol. Biol. 15:49-58); and maize chlorotic mottle virus leader (MCMV; Lommel et al. (1991) Virology 81:382). See also, Della-Cioppa et al. (1987) Plant Physiology 84:965. Leader sequence comprising plant intron sequence, including intron sequence from the maize alcohol dehydrogenase 1 (ADH1) gene, the castor bean catalase gene, or the Arabidopsis tryptophan pathway gene PAT1 has also been shown to increase translational efficiency in plants (Callis et al. (1987) Genes Dev. 1:1183-1200; Mascarenhas et al. (1990) Plant Mol. Biol. 15:913-920).

[0146] In some embodiments of the present invention, nucleotide sequence corresponding to nucleotides 1222-1775 of the maize alcohol dehydrogenase 1 gene (SEQ ID NO:4; ADH1; GenBank Accession Number X04049) is inserted upstream of the polynucleotide encoding the antigenic FMDV polypeptide of interest, or fragment or variant thereof, to enhance the efficiency of its translation. In another embodiment, the expression cassette contains the leader from the Lemna gibba ribulose-bis-phosphate carboxylase small subunit 5B gene (RbcS leader; see Buzby et al. (1990) Plant Cell 2:805-814).

[0147] It is recognized that any of the expression-enhancing nucleotide sequence modifications described above can be used in the present invention, including any single modification or any possible combination of modifications. The phrase "modified for enhanced expression" in duckweed, as used herein, refers to a polynucleotide sequence that contains any one or any combination of these modifications.

[0148] Transformed Duckweed Plants and Duckweed Nodule Cultures or Transformed Microalgae

[0149] The present invention provides transformed duckweed plants expressing an antigenic FMDV polypeptide of interest, or fragment or variant thereof. The term "duckweed" refers to members of the family Lemnaceae. This family currently is divided into five genera and 38 species of duckweed as follows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S. punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa. microscopica, Wa. neglecta); genus Wolfiella (Wl. caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda, Wl. rotunda, and Wl. neotropica) and genus Landoltia (L. punctata). Any other genera or species of Lemnaceae, if they exist, are also aspects of the present invention. Lemna species can be classified using the taxonomic scheme described by Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae--A Monograph Study (Geobatanischen Institut ETH, Stiftung Rubel, Zurich).

[0150] As used herein, "plant" includes whole plants, plant organs (e.g., fronds (leaves), stems, roots, etc.), seeds, plant cells, and progeny of same. Parts of transgenic plants are to be understood within the scope of the invention to comprise, e.g., plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, tissues, plant calli, embryos as well as flowers, ovules, stems, fruits, leaves, roots, root tips, nodules, and the like originating in transgenic plants or their progeny previously transformed with a polynucleotide of interest and therefore consisting at least in part of transgenic cells. As used herein, the term "plant cell" includes cells of seeds, embryos, ovules, meristematic regions, callus tissue, leaves, fronds, roots, nodules, shoots, anthers, and pollen.

[0151] As used herein, "duckweed nodule" means duckweed tissue comprising duckweed cells where at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the cells are differentiated cells. As used herein, "differentiated cell," means a cell with at least one phenotypic characteristic (e.g., a distinctive cell morphology or the expression of a marker nucleic acid or protein) that distinguishes it from undifferentiated cells or from cells found in other tissue types. The differentiated cells of the duckweed nodule culture described herein form a tiled smooth surface of interconnected cells fused at their adjacent cell walls, with nodules that have begun to organize into frond primordium scattered throughout the tissue. The surface of the tissue of the nodule culture has epidermal cells connected to each other via plasmadesmata.

[0152] The growth habit of the duckweeds is ideal for culturing methods. The plant rapidly proliferates through vegetative budding of new fronds, in a macroscopic manner analogous to asexual propagation in yeast. This proliferation occurs by vegetative budding from meristematic cells. The meristematic region is small and is found on the ventral surface of the frond. Meristematic cells lie in two pockets, one on each side of the frond midvein. The small midvein region is also the site from which the root originates and the stem arises that connects each frond to its mother frond. The meristematic pocket is protected by a tissue flap. Fronds bud alternately from these pockets. Doubling times vary by species and are as short as 20-24 hours (Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Chang et al. (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Datko and Mudd (1970) Plant Physiol. 65:16; Venkataraman et al. (1970) Z. Pflanzenphysiol. 62: 316). Intensive culture of duckweed results in the highest rates of biomass accumulation per unit time (Landolt and Kandeler (1987) The Family of Lemnaceae--A Monographic Study Vol. 2: Phytochemistry, Physiology, Application, Bibliography (Veroffentlichungen des Geobotanischen Institutes ETH, Stiftung Rubel, Zurich)), with dry weight accumulation ranging from 6-15% of fresh weight (Tillberg et al. (1979) Physiol. Plant. 46:5; Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Stomp, unpublished data). Protein content of a number of duckweed species grown under varying conditions has been reported to range from 15-45% dry weight (Chang et al. (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Chang and Chui (1978) Z. Pflanzenphysiol. 89:91; Porath et al. (1979) Aquatic Botany 7:272; Appenroth et al. (1982) Biochem. Physiol. Pflanz. 177:251). Using these values, the level of protein production per liter of medium in duckweed is on the same order of magnitude as yeast gene expression systems.

[0153] The present invention also provides transformed microalgae plants expressing an FMDV polypeptide of interest, or fragment or variant thereof. The term "microalgae" or "microalga" refers to members of the family Thraustochytriaceae. This family currently is divided into four genera: Schizochytrium, Thraustochytrium, Labyrinthuloides, and Japonochytrium.

[0154] The transformed duckweed plants or microalgae of the invention can be obtained by introducing an expression construct comprising a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof, into the duckweed plant or microalga of interest.

[0155] The term "introducing" in the context of a polynucleotide, for example, an expression construct comprising a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof, is intended to mean presenting to the duckweed plant or microalga the polynucleotide in such a manner that the polynucleotide gains access to the interior of a cell of the duckweed plant or microalga. Where more than one polynucleotide is to be introduced, these polynucleotides can be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and can be located on the same or different transformation vectors. Accordingly, these polynucleotides can be introduced into the duckweed or microalga host cell of interest in a single transformation event, in separate transformation events, or, for example, as part of a breeding protocol. The compositions and methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a duckweed plant or microalga, only that the polynucleotide(s) gains access to the interior of at least one cell of the duckweed plant or microalga. Methods for introducing polynucleotides into plants or algae are known in the art including, but not limited to, transient transformation methods, stable transformation methods, and virus-mediated methods.

[0156] "Transient transformation" in the context of a polynucleotide such as a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof, is intended to mean that a polynucleotide is introduced into the duckweed plant or microalga and does not integrate into the genome of the duckweed plant or microalga.

[0157] By "stably introducing" or "stably introduced" in the context of a polynucleotide (such as a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof) introduced into a duckweed plant or microalga is intended the introduced polynucleotide is stably incorporated into the duckweed or microalga genome, and thus the duckweed plant or microalga is stably transformed with the polynucleotide.

[0158] "Stable transformation" or "stably transformed" is intended to mean that a polynucleotide, for example, a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof, introduced into a duckweed plant or microalga integrates into the genome of the plant or alga and is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. In some embodiments, successive generations include progeny produced vegetatively (i.e., asexual reproduction), for example, with clonal propagation. In other embodiments, successive generations include progeny produced via sexual reproduction.

[0159] An expression construct comprising a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof, can be introduced into a duckweed plant or microalga of interest using any transformation protocol known to those of skill in art. Suitable methods of introducing nucleotide sequences into duckweed plants or plant cells or nodules or microalgae include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840, both of which are herein incorporated by reference), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782 (each of which is herein incorporated by reference); and Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926). The cells that have been transformed may be grown into plants in accordance with conventional ways.

[0160] As noted above, stably transformed duckweed or microalgae can be obtained by any gene transfer method known in the art, such as one of the gene transfer methods disclosed in U.S. Pat. No. 6,040,498 or U.S. Patent Application Publication Nos. 2003/0115640, 2003/0033630 or 2002/0088027. Duckweed plant or nodule cultures or microalga can be efficiently transformed with an expression cassette containing a nucleic acid sequence as described herein by any one of a number of methods including Agrobacterium-mediated gene transfer, ballistic bombardment or electroporation. The Agrobacterium used can be Agrobacterium tumefaciens or Agrobacterium rhizogenes. Stable duckweed or microalga transformants can be isolated by transforming the duckweed or microalga cells with both the nucleic acid sequence of interest and a gene that confers resistance to a selection agent, followed by culturing the transformed cells in a medium containing the selection agent. See, for example, U.S. Pat. No. 6,040,498, the contents of which are herein incorporated by reference in their entirety.

[0161] The stably transformed duckweed plants or microalgae utilized in these methods should exhibit normal morphology and be fertile by sexual reproduction and/or able to reproduce vegetatively (i.e., asexual reproduction), for example, with clonal propagation. Preferably, transformed duckweed plants or microalgae of the present invention contain a single copy of the transferred nucleic acid comprising a polynucleotide encoding an antigenic FMDV polypeptide, or fragment or variant thereof, and the transferred nucleic acid has no notable rearrangements therein. It is recognized that the transformed duckweed plants or microalgae of the invention may contain the transferred nucleic acid present in low copy numbers (i.e., no more than twelve copies, no more than eight copies, no more than five copies, alternatively, no more than three copies, as a further alternative, fewer than three copies of the nucleic acid per transformed cell).

[0162] Transformed plants or microalgae expressing an antigenic FMDV polypeptide, or fragment or variant thereof, can be cultured under suitable conditions for expressing the antigenic FMDV polypeptide, or fragment or variant thereof. The antigenic FMDV polypeptide, or fragment or variant thereof, can then be harvested from the duckweed plant or microalgae, the culture medium, or the duckweed plant or microalgae and the culture medium, and, where desired, purified using any conventional isolation and purification method known in the art, as described elsewhere herein. The antigenic FMDV polypeptide, or fragment or variant thereof, can then be formulated as a vaccine for therapeutic applications, as described elsewhere herein.

Methods of Preparing an FMDV Polypeptide

[0163] As described fully herein, in an embodiment, a method of producing an FMDV polypeptide comprises: (a) culturing within a duckweed or microalgae culture medium a duckweed or microalga culture, wherein the duckweed or microalga culture is stably transformed to express the polypeptide, and wherein the polypeptide is expressed from a nucleotide sequence comprising a coding sequence for said polypeptide; and (b) collecting the antigenic polypeptide from said culture medium. The term collecting includes, but is not limited to, harvesting from the culture medium or purifying.

[0164] After production of the recombinant polypeptide in duckweed or microalgae, any method available in the art may be used for protein purification. The various steps include freeing the protein from the nonprotein or plant or microalga material, followed by the purification of the protein of interest from other proteins. Initial steps in the purification process include centrifugation, filtration or a combination thereof. Proteins secreted within the extracellular space of tissues can be obtained using vacuum or centrifugal extraction. Minimal processing could also involve preparation of crude products. Other methods include maceration and extraction in order to permit the direct use of the extract.

[0165] Such methods to purify the protein of interest can exploit differences in protein size, physicochemical properties, and binding affinity. Such methods include chromatography, including procainamide affinity, size exclusion, high pressure liquid, reversed-phase, and anion-exchange chromatography, affinity tags, filtration, etc. In particular, immobilized Ni-ion affinity chromatography can be used to purify the expressed protein. See, Favacho et al. (2006) Protein expression and purification 46:196-203. See also, Zhou et al. (2007) The Protein J 26:29-37; Wang et al. (2006) Vaccine 15:2176-2185; and WO/2009/076778; all of which are herein incorporated by reference. Protectants may be used in the purification process such as osmotica, antioxidants, phenolic oxidation inhibitors, protease inhibitors, and the like.

Methods of Use

[0166] In an embodiment, the subject matter disclosed herein is directed to a method of vaccinating an ovine, bovine, caprine, or porcine comprising administering to the ovine, bovine, caprine, or porcine an effective amount of a vaccine which may comprise an effective amount of a recombinant FMDV antigen and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.

[0167] In one embodiment of the present invention, the method comprises a single administration of a vaccine composition formulated with an emulsion according to the invention. For example, in one embodiment, the immunological or vaccine composition comprises duckweed-expressed FMDV antigens, including polypeptides and VLPs (virus-like particles). Electron microscopy indicates the duckweed transformed with MerE expression vectors likely produce FMDV VLP, and so immunological or vaccine compositions according to the instant invention encompass those comprising FMDV VLP.

[0168] In an embodiment, the subject matter disclosed herein is directed to a method of vaccinating an ovine, bovine, caprine, or porcine comprising administering to the ovine, bovine, caprine, or porcine an ovine, bovine, caprine, or porcine FMDV antigen produced in a plant or alga, and plant material from the genus Lemna or microalga material from schizochytrium.

[0169] In an embodiment, the subject matter disclosed herein is directed to a method of eliciting an immune response comprising administering to the ovine, bovine, caprine, or porcine a vaccine comprising an ovine, bovine, caprine, or porcine FMDV antigen expressed in a plant or alga, wherein an immune response is elicited.

[0170] In an embodiment, the subject matter disclosed herein is directed to a method of eliciting an immune response comprising administering to the ovine, bovine, caprine, or porcine a vaccine comprising an ovine, bovine, caprine, or porcine FMDV antigen produced in a plant or alga, and plant material from the genus Lemna or microalga material from schizochytrium., wherein an immune response is elicited.

[0171] In an embodiment, the subject matter disclosed herein is directed to a method of preparing a stably transformed duckweed plant or microalga culture comprising, (a) introducing into the plant or microalga a genetic construct comprising an FMDV antigen gene; and (b) cultivating the plant or microalga. Methods for transformation of duckweed or microalga are available in the art.

[0172] In an embodiment, the subject matter disclosed herein is directed to a method of preparing a vaccine or composition comprising isolating an FMDV antigen produced by a duckweed or microalgal expression system and optionally combining with a pharmaceutically or veterinarily acceptable carrier, excipient or vehicle.

[0173] In an embodiment, the subject matter disclosed herein is directed to a method of preparing a vaccine or composition comprising combining an FMDV antigen produced by a Lemna expression system and plant material from the genus Lemna and optionally a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.

[0174] In another embodiment, the subject matter disclosed herein is directed to a method of preparing a vaccine or composition comprising combining an FMDV antigen produced by a Schizochytrium expression system and Schizochytrium material and optionally a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.

[0175] The administering may be subcutaneously or intramuscularly. The administering may be needle free (for example Pigjet or Bioject).

[0176] In one embodiment of the invention, a prime-boost regimen can be employed, which is comprised of at least one primary administration and at least one booster administration using at least one common polypeptide, antigen, epitope or immunogen. Typically the immunological composition or vaccine used in primary administration is different in nature from those used as a booster. However, it is noted that the same composition can be used as the primary administration and the boost. This administration protocol is called "prime-boost".

[0177] A prime-boost according to the present invention can include a recombinant viral vector is used to express an FMDV coding sequence or fragments thereof encoding an antigenic polypeptide or fragment or variant thereof. Specifically, the viral vector can express an FMDV gene or fragment thereof that encodes an antigenic polypeptide. Viral vector contemplated herein includes, but not limited to, poxvirus [e.g., vaccinia virus or attenuated vaccinia virus, avipox virus or attenuated avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC, TROVAC; see e.g., U.S. Pat. No. 5,505,941, U.S. Pat. No. 5,494,8070), raccoonpox virus, swinepox virus, etc.], adenovirus (e.g., human adenovirus, canine adenovirus), herpesvirus (e.g. canine herpesvirus, herpesvirus of turkey, Marek's disease virus, infectious laryngotracheitis virus, feline herpesvirus, laryngotracheitis virus (ILTV), bovine herpesvirus, swine herpesvirus), baculovirus, retrovirus, etc. In another embodiment, the avipox expression vector may be a canarypox vector, such as, ALVAC. In yet another embodiment, the avipox expression vector may be a fowlpox vector, such as, TROVAC. The FMDV antigen of the invention to be expressed is inserted under the control of a specific poxvirus promoter, e.g., the entomopoxvirus Amsacta moorei 42K promoter (Barcena, Lorenzo et al. 2000), the vaccinia promoter 7.5 kDa (Cochran et al., 1985), the vaccinia promoter I3L (Riviere et al., 1992), the vaccinia promoter HA (Shida, 1986), the cowpox promoter ATI (Funahashi et al., 1988), the vaccinia promoter H6 (Taylor et al., 1988b; Guo et al., 1989; Perkus et al., 1989), inter alia.

[0178] In another embodiment, the avipox expression vector may be a canarypox vector, such as, ALVAC. The FMDV antigen, epitope or immunogen may be FMDV P1-3C. The FMDV viral vector may be a canarypox virus such as vCP2186, vCP2181, or vCP2176, or a fowlpox virus such as vFP2215 (see U.S. Pat. No. 7,527,960).

[0179] In another aspect of the prime-boost protocol of the invention, a composition comprising the FMDV antigen of the invention is administered followed by the administration of vaccine or composition comprising a recombinant viral vector that contains and expresses the FMDV antigen in vivo, or an inactivated viral vaccine or composition comprising the FMDV antigen, or a DNA plasmid vaccine or composition that contains or expresses the FMDV antigen. Likewise, a prime-boost protocol may comprise the administration of vaccine or composition comprising a recombinant viral vector that contains and expresses an FMDV antigen in vivo, or an inactivated viral vaccine or composition comprising an FMDV antigen, or a DNA plasmid vaccine or composition that contains or expresses an FMDV antigen, followed by the administration of a composition comprising the FMDV antigen of the invention. It is further noted that both the primary and the secondary administrations may comprise the composition comprising the FMDV antigen of the invention.

[0180] A prime-boost protocol comprises at least one prime-administration and at least one boost administration using at least one common polypeptide and/or variants or fragments thereof. The vaccine used in prime-administration may be different in nature from those used as a later booster vaccine. The prime-administration may comprise one or more administrations. Similarly, the boost administration may comprise one or more administrations.

[0181] The dose volume of compositions for target species that are mammals, e.g., the dose volume of ovine, bovine, caprine or porcine compositions, based on viral vectors, e.g., non-poxvirus-viral-vector-based compositions, is generally between about 0.1 to about 5.0 ml, between about 0.1 to about 3.0 ml, and between about 0.5 ml to about 2.5 ml.

[0182] The efficacy of the vaccines may be tested about 2 to 4 weeks after the last immunization by challenging animals, such as ovine, bovine, caprine or porcines, with a virulent strain of FMDV, advantageously the FMDV O1 Manisa, O1 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran '96, A22 Iraq, SAT2 Saudi Arabia strains.

[0183] Still other strains may include FMDV strains A10-61, A5, A12, A24/Cruzeiro, C3/Indaial, O1, C1-Santa Pau, C1-C5, A22/550/Azerbaijan/65, SAT1-SAT3, A, A/TNC/71/94, A/IND/2/68, A/IND/3/77, A/IND/5/68, A/IND/7/82, A/IND/16/82, A/IND/17177, A/IND/17/82, A/IND/19/76, A/IND/20/82, A/IND/22/82, A/IND/25/81, A/IND/26/82, A/IND/54/79, A/IND/57/79, A/IND/73/79, A/IND/85/79, A/IND/86/79, A/APA/25/84, A/APN/41/84, A/APS/44/05, A/APS/50/05, A/APS/55/05, A/APS/66/05, A/APS/68/05, A/BIM/46/95, A/GUM/33/84, A/ORS/66/84, A/ORS/75/88, A/TNAn/60/947/Asia/1, A/IRN/05, Asia/IRN/05, O/HK/2001, O/UKG/3952/2001, O/UKG/4141/2001, Asia 1/HNK/CHA/05 (GenBank accession number EF149010, herein incorporated by reference), Asia I/XJ (Li, ZhiYong et al. Chin Sci Bull, 2007), HK/70 (Chin Sci Bull, 2006, 51(17): 2072-2078), O/UKG/7039/2001, O/UKG/9161/2001, O/UKG/7299/2001, O/UKG/4014/2001, O/UKG/4998/2001, O/UKG/9443/2001, O/UKG/5470/2001, O/UKG/5681/2001, O/ES/2001, HKN/2002, O5India, O/BKF/2/92, K/37/84/A, KEN/1/76/A, GAM/51/98/A, A10/Holland, O/KEN/1/91, O/IND49/97, O/IND65/98, O/IND64/98, O/IND48/98, O/IND47/98, O/IND82/97, O/IND81/99, O/IND81/98, O/IND79/97, O/IND78/97, O/IND75/97, O/IND74/97, O/IND70/97, O/IND66/98, O/IND63/97, O/IND61/97, O/IND57/98, O/IND56/98, O/IND55/98, O/IND54/98, O/IND469/98, O/IND465/97, O/IND464/97, O/IND424/97, O/IND423/97, O/IND420/97, O/IND414/97, O/IND411/97, O/IND410/97, O/IND409/97, O/IND407/97, O/IND399/97, O/IND39/97, O/IND391/97, O/IND38/97, O/IND384/97, O/IND380/97, O/IND37/97, O/IND352/97, O/IND33/97, O/IND31/97, O/IND296/97, O/IND23/99, O/IND463/97, O/IND461/97, O/IND427/98, O/IND28/97, O/IND287/99, O/IND285/99, O/IND282/99, O/IND281/97, O/IND27/97, O/IND278/97, O/IND256/99, O/IND249/99, O/IND210/99, O/IND208/99, O/IND207/99, O/IND205/99, O/IND185/99, O/IND175/99, O/IND170/97, O/IND164/99, O/IND160/99, O/IND153/99, O/IND148/99, O/IND146/99, O/SKR/2000, A22/India/17/77.

[0184] Further details of these FMDV strains may be found on the European Bioinformatics Information (EMBL-EBI) web pages, and all of the associated nucleotide sequences are herein incorporated by reference. The inventors contemplate that all FMDV strains, both herein listed, and those yet to be identified, could be expressed according to the teachings of the present disclosure to produce, for example, effective vaccine compositions. Both homologous and heterologous strains are used for challenge to test the efficacy of the vaccines. The animal may be challenged intradermally, subcutaneously, spray, intra-nasally, intra-ocularly, intra-tracheally, and/or orally.

[0185] The prime-boost administrations may be advantageously carried out 2 to 6 weeks apart, for example, about 3 weeks apart. According to one embodiment, a semi-annual booster or an annual booster, advantageously using the viral vector-based vaccine, is also envisaged. The animals are advantageously at least 6 to 8 weeks old at the time of the first administration.

[0186] The compositions comprising the recombinant antigenic polypeptides of the invention used in the prime-boost protocols are contained in a pharmaceutically or veterinary acceptable vehicle, diluent or excipient. The protocols of the invention protect the animal from ovine, bovine, caprine or porcine FMDV and/or prevent disease progression in an infected animal.

[0187] The various administrations are preferably carried out 1 to 6 weeks apart, and more particularly about 3 weeks apart. According to a preferred mode, an annual booster, preferably using the viral vector-based immunological composition of vaccine, is also envisaged. The animals are preferably at least one-day-old at the time of the first administration.

[0188] It should be understood by one of skill in the art that the disclosure herein is provided by way of example and the present invention is not limited thereto. From the disclosure herein and the knowledge in the art, the skilled artisan can determine the number of administrations, the administration route, and the doses to be used for each injection protocol, without any undue experimentation.

[0189] The present invention contemplates at least one administration to an animal of an efficient amount of the therapeutic composition made according to the invention. The animal may be male, female, pregnant female and newborn. This administration may be via various routes including, but not limited to, intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal or oral administration. The therapeutic composition according to the invention can also be administered by a needleless apparatus (as, for example with a Pigjet, Dermojet, Biojector, Avijet (Merial, Ga., USA), Vetjet or Vitajet apparatus (Bioject, Oregon, USA)). Another approach to administering plasmid compositions is to use electroporation (see, e.g. Tollefsen et al., 2002; Tollefsen et al., 2003; Babiuk et al., 2002; PCT Application No. WO99/01158). In another embodiment, the therapeutic composition is delivered to the animal by gene gun or gold particle bombardment.

[0190] In one embodiment, the invention provides for the administration of a therapeutically effective amount of a formulation for the delivery and expression of an FMDV antigen or epitope in a target cell. Determination of the therapeutically effective amount is routine experimentation for one of ordinary skill in the art. In one embodiment, the formulation comprises an expression vector comprising a polynucleotide that expresses an FMDV antigen or epitope and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. In another embodiment, the pharmaceutically or veterinarily acceptable carrier, vehicle or excipient facilitates transfection or other means of transfer of polynucleotides to a host animal and/or improves preservation of the vector or protein in a host.

[0191] In one embodiment, the subject matter disclosed herein provides a detection method for differentiation between infected and vaccinated animals (DIVA).

[0192] It is disclosed herein that the use of the vaccine or composition of the present invention allows the detection of FMDV infection in an animal. It is disclosed herein that the use of the vaccine or composition of the present invention allows the detection of the infection in animals by differentiating between infected and vaccinated animals (DIVA). A method is disclosed herein for diagnosing the infection of FMDV in an animal using an FMDV non-structural protein (e.g. a FMDV 3ABC or 3D-specific ELISA).

Article of Manufacture

[0193] In an embodiment, the subject matter disclosed herein is directed to a kit for performing a method of eliciting or inducing an immune response which may comprise any one of the recombinant FMDV immunological compositions or vaccines, or inactivated FMDV immunological compositions or vaccines, recombinant FMDV viral compositions or vaccines, and instructions for performing the method.

[0194] Another embodiment of the invention is a kit for performing a method of inducing an immunological or protective response against FMDV in an animal comprising a composition or vaccine comprising an FMDV antigen of the invention and a recombinant FMDV viral immunological composition or vaccine, and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.

[0195] Another embodiment of the invention is a kit for performing a method of inducing an immunological or protective response against FMDV in an animal comprising a composition or vaccine comprising an FMDV antigen of the invention and an inactivated FMDV immunological composition or vaccine, and instructions for performing the method of delivery in an effective amount for eliciting an immune response in the animal.

[0196] Yet another aspect of the present invention relates to a kit for prime-boost vaccination according to the present invention as described above. The kit may comprise at least two vials: a first vial containing a vaccine or composition for the prime-vaccination according to the present invention, and a second vial containing a vaccine or composition for the boost-vaccination according to the present invention. The kit may advantageously contain additional first or second vials for additional primo-vaccinations or additional boost-vaccinations.

[0197] The following embodiments are encompassed by the invention. In an embodiment, a composition comprising an FMDV antigen or fragment or variant thereof and a pharmaceutical or veterinarily acceptable carrier, excipient, or vehicle is disclosed. In another embodiment, the composition described above wherein the FMDV antigen or fragment or variant thereof comprises an immunogenic fragment comprising at least 15 amino acids of an ovine, bovine, caprine, or porcine FMDV antigen is disclosed. In yet another embodiment, the above compositions wherein the FMDV antigen or fragment or variant thereof is produced in duckweed or microalgae are disclosed. In an embodiment, the above compositions wherein the FMDV antigen or fragment or variant thereof is partially purified are disclosed. In an embodiment, the above compositions wherein the FMDV antigen or fragment or variant thereof is substantially purified are disclosed.

[0198] In an embodiment, the above compositions wherein the FMDV antigen or fragment or variant thereof is an ovine, bovine, caprine, or porcine FMDV polypeptide are disclosed. In an embodiment, the above compositions wherein the FMDV polypeptide is a P1-3C polypeptide, P1 polypeptide, VP0 polypeptide, VP1 polypeptide, VP3 polypeptide, VP2 polypeptide, VP4 polypeptide, 2A polypeptide, 2B1 polypeptide, or 3C polypeptide are disclosed. In an embodiment, the above compositions wherein the FMDV antigen or fragment or variant thereof has at least 80% sequence identity to the sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, 29 are disclosed. In one embodiment, the above compositions wherein the FMDV antigen is encoded by a polynucleotide having at least 70% sequence identity to the sequence as set forth in SEQ ID NOs:1, 2, 9, 11, 13, 14, 15, 16, 18, 19, 21, 22, or 24, 25, 27, 28, 30-35 are disclosed. In an embodiment, the above compositions wherein the pharmaceutical or veterinarily acceptable carrier, excipient, or vehicle is a water-in-oil emulsion or an oil-in-water emulsion are disclosed. In another embodiment, a method of vaccinating an animal susceptible to ovine, bovine, caprine, or porcine FMDV comprising administering the compositions above to the animal is disclosed. In an embodiment, a method of vaccinating an animal susceptible to ovine, bovine, caprine, or porcine FMDV comprising a prime-boost regime is disclosed. In an embodiment, a substantially purified antigenic polypeptide expressed in duckweed or microalga, wherein the polypeptide comprises: an amino acid sequence having at least 80% sequence identity to a polypeptide having the sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, 29 is disclosed. In any embodiment the animal is preferably an ovine, a bovine, a porcine, or a caprine. In one embodiment, a method of diagnosing FMDV infection in an animal is disclosed. In yet another embodiment, a kit for prime-boost vaccination comprising at least two vials, wherein a first vial containing the composition of the present invention, and a second vial containing a composition for the boost-vaccination comprising a composition comprising a recombinant viral vector, or a composition comprising an inactivated viral composition, or a DNA plasmid composition that contains or expresses the FMDV antigen is disclosed.

[0199] The pharmaceutically or veterinarily acceptable carriers or vehicles or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Other pharmaceutically or veterinarily acceptable carrier or vehicle or excipients that can be used for methods of this invention include, but are not limited to, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier or vehicle or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); advantageously, the carrier, vehicle or excipient may facilitate transfection and/or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.

[0200] The cationic lipids containing a quaternary ammonium salt which are advantageously but not exclusively suitable for plasmids, are advantageously those having the following formula:

##STR00001##

[0201] in which R1 is a saturated or unsaturated straight-chain aliphatic radical having 12 to 18 carbon atoms, R2 is another aliphatic radical containing 2 or 3 carbon atoms and X is an amine or hydroxyl group, e.g. the DMRIE. In another embodiment the cationic lipid can be associated with a neutral lipid, e.g. the DOPE.

[0202] Among these cationic lipids, preference is given to DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium; WO96/34109), advantageously associated with a neutral lipid, advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr, 1994), to form DMRIE-DOPE.

[0203] Advantageously, the plasmid mixture with the adjuvant is formed extemporaneously and advantageously contemporaneously with administration of the preparation or shortly before administration of the preparation; for instance, shortly before or prior to administration, the plasmid-adjuvant mixture is formed, advantageously so as to give enough time prior to administration for the mixture to form a complex, e.g. between about 10 and about 60 minutes prior to administration, such as approximately 30 minutes prior to administration.

[0204] When DOPE is present, the DMRIE:DOPE molar ratio is advantageously about 95:about 5 to about 5:about 95, more advantageously about 1:about 1, e.g., 1:1.

[0205] The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be between about 50:about 1 and about 1:about 10, such as about 10:about 1 and about 1:about 5, and about 1:about 1 and about 1:about 2, e.g., 1:1 and 1:2.

[0206] In another embodiment, pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle may be a water-in-oil emulsion. Examples of suitable water-in-oil emulsions include oil-based water-in-oil vaccinal emulsions which are stable and fluid at 4.degree. C. containing: from 6 to 50 v/v % of an antigen-containing aqueous phase, preferably from 12 to 25 v/v %, from 50 to 94 v/v % of an oil phase containing in total or in part a non-metabolizable oil (e.g., mineral oil such as paraffin oil) and/or metabolizable oil (e.g., vegetable oil, or fatty acid, polyol or alcohol esters), from 0.2 to 20 p/v % of surfactants, preferably from 3 to 8 p/v %, the latter being in total or in part, or in a mixture either polyglycerol esters, said polyglycerol esters being preferably polyglycerol (poly)ricinoleates, or polyoxyethylene ricin oils or else hydrogenated polyoxyethylene ricin oils. Examples of surfactants that may be used in a water-in-oil emulsion include ethoxylated sorbitan esters (e.g., polyoxyethylene (20) sorbitan monooleate (TWEEN 80.RTM.), available from AppliChem, Inc., Cheshire, Conn.) and sorbitan esters (e.g., sorbitan monooleate (SPAN 80.RTM.), available from Sigma Aldrich, St. Louis, Mo.). In addition, with respect to a water-in-oil emulsion, see also U.S. Pat. No. 6,919,084, e.g., Example 8 thereof, incorporated herein by reference. In some embodiments, the antigen-containing aqueous phase comprises a saline solution comprising one or more buffering agents. An example of a suitable buffering solution is phosphate buffered saline. In an advantageous embodiment, the water-in-oil emulsion may be a water/oil/water (W/O/W) triple emulsion (U.S. Pat. No. 6,358,500). Examples of other suitable emulsions are described in U.S. Pat. No. 7,371,395.

[0207] The immunological compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on page 183 of the same work, (4) cation lipids containing a quaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.

[0208] The oil in water emulsion (3), which is especially appropriate for viral vectors, can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters.

[0209] The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121.

[0210] Among the type (1) adjuvant polymers, preference is given to polymers of crosslinked acrylic or methacrylic acid, especially crosslinked by polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). One skilled in the art can also refer to U.S. Pat. No. 2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, preferably no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can also contain other substituents, such as methyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are especially suitable. They are crosslinked by allyl saccharose or by allyl pentaerythritol. Among them, reference is made to Carbopol 974P, 934P and 971P.

[0211] As to the maleic anhydride-alkenyl derivative copolymers, preference is given to EMA (Monsanto), which are straight-chain or crosslinked ethylene-maleic anhydride copolymers and they are, for example, crosslinked by divinyl ether. Reference is also made to J. Fields et al., 1960.

[0212] With regard to structure, the acrylic or methacrylic acid polymers and EMA are preferably formed by basic units having the following formula:

##STR00002##

in which: [0213] R1 and R2, which can be the same or different, represent H or CH3 [0214] x=0 or 1, preferably x=1 [0215] y=1 or 2, with x+y=2.

[0216] For EMA, x=0 and y=2 and for carbomers x=y=1.

[0217] These polymers are soluble in water or physiological salt solution (20 g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda (NaOH), to provide the adjuvant solution in which the expression vector(s) can be incorporated. The polymer concentration in the final immunological or vaccine composition can range between about 0.01 to about 1.5% w/v, about 0.05 to about 1% w/v, and about 0.1 to about 0.4% w/v.

[0218] The cytokine or cytokines (5) can be in protein form in the immunological or vaccine composition, or can be co-expressed in the host with the immunogen or immunogens or epitope(s) thereof. Preference is given to the co-expression of the cytokine or cytokines, either by the same vector as that expressing the immunogen or immunogens or epitope(s) thereof, or by a separate vector thereof.

[0219] The invention comprehends preparing such combination compositions; for instance by admixing the active components, advantageously together and with an adjuvant, carrier, cytokine, and/or diluent.

[0220] Cytokines that may be used in the present invention include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interferon .alpha. (IFN.alpha.), interferon .beta.(IFN.beta.), interferon .gamma., (IFN.gamma.), interleukin-1.alpha. (IL-1.alpha.), interleukin-1.beta. (IL-1.beta.), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor .alpha. (TNF.alpha.), tumor necrosis factor .beta. (TNF.beta.), and transforming growth factor .beta. (TGF.beta.). It is understood that cytokines can be co-administered and/or sequentially administered with the immunological or vaccine composition of the present invention. Thus, for instance, the vaccine of the instant invention can also contain an exogenous nucleic acid molecule that expresses in vivo a suitable cytokine, e.g., a cytokine matched to this host to be vaccinated or in which an immunological response is to be elicited (for instance, a bovine cytokine for preparations to be administered to bovines).

[0221] Advantageously, the immunological composition and/or vaccine according to the invention comprise or consist essentially of or consist of an effective quantity to elicit a therapeutic response of one or more Duckweed-expressed polypeptides as discussed herein; and, an effective quantity can be determined from this disclosure, including the documents incorporated herein, and the knowledge in the art, without undue experimentation.

[0222] In the case of immunological composition and/or vaccine based on a Duckweed-expressed polypeptides, a dose may include, about in 1 .mu.g to about 2000 .mu.g, advantageously about 50 .mu.g to about 1000 .mu.g and more advantageously from about 100 .mu.g to about 500 .mu.g of FMDV antigen, epitope or immunogen. The dose volumes can be between about 0.1 and about 10 ml, advantageously between about 0.2 and about 5 ml.

[0223] The invention will now be further described by way of the following non-limiting examples.

EXAMPLES

[0224] Construction of DNA inserts, plasmids and recombinant viral or plant vectors was carried out using the standard molecular biology techniques described by J. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

Example 1

Generation and Screening of FMDV-Expressing Duckweed Lines

[0225] Duckweed optimized FMDV P1 and 3C sequences were produced and cloned into the parental plasmid to generate the MerE vectors depicted in FIGS. 6-9. Four independent constructs were designed for the FMDV project. Table 3 summarizes the number of transgenic lines that were generated and screened and FIG. 3 provides a schematic representation of gene structure for the FMDV inserts. Three lines express the P1 capsid+3C protease (MerE01, 3, 4), whereas the other express the P1 capsid antigen alone (MerE02). ELISA and Agilent analyses were used to quantify the expression of the FMDV antigens. Western blots were performed to verify the correct size of expressed proteins (FIG. 12). The highest FMDV serotype A24-expressing duckweed lines, as determined by mRNA analysis and by western blot, were grown in scale vessels to provide biomass for use in characterization and animal studies.

TABLE-US-00003 TABLE 3 Line generation and further screening of FMDV "MerE" lines # of lines # of lines Construct Description generated screened MerE01 P1-3C 16 16 MerE02 P1 alone (Optimized 100 100 5' UTR) MerE03 P1-2A + 3C 20 20 MerE04 P1-2A + 3C (Optimized 8 8 5' UTR)

[0226] Screening. 144 transgenic FMDV lines were developed. After 144 FMDV expressing lines were developed, they were screened to determine the relative levels of FMDV expression in the tissue. FMDV mRNA levels were measured via RNA dot blot (FIG. 10) and real time rtPCR (FIG. 11). FMDV proteins levels were measured using western blot and ELISA. FMDV-expressing duckweed lines were screened via RNA dot blot using a labeled P1 region to probe the blot. High expressing lines were confirmed by real time rtPCR, and there was reasonable agreement between the mRNA quantification methods. The results indicate that the FMDV P1 is highly expressed in duckweed.

[0227] Methods. The duckweed plants were grown for two weeks in small research vessels and the resulting tissue was collected and snap frozen in liquid N.sub.2. Total RNA was extracted from 100 mg of frozen tissue samples using the Qiagen RNeasy 96-well RNA extraction kit (Qiagen, Valencia, Calif., #74181). RNA was quantified, vacuum transferred to nylon membranes and probed with a gene fragment from the P1 region of the FMDV gene sequence. Crude tissue extraction from lines containing FMDV antigens were prepared. All steps were taken place at 4.degree. C. One hundred grams of frozen biomass was mixed with 200 ml extraction buffer (50 mM NaPO.sub.4, 0.3M NaCl, 10 mm EDTA, pH 7.4) and then homogenized in a Waring Blender with a 20 second burst for 4 times and 10-20 seconds cooling in between. The homogenate was centrifuged at 10,000.times.g for 30 min at 4.degree. C., clarified by passing through a cheese cloth to remove any large debris and finally cellulose acetate filter (0.22 .mu.m). The resulting homogenate was stored at 4.degree. C. or on ice for immediate testing. The remaining homogenate was frozen in aliquots at -80.degree. C. for further analysis. Total soluble protein (TSP) was determined using the Bradford assay with bovine serum albumin as a standard. Protein level analysis was performed for P1-expressing duckweed lines. Duplicate samples were read from two separate extractions (total of 4 measurements).

[0228] RNA Results. A broad range of RNA expression levels was observed between the various FMDV transgenic lines (FIG. 11). RNA expression from transgenic lines showing the strongest hybridization signals were then confirmed by real time rtPCR. Relative expression of these lines was shown to be comparable between the dot-blot and PCR methods. The top lines were then selected for further western blot characterization (FIG. 12).

[0229] Protein Results. The ELISA results are summarized in Table 4 and the densitometry and Agilent results are summarized in Table 5. FIG. 12 depicts the WB results, with arrows indicating the FMDV bands. Lane 1 (both gels)--marker, 2 (both gels)--sucrose-purified FMDV A24 inactivated virus, 3 (both gels)--duckweed wild type lysate. Lanes 4-6 of MerE01-expressing duckweed lysates (three lines--6, 8 and 10). Lanes 4-12 MerE02-expressing duckweed lysates (nine lines--2, 3, 16, 20, 23, 24, 25, 26 and 31). MerE01-expressing duckweed lines 6 and 10 appear to produce a properly cleaved protein (this line contains the 3C protease). MerE02-expressing duckweed lines (lacking the protease) appear to produce significant amounts of the uncleaved P1 protein, as well as higher MW aggregate species.

TABLE-US-00004 TABLE 4 ELISA Quantification Results for the MerE01 Lines. Average Antigen Conc. Average TSP Duckweed line (.mu.g/ml) (mg/ml) % TSP MerE01-6 11.35-16.84 5.0 0.23-0.34 MerE01-10 0.79-3.4 5.0 0.02-0.07

TABLE-US-00005 TABLE 5 Expression Level of Duckweed-FMDV MerE01 Lines. Average Antigen Conc. Duckweed line (.mu.g/ml).sup.1 Average % TSP.sup.2 MerE02-2 54.6 .+-. 4.3 2.35 MerE02-3 36.8 .+-. 6.6 1.70 MerE02-26 91.6 .+-. 9.0 3.95 .sup.1WT background band at the same MW (around 99-100 kDa) less than 5 .mu.g/ml. .sup.2Average total soluble protein between 2.1 and 2.3 mg/ml by Agilent analysis.

[0230] Inventors observed expression of the 3C protease was exerting toxic effects on the recombinant duckweed, causing reduced growth and antigen production, as compared to, for example, recombinant duckweed expressing only the P1 polypeptide. Therefore, new constructs were produced, some of which comprised a 3C expression cassette driven by an inducible promoter. Unhindered by the toxic effects of 3C expression, the duckweed grew and expressed robust levels of P1. Then, after the culture reached an optimum density, 3C expression was induced so it could cleave the established pools of P1 into the various subunits. The subunits were then able to assemble into protomers, pentamers, and finally, the FMD viral particles (schematized in FIG. 16).

[0231] These new FMDV duckweed expression constructs included MerF01 (SEQ ID NO:30), which incorporated P1-3C expressed driven by the Super Promoter in standard single gene expression vector (EC1.0, analogous to approach taken to produce MerE01). MerF02 (SEQ ID NO:31) used the Lemna gibba NPR promoter (ABA inducible promoter) to express P1-3C and MerF03 (SEQ ID NO:32) expressed P1-2A and 3C with separate promoters in a single vector: P1-2A expression driven by Super Promoter and 3C expression driven by Lemna minor R-histone promoter. MerF04 (SEQ ID NO:33) expressed P1-2A driven by Super promoter and expressed 3C driven by Lemna gibba NPR promoter. MerF05 (SEQ ID NO:34) expressed P1-2A (SpUbq promoter) and 3C (Lemna minor R-histone promoter), and MerF06 (SEQ ID NO:35) expressed P1-2A (SpUbq promoter) and 3C (Lemna gibba NPR promoter).

[0232] Antigen preparation. Crude extract was produced using a Waring Blender or bead beater (1:4 biomass to buffer ratio, PBS pH7.2), and the lysates were clarified by centrifugation (.about.10K.times.g). To produce the concentrate, the clarified lysate was filtered through a sterile 0.22 .mu.m filter. This material was then concentrated using 30 KDa centricon filters (5-10.times.), and subjected to in vitro characterization or animal study.

Example 2

Expression of FMDV Antigens in Schizochytrium

[0233] Codon-optimized FMDV P1 and 3C genes are cloned into the expression vector pAB0018 (ATCC deposit no. PTA9616). The specific nucleic acid sequence of FMDV gene is optimized for expression in Schizochytrium sp. Additionally, the expression vector contains a selection marker cassette conferring resistance to Schizochytrium transformants, a promoter from the Schizochytrium native gene to drive expression of the transgene, and a terminator.

[0234] Schizochytrium sp. (ATCC 20888) is used as a host for transformation with the expression vector containing the FDMV gene using electroporation method. Cryostocks of transgenic strains of Schizochytrium are grown in M50-20 (described in US 2008/0022422) to confluency. The propagated Schizochytrium cultures are transferred to 50 mL conical tubes and centrifuged at 3000 g for 15 min or 100,000 g for 1 hour. The resulting pellet and the soluble fraction are used for expression analysis and in animal challenge study.

Example 3

Vaccination of Pigs--Safety Assessment

[0235] Three (3) groups of five (5) pigs were vaccinated on days 0 and 21 (D0 and D21) according to the study design (Table 6). Details of the TS6 adjuvant (emulsions) may be found in U.S. Pat. No. 7,608,279 B2 and U.S. Pat. No. 7,371,395 B2 (both to Merial Limited).

[0236] Assessment of Safety. No adverse general/systemic reactions were observed after vaccination, though transient, slight to moderate increases of rectal temperature were observed in all groups. Locally, slight to moderate reactions were observed for the duckweed groups. The vaccines were globally acceptable for all groups.

TABLE-US-00006 TABLE 6 Vaccination of pigs - study design Group Antigen Dilution Adjuvant G1 MerE01 Not diluted TS6 G2 MerE01 1/10 diluted TS6 G3 -- Control --

Example 4

Vaccination of Cattle

[0237] Seventeen (17) conventional cattle, free from FMDV, but not previously immunized against FMDV, were used for this study (study design summarized in Table 7). At D-1, the cattle were allocated into to three groups of 5 animals, and 1 group of 2 animals. Vaccination was performed on D0, via sub-cutaneous route on the left side of the neck. Challenge was administered on D21, and the final observations were made on D29. Tested vaccines expressing FMDV A24 P1-3C were formulated in TS6 adjuvant as described above. Separate vials of antigens and adjuvant were stored at 5.degree. C. prior to administration. The contents of the vials were reconstituted extemporaneously by mixing antigen with adjuvant accordingly. The volume of a dose of the reconstituted vaccines was 2 mL. The challenge strain was FMD type A24 virus prepared to obtain 10 000 ID50 per 0.2 mL. The challenge strain was diluted in Hanks MEM 2% fetal bovine serum with antibiotics.

TABLE-US-00007 TABLE 7 cattle vaccine summary Group Antigen Dose # Cattle A Duckweed expressed FMDV 2 ml 5 B Experimental recombinant vaccine 2 ml 5 1 expressed FMDV C Experimental recombinant vaccine 2 ml 5 2 expressed FMDV Control NA NA 2

[0238] On D21, all animals were tranquilized by administration of Xylazine (0.03-0.10 mg per kg BW I.V. (0.15-0.5 ml per 100 kg BW I.V.) and challenged with 10 000 ID.sub.50of virus by intra-dermal route, into two locations of the tongue, 0.1 ml per location. The general well being of the animals was checked daily from D1 to D21. Any clinical observation and treatments administered (commercial name, active ingredient, preemption date, volume, route) were noted.

[0239] Necropsy results (Number of feet with at least 1 vesicle, min=0, max=4) for the animals were as follows: duckweed (4, 3, 2, 1, 1), experimental recombinant vaccine 1 (0, 0, 1, 1, 1), experimental recombinant vaccine 2 (3, 4, 4, 4, 4) and the Controls (4, 4). The challenge study was validated since all 2 control cattle showed FMD clinical signs, in addition, experimental vaccine 2 also exhibited clear FMD signs, and can be served as negative control. Even though the cattle in duckweed group were not well protected, clinical symptom was eased in two cattle. There were two cattle in experimental vaccine 1 group were considered to be protected, the result was consistent with other reports using the similar expression system and suggested that intact VLP was immunogenic and was able to provide protection against virulent FMDV.

[0240] It is likely the duckweed vaccine will be improved by 1) increasing percentage of intact VLP, and 2) improving the concentration/purification strategies, so that the antigen will be more accessible to the immune system in order to achieve greater protection.

Example 5

Characterization of FMDV Antigens by Sandwich ELISA

[0241] In vitro characterization of duckweed expressed FMDV was conducted with a sandwich ELISA using FA24 005E9G (monoclonal antibody against FMDV A24) and biotinylated M3, a 12S-specific llama single chain domain antibody.

[0242] Results (FIG. 17) indicated that positive optical density at 450 nm/630 nm for 1.times. crude extract, 5.times., and 10.times. concentrated crude extract, which was in good agreement with inactivated FMDV A24 as positive control, whereas crude extract prepared from duckweed wild type remained undetectable. ELISA titer was determined as the decimal logarithm per milliliter of the dilution for which 50% of the maximum OD was obtained. Table 8 demonstrated that ELISA titer for 5.times. was higher than 1.times. extract, however, equivalent to 10.times. extract, 5.times. concentrated duckweed crude extract was used in the cattle challenge study.

TABLE-US-00008 TABLE 8 Summary of ELISA titer Titre Sample log10OD50/ml Inactivated FMDV A24 5.46 MerE01 1x crude extract 2.79 MerE01 5x concentrated crude extract 3.34 MerE01 10x concentrated crude extract 3.21 Duckweed wild type 1x crude extract Negative

[0243] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

[0244] All documents cited or referenced in the application cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

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Proc Natl Acad Sci USA 93(21): 11307-12. [0287] Sambrook, J. and D. W. Russell (2001). Molecular Cloning: a laboratory manual/Joseph Sambrook, David W. Russell. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press. [0288] Schneider, K., F. Puehler, et al. (2000). "cDNA cloning of biologically active chicken interleukin-18." J Interferon Cytokine Res 20(10): 879-83. [0289] Shida, H. (1986). "Nucleotide sequence of the vaccinia virus hemagglutinin gene." Virology 150(2): 451-62. [0290] Smith, G. E., M. D. Summers, et al. (1983). "Production of human beta interferon in insect cells infected with a baculovirus expression vector." Mol Cell Biol 3(12): 2156-65. [0291] Snedecor, G. W. & COCHRAN, W. G. (1971) Transformation de proportions en Arcsinus. In Methodes Statistiques. 6th edn. Eds H. Boelle, E. Camhaji. Association de Coordination Technique Agricole. pp 366-367 [0292] Stickl, H. and V. Hochstein-Mintzel (1971). "[Intracutaneous smallpox vaccination with a weak pathogenic vaccinia virus ("MVA virus")]." Munch Med Wochenschr 113(35): 1149-53. [0293] Stittelaar, K. J., L. S. Wyatt, et al. (2000). "Protective immunity in macaques vaccinated with a modified vaccinia virus Ankara-based measles virus vaccine in the presence of passively acquired antibodies." J Virol 74(9): 4236-43. [0294] Sutter, G. and B. Moss (1992). "Nonreplicating vaccinia vector efficiently expresses recombinant genes." Proc Natl Acad Sci USA 89(22): 10847-51. [0295] Sutter, G., L. S. Wyatt, et al. (1994). "A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to FMDV virus." Vaccine 12(11): 1032-40. [0296] Tang, D. C., M. DeVit, et al. (1992). "Genetic immunization is a simple method for eliciting an immune response." Nature 356(6365): 152-4. [0297] Taylor, J., R. Weinberg, et al. (1988). "Protective immunity against avian FMDV induced by a fowlpox virus recombinant." Vaccine 6(6): 504-8. [0298] Thompson, J. D., D. G. Higgins, et al. (1994). "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice." Nucleic Acids Res 22(22): 4673-80. [0299] Ulmer, J. B., J. J. Donnelly, et al. (1993). "Heterologous protection against FMDV by injection of DNA encoding a viral protein." Science 259(5102): 1745-9. [0300] Van der Zee, R., W. Van Eden, et al. (1989). "Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides." Eur J Immunol 19(1): 43-7. [0301] van Ooyen, A., J. van den Berg, et al. (1979). "Comparison of total sequence of a cloned rabbit beta-globin gene and its flanking regions with a homologous mouse sequence." Science 206(4416): 337-44. [0302] Vialard, J., M. Lalumiere, et al. (1990). "Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene." J Virol 64(1): 37-50. [0303] Xin, K. Q., K. Hamajima, et al. (1999). "IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine." Vaccine 17(7-8): 858-66.

Sequence CWU 1

1

3913378DNAArtificial SequencepHM1119.1 FMDV P1-3C optimized for mammalian expression 1atgggagctg ggcaatccag cccagcaacc ggctcgcaga accagtctgg caacactggc 60agcataatca acaactacta catgcaacag taccagaact ccatggacac acagttggga 120gacaatgcca tcagtggagg ctccaacgag ggctccacgg acacaacttc aacacacaca 180accaacactc aaaacaatga ctggttctcg aagctcgcca gttcagcttt taccggtctg 240ttcggtgcac tgctcgccga caagaagaca gaggaaacga cacttcttga ggaccgcatc 300ctcaccaccc gcaacgggca caccacctcg acgacccaat cgagtgtggg tgtcacacac 360gggtactcca cagaggagga ccacgttgct gggcccaaca catcgggcct ggagacgcga 420gtggtgcagg cagagagatt ctacaaaaag tacttgtttg actggacaac ggacaaggca 480tttggacacc tggaaaagct ggagctcccg tccgaccacc acggtgtctt tggacacttg 540gtggactcgt acgcctatat gagaaatggc tgggatgttg aggtgtccgc tgttggcaac 600cagttcaacg gcgggtgcct cctggtggcc atggtacctg aatggaagga atttgacaca 660cgggagaaat accaactcac ccttttcccg caccagttta ttagccccag aactaacatg 720actgcccaca tcacggtccc ctaccttggt gtgaacaggt atgatcagta caagaagcat 780aagccctgga cattggttgt catggtcgtg tcgccactta cggtcaacaa cactagtgcg 840gcacaaatca aggtctacgc caacatagct ccgacctatg ttcacgtggc cggtgaactc 900ccctcgaaag aggggatttt cccggttgca tgtgcggacg gttacggagg attggtgacg 960acagacccga agacagctga ccctgcttat ggcaaggtgt acaacccgcc taggactaac 1020taccctgggc gcttcaccaa cctgttggac gtggccgaag cgtgtcccac tttcctctgc 1080tttgacgacg ggaaaccgta cgtcaccacg cggacggatg acacccgact tttggccaag 1140tttgaccttt cccttgccgc aaaacatatg tccaacacat acctgtcagg gattgctcag 1200tactacacac agtactctgg caccatcaat ttgcatttca tgtttacagg ttccactgat 1260tcaaaggccc gatacatggt ggcctacatc ccacctgggg tggagacacc accggacaca 1320cctgaaaggg ctgcccactg cattcacgct gaatgggaca ctggactaaa ctccaaattc 1380actttctcaa tcccgtacgt atccgccgcg gattacgcgt acacagcgtc tgacacggca 1440gaaacaatca acgtacaggg atgggtctgc atctaccaaa ttacacacgg gaaggctgaa 1500aatgacacct tggtcgtgtc ggttagcgcc ggcaaagact ttgagttgcg cctcccgatt 1560gacccccgcc agcagaccac cgctaccggg gaatcagcag acccggtcac caccaccgtg 1620gagaactacg gcggtgagac acaaatccag agacgtcacc acacggacat tggtttcatc 1680atggacagat ttgtgaagat ccaaagcttg agcccaacac atgtcattga cctcatgcag 1740gctcaccaac acggtctggt gggtgccttg ctgcgtgcag ccacgtacta cttttctgac 1800ctggaaattg ttgtacggca cgaaggcaat ctgacctggg tgcccaacgg cgcccctgaa 1860tcagccctgt tgaacaccag caaccccact gcctacaaca aggcaccatt cacgagactc 1920gctctcccct acactgcgcc gcaccgtgtg ctggcaacag tgtacaacgg gacgagtaag 1980tatgctgtgg gtggttcagg cagaagaggc gacatggggt ctctcgcggc gcgagtcgtg 2040aaacagcttc ctgcttcatt taactacggt gcaatcaagg ccgacgccat ccacgaactt 2100ctcgtgcgca tgaaacgggc cgagctctac tgccccagac cgctgttggc aatagaggtg 2160tcttcgcaag acaggcacaa gcaaaagatc attgcaccag caaagcagct tctgaatttt 2220gacctgctca agttggccgg agacgttgag tccaaccccg ggccattctt ctttgctgac 2280gttaggtcaa acttttcaaa gttggtagac acaatcaacc agatgcagga ggacatgtcc 2340acaaaacacg ggcccgactt caaccggttg gtgtccgcat ttgaggaatt ggccactgga 2400gttaaagcta tcaggaccgg tctcgacgag gccaaaccct ggtacaagct tatcaaactc 2460ctaagccgcc tgtcgtgcat ggccgctgtg gcagcacggt ccaaggaccc agtccttgtg 2520gccatcatgc tggccgacac cggtctcgag cgtcagagac ctctgaaagt gagagctaag 2580ctcccacagc aggaaggacc ttacgctggc ccgttggaga gacagaaacc gctgaaagtg 2640aaagcaaaag ccccggtcgt caaggaagga ccttacgagg gaccggtgaa gaagcctgtc 2700gctttgaaag tgaaagctaa gaacttgata gtcactgaga gtggtgcccc accgaccgac 2760ttgcaaaaga tggtcatggg caacacaaag cctgttgagc tcatccttga cgggaagaca 2820gtagccatct gttgtgctac tggagtgttt ggcactgctt acctcgtgcc tcgtcatctt 2880ttcgcagaga agtatgacaa gatcatgctg gatggcagag ccatgacaga cagtgactac 2940agagtgtttg agtttgagat taaagtaaaa ggacaggaca tgctctcaga cgctgcgctc 3000atggtgctcc accgtgggaa ccgcgtgaga gatatcacga aacactttcg tgatacagca 3060agaatgaaga aaggcacccc cgtcgtcggt gtggtcaaca acgccgacgt tgggagactg 3120attttctctg gtgaggccct cacctacaag gatattgtag tgtgcatgga cggagacacc 3180atgcctggcc tctttgccta caaagccgcc accaaggcag gctactgtgg aggagccgtt 3240ctcgccaagg acggggccga cactttcatc gtcggcactc actccgcagg aggcaatgga 3300gttggatact gctcatgcgt ttccaggtcc atgcttctca gaatgaaggc acacgttgac 3360cctgaaccac aacacgag 337823381DNAArtificial SequenceFMDV - P1-3C (MerE01 - MerE04) optimized for Lemna expression 2atgggcgccg ggcagtcctc gcctgcgacg gggagccaga accagtcggg caacaccggc 60tcgatcatca acaactacta catgcagcaa taccagaaca gcatggacac ccagctcggc 120gataacgcca tctccggcgg gtccaacgag ggctcgaccg acacgacctc cacccacacc 180acgaacaccc agaacaatga ctggttcagc aagctggcct ccagcgcctt cacgggcctg 240ttcggggcgc tgctcgcgga caagaagacc gaggagacca cgctgctcga ggaccggatc 300ctcaccacgc gcaacgggca cacgaccagc acgacccagt ccagcgtcgg ggtgacccac 360ggctactcca cggaggagga ccacgtcgcc gggccgaaca cgagcggcct cgagacccgc 420gtggtccagg cggagcgctt ctacaagaag tacctgttcg actggaccac ggacaaggcg 480ttcgggcacc tcgagaagct ggagctgccg agcgaccacc acggcgtctt cggccacctc 540gtggactcct acgcgtacat gagaaatggg tgggacgtcg aggtcagcgc cgtcggcaac 600cagttcaacg gcgggtgcct gctcgtggcg atggtcccgg agtggaagga gttcgacacc 660cgcgagaagt atcagctgac cctcttcccc caccagttca tcagcccgcg caccaacatg 720accgcccaca tcacggtgcc gtacctcggg gtgaaccgct acgaccagta caagaagcac 780aagccctgga ccctcgtggt catggtggtc tccccgctga ctgtgaacaa cacgtccgcc 840gcgcagatca aagtctacgc caacatcgcc cccacctacg tgcacgtcgc cggcgagctg 900ccctccaagg agggcatctt ccccgtcgcc tgcgccgacg ggtacggggg cctggtgacc 960accgacccca agacggccga cccggcctac ggcaaagtgt acaacccgcc caggacgaac 1020taccccggtc gcttcaccaa cctcctggac gtggcggagg cctgcccgac cttcctgtgc 1080ttcgacgacg ggaagcccta cgtcaccacg cgcaccgacg acacgcgcct gctcgccaag 1140ttcgacctca gcctggccgc taagcacatg agcaacacct acctcagcgg catcgcccaa 1200tactataccc aatactcggg caccatcaac ctgcacttca tgttcacggg cagcaccgac 1260agcaaggccc ggtacatggt ggcctacatc ccgccgggcg tggagacccc tcccgacacg 1320cccgagcggg ctgcgcactg catccacgcc gagtgggaca ccggcctcaa cagcaagttc 1380acgttcagca tcccctacgt gtccgccgcg gattacgctt acaccgcctc ggacacggcc 1440gagacgatca acgtccaggg ctgggtctgc atctaccaga tcactcacgg caaggccgag 1500aacgacaccc tcgtcgtgag cgtctccgcc gggaaggact tcgagctgag gctgcccatc 1560gaccccaggc agcagaccac ggcgaccggg gagtccgccg accccgtgac cacgaccgtg 1620gagaactacg gcggggagac tcagatccag cggcgccacc acaccgacat cggcttcatc 1680atggaccgct tcgtgaagat ccagtccctg tcgcccaccc acgttatcga cctcatgcag 1740gcccaccagc acgggctcgt gggtgccctc ctgcgcgcgg ccacctacta cttcagcgac 1800ctcgagatcg ttgtccgcca cgaggggaac ctcacctggg tcccgaacgg tgcccccgag 1860agcgccctgc tcaacacctc caaccccacg gcgtacaaca aggccccctt cacgcgcctc 1920gccctgcctt acaccgcgcc ccaccgcgtg ctggccacgg tgtacaacgg gacctccaag 1980tacgccgtgg gcgggagcgg ccgcagaggc gacatgggga gcctcgccgc tagggtcgtg 2040aagcagctcc cggcctcctt caactacggc gccatcaagg ccgacgccat ccacgaactc 2100ctggtccgca tgaagcgcgc cgaactctac tgcccccggc ccctcctggc catcgaggtc 2160tcctcgcagg accgccacaa gcagaagatc atcgccccgg ccaagcagct gctcaacttc 2220gacctgctca agctcgcggg ggacgtcgag tcgaaccccg gtcccttctt cttcgccgac 2280gtgcggtcca acttctcgaa gctcgtggac accatcaacc agatgcagga ggacatgagc 2340accaagcacg ggccggactt caaccgcctc gtctcggcct tcgaggaact cgccaccggg 2400gtcaaggcca tcaggacggg cctggacgag gcgaagccgt ggtacaagct catcaagctc 2460ctgtcgcgcc tctcctgcat ggcggctgtc gcggcccgct ccaaggaccc cgtcctcgtc 2520gccatcatgc tcgcggacac cgggctcgag cgccagcggc ccctgaaagt gcgcgccaag 2580ctgccgcagc aggagggccc ctacgccggc ccgctcgaga gacagaagcc gctgaaagtc 2640aaggccaagg cgcccgtggt caaggagggg ccctacgagg gccccgtcaa gaagccggtg 2700gcgctgaaag tgaaggccaa gaacctcatc gtgaccgagt ccggtgcgcc tcccaccgac 2760ctgcagaaga tggtgatggg caacaccaag cccgtcgagt tgatcctcga cgggaagacc 2820gtggcgatct gctgcgccac cggcgtgttc ggcaccgcct acctcgtccc gagacacctg 2880ttcgccgaga agtatgacaa gatcatgctg gacgggcggg ccatgaccga ctcggactac 2940cgggtcttcg agttcgagat caaagtgaag ggccaggata tgctctccga cgccgcgctg 3000atggtgctcc acagaggcaa ccgcgtgcgg gacatcacca agcacttcag ggacaccgcg 3060cgcatgaaga aggggacccc tgtggtcggg gtcgtgaaca acgccgacgt cgggcgcctc 3120atcttctccg gcgaggcgct gacctacaag gacatcgtcg tgtgcatgga cggggacacg 3180atgccggggc tcttcgccta caaggccgcg accaaggccg ggtactgcgg gggtgcggtg 3240ctcgccaagg acggcgccga cacgttcatc gtgggcaccc actccgccgg cggtaacggc 3300gtcggctact gctcctgcgt gtcccggtcc atgctgctgc ggatgaaggc ccacgttgac 3360cccgagccgc agcacgagta a 338131126PRTArtificial SequenceFMDV - P1-3C (MerE01 - MerE04) translation of SEQ ID NOs1 and 2 3Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser1 5 10 15Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu65 70 75 80Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110Gln Ser Ser Val Gly Val Thr His Gly Tyr Ser Thr Glu Glu Asp His 115 120 125Val Ala Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140Glu Arg Phe Tyr Lys Lys Tyr Leu Phe Asp Trp Thr Thr Asp Lys Ala145 150 155 160Phe Gly His Leu Glu Lys Leu Glu Leu Pro Ser Asp His His Gly Val 165 170 175Phe Gly His Leu Val Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205Val Ala Met Val Pro Glu Trp Lys Glu Phe Asp Thr Arg Glu Lys Tyr 210 215 220Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met225 230 235 240Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255Tyr Lys Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270Leu Thr Val Asn Asn Thr Ser Ala Ala Gln Ile Lys Val Tyr Ala Asn 275 280 285Ile Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300Gly Ile Phe Pro Val Ala Cys Ala Asp Gly Tyr Gly Gly Leu Val Thr305 310 315 320Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Lys Val Tyr Asn Pro 325 330 335Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Val 355 360 365Thr Thr Arg Thr Asp Asp Thr Arg Leu Leu Ala Lys Phe Asp Leu Ser 370 375 380Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Ile Ala Gln385 390 395 400Tyr Tyr Thr Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro 420 425 430Gly Val Glu Thr Pro Pro Asp Thr Pro Glu Arg Ala Ala His Cys Ile 435 440 445His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455 460Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr Ala465 470 475 480Glu Thr Ile Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 485 490 495Gly Lys Ala Glu Asn Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 500 505 510Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Gln Gln Thr Thr Ala 515 520 525Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540Gly Glu Thr Gln Ile Gln Arg Arg His His Thr Asp Ile Gly Phe Ile545 550 555 560Met Asp Arg Phe Val Lys Ile Gln Ser Leu Ser Pro Thr His Val Ile 565 570 575Asp Leu Met Gln Ala His Gln His Gly Leu Val Gly Ala Leu Leu Arg 580 585 590Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg His Glu 595 600 605Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ser Ala Leu Leu 610 615 620Asn Thr Ser Asn Pro Thr Ala Tyr Asn Lys Ala Pro Phe Thr Arg Leu625 630 635 640Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn 645 650 655Gly Thr Ser Lys Tyr Ala Val Gly Gly Ser Gly Arg Arg Gly Asp Met 660 665 670Gly Ser Leu Ala Ala Arg Val Val Lys Gln Leu Pro Ala Ser Phe Asn 675 680 685Tyr Gly Ala Ile Lys Ala Asp Ala Ile His Glu Leu Leu Val Arg Met 690 695 700Lys Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Ile Glu Val705 710 715 720Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln 725 730 735Leu Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 740 745 750Pro Gly Pro Phe Phe Phe Ala Asp Val Arg Ser Asn Phe Ser Lys Leu 755 760 765Val Asp Thr Ile Asn Gln Met Gln Glu Asp Met Ser Thr Lys His Gly 770 775 780Pro Asp Phe Asn Arg Leu Val Ser Ala Phe Glu Glu Leu Ala Thr Gly785 790 795 800Val Lys Ala Ile Arg Thr Gly Leu Asp Glu Ala Lys Pro Trp Tyr Lys 805 810 815Leu Ile Lys Leu Leu Ser Arg Leu Ser Cys Met Ala Ala Val Ala Ala 820 825 830Arg Ser Lys Asp Pro Val Leu Val Ala Ile Met Leu Ala Asp Thr Gly 835 840 845Leu Glu Arg Gln Arg Pro Leu Lys Val Arg Ala Lys Leu Pro Gln Gln 850 855 860Glu Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Lys Val865 870 875 880Lys Ala Lys Ala Pro Val Val Lys Glu Gly Pro Tyr Glu Gly Pro Val 885 890 895Lys Lys Pro Val Ala Leu Lys Val Lys Ala Lys Asn Leu Ile Val Thr 900 905 910Glu Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met Gly Asn 915 920 925Thr Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val Ala Ile Cys 930 935 940Cys Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro Arg His Leu945 950 955 960Phe Ala Glu Lys Tyr Asp Lys Ile Met Leu Asp Gly Arg Ala Met Thr 965 970 975Asp Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile Lys Val Lys Gly Gln 980 985 990Asp Met Leu Ser Asp Ala Ala Leu Met Val Leu His Arg Gly Asn Arg 995 1000 1005Val Arg Asp Ile Thr Lys His Phe Arg Asp Thr Ala Arg Met Lys 1010 1015 1020Lys Gly Thr Pro Val Val Gly Val Val Asn Asn Ala Asp Val Gly 1025 1030 1035Arg Leu Ile Phe Ser Gly Glu Ala Leu Thr Tyr Lys Asp Ile Val 1040 1045 1050Val Cys Met Asp Gly Asp Thr Met Pro Gly Leu Phe Ala Tyr Lys 1055 1060 1065Ala Ala Thr Lys Ala Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys 1070 1075 1080Asp Gly Ala Asp Thr Phe Ile Val Gly Thr His Ser Ala Gly Gly 1085 1090 1095Asn Gly Val Gly Tyr Cys Ser Cys Val Ser Arg Ser Met Leu Leu 1100 1105 1110Arg Met Lys Ala His Val Asp Pro Glu Pro Gln His Glu 1115 1120 11254554DNAArtificial SequenceZea mays alcohol dehydrogenase - Nucleotides 1222-1775 4gatcaagtgc aaaggtccgc cttgtttctc ctctgtctct tgatctgact aatcttggtt 60tatgattcgt tgagtaattt tggggaaagc ttcgtccaca gttttttttt cgatgaacag 120tgccgcagtg gcgctgatct tgtatgctat cctgcaatcg tggtgaactt atgtctttta 180tatccttcac taccatgaaa agactagtaa tctttctcga tgtaacatcg tccagcactg 240ctattaccgt gtggtccatc cgacagtctg gctgaacaca tcatacgata ttgagcaaag 300atctatcttc cctgttcttt aatgaaagac gtcattttca tcagtatgat ctaagaatgt 360tgcaacttgc aaggaggcgt ttctttcttt gaatttaact aactcgttga gtggccctgt 420ttctcggacg taaggccttt gctgctccac acatgtccat tcgaatttta ccgtgtttag 480caagggcgaa aagtttgcat cttgatgatt tagcttgact atgcgattgc tttcctggac 540ccgtgcagct gcgg 554521PRTArtificial SequenceArabidopsis thaliana basic endochitinase signal peptide 5Met Lys Thr Asn Leu Phe Leu Phe Leu Ile Phe Ser Leu Leu Leu Ser1 5 10 15Leu Ser Ser Ala Glu 20631PRTArtificial SequenceRice alpha-amylase signal sequence peptide (translation of SEQ ID NO8) 6Met Gln Val Leu Asn Thr Met Val Asn Lys His Phe Leu Ser Leu Ser1 5 10 15Val Leu Ile Val Leu Leu Gly Leu Ser Ser Asn Leu Thr Ala Gly

20 25 30731PRTArtificial SequenceModified rice alpha-amylase signal sequence peptide 7Met Gln Val Leu Asn Thr Met Val Asn Lys His Phe Leu Ser Leu Ser1 5 10 15Val Leu Ile Val Leu Thr Val Leu Ser Ser Asn Leu Thr Ala Gly 20 25 30896DNAArtificial SequenceRice alpha-amylase signal sequence nucleic acid 8accatgcagg tcctgaacac gatggtcaac aagcacttcc tctccctgtc cgtcctcatc 60gtcctcctcg ggctgagcag caacctcacc gccggc 9692208DNAArtificial SequenceFMDV - P1 DNA optimized for Lemna expression 9atgggcgccg ggcagtcctc gcctgcgacg gggagccaga accagtcggg caacaccggc 60tcgatcatca acaactacta catgcagcaa taccagaaca gcatggacac ccagctcggc 120gataacgcca tctccggcgg gtccaacgag ggctcgaccg acacgacctc cacccacacc 180acgaacaccc agaacaatga ctggttcagc aagctggcct ccagcgcctt cacgggcctg 240ttcggggcgc tgctcgcgga caagaagacc gaggagacca cgctgctcga ggaccggatc 300ctcaccacgc gcaacgggca cacgaccagc acgacccagt ccagcgtcgg ggtgacccac 360ggctactcca cggaggagga ccacgtcgcc gggccgaaca cgagcggcct cgagacccgc 420gtggtccagg cggagcgctt ctacaagaag tacctgttcg actggaccac ggacaaggcg 480ttcgggcacc tcgagaagct ggagctgccg agcgaccacc acggcgtctt cggccacctc 540gtggactcct acgcgtacat gagaaatggg tgggacgtcg aggtcagcgc cgtcggcaac 600cagttcaacg gcgggtgcct gctcgtggcg atggtcccgg agtggaagga gttcgacacc 660cgcgagaagt atcagctgac cctcttcccc caccagttca tcagcccgcg caccaacatg 720accgcccaca tcacggtgcc gtacctcggg gtgaaccgct acgaccagta caagaagcac 780aagccctgga ccctcgtggt catggtggtc tccccgctga ctgtgaacaa cacgtccgcc 840gcgcagatca aagtctacgc caacatcgcc cccacctacg tgcacgtcgc cggcgagctg 900ccctccaagg agggcatctt ccccgtcgcc tgcgccgacg ggtacggggg cctggtgacc 960accgacccca agacggccga cccggcctac ggcaaagtgt acaacccgcc caggacgaac 1020taccccggtc gcttcaccaa cctcctggac gtggcggagg cctgcccgac cttcctgtgc 1080ttcgacgacg ggaagcccta cgtcaccacg cgcaccgacg acacgcgcct gctcgccaag 1140ttcgacctca gcctggccgc taagcacatg agcaacacct acctcagcgg catcgcccaa 1200tactataccc aatactcggg caccatcaac ctgcacttca tgttcacggg cagcaccgac 1260agcaaggccc ggtacatggt ggcctacatc ccgccgggcg tggagacccc tcccgacacg 1320cccgagcggg ctgcgcactg catccacgcc gagtgggaca ccggcctcaa cagcaagttc 1380acgttcagca tcccctacgt gtccgccgcg gattacgctt acaccgcctc ggacacggcc 1440gagacgatca acgtccaggg ctgggtctgc atctaccaga tcactcacgg caaggccgag 1500aacgacaccc tcgtcgtgag cgtctccgcc gggaaggact tcgagctgag gctgcccatc 1560gaccccaggc agcagaccac ggcgaccggg gagtccgccg accccgtgac cacgaccgtg 1620gagaactacg gcggggagac tcagatccag cggcgccacc acaccgacat cggcttcatc 1680atggaccgct tcgtgaagat ccagtccctg tcgcccaccc acgttatcga cctcatgcag 1740gcccaccagc acgggctcgt gggtgccctc ctgcgcgcgg ccacctacta cttcagcgac 1800ctcgagatcg ttgtccgcca cgaggggaac ctcacctggg tcccgaacgg tgcccccgag 1860agcgccctgc tcaacacctc caaccccacg gcgtacaaca aggccccctt cacgcgcctc 1920gccctgcctt acaccgcgcc ccaccgcgtg ctggccacgg tgtacaacgg gacctccaag 1980tacgccgtgg gcgggagcgg ccgcagaggc gacatgggga gcctcgccgc tagggtcgtg 2040aagcagctcc cggcctcctt caactacggc gccatcaagg ccgacgccat ccacgaactc 2100ctggtccgca tgaagcgcgc cgaactctac tgcccccggc ccctcctggc catcgaggtc 2160tcctcgcagg accgccacaa gcagaagatc atcgccccgg ccaagcag 220810736PRTArtificial SequenceFMDV - P1 protein (translation of SEQ ID NOs9 and 13) 10Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser1 5 10 15Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu65 70 75 80Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110Gln Ser Ser Val Gly Val Thr His Gly Tyr Ser Thr Glu Glu Asp His 115 120 125Val Ala Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140Glu Arg Phe Tyr Lys Lys Tyr Leu Phe Asp Trp Thr Thr Asp Lys Ala145 150 155 160Phe Gly His Leu Glu Lys Leu Glu Leu Pro Ser Asp His His Gly Val 165 170 175Phe Gly His Leu Val Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205Val Ala Met Val Pro Glu Trp Lys Glu Phe Asp Thr Arg Glu Lys Tyr 210 215 220Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met225 230 235 240Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255Tyr Lys Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270Leu Thr Val Asn Asn Thr Ser Ala Ala Gln Ile Lys Val Tyr Ala Asn 275 280 285Ile Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300Gly Ile Phe Pro Val Ala Cys Ala Asp Gly Tyr Gly Gly Leu Val Thr305 310 315 320Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Lys Val Tyr Asn Pro 325 330 335Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 340 345 350Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Val 355 360 365Thr Thr Arg Thr Asp Asp Thr Arg Leu Leu Ala Lys Phe Asp Leu Ser 370 375 380Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Ile Ala Gln385 390 395 400Tyr Tyr Thr Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro 420 425 430Gly Val Glu Thr Pro Pro Asp Thr Pro Glu Arg Ala Ala His Cys Ile 435 440 445His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455 460Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr Ala465 470 475 480Glu Thr Ile Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 485 490 495Gly Lys Ala Glu Asn Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 500 505 510Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Gln Gln Thr Thr Ala 515 520 525Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540Gly Glu Thr Gln Ile Gln Arg Arg His His Thr Asp Ile Gly Phe Ile545 550 555 560Met Asp Arg Phe Val Lys Ile Gln Ser Leu Ser Pro Thr His Val Ile 565 570 575Asp Leu Met Gln Ala His Gln His Gly Leu Val Gly Ala Leu Leu Arg 580 585 590Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg His Glu 595 600 605Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ser Ala Leu Leu 610 615 620Asn Thr Ser Asn Pro Thr Ala Tyr Asn Lys Ala Pro Phe Thr Arg Leu625 630 635 640Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr Val Tyr Asn 645 650 655Gly Thr Ser Lys Tyr Ala Val Gly Gly Ser Gly Arg Arg Gly Asp Met 660 665 670Gly Ser Leu Ala Ala Arg Val Val Lys Gln Leu Pro Ala Ser Phe Asn 675 680 685Tyr Gly Ala Ile Lys Ala Asp Ala Ile His Glu Leu Leu Val Arg Met 690 695 700Lys Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Ile Glu Val705 710 715 720Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala Lys Gln 725 730 73511639DNAArtificial SequenceFMDV - 3C DNA optimized for Lemna expression 11tccggtgcgc ctcccaccga cctgcagaag atggtgatgg gcaacaccaa gcccgtcgag 60ttgatcctcg acgggaagac cgtggcgatc tgctgcgcca ccggcgtgtt cggcaccgcc 120tacctcgtcc cgagacacct gttcgccgag aagtatgaca agatcatgct ggacgggcgg 180gccatgaccg actcggacta ccgggtcttc gagttcgaga tcaaagtgaa gggccaggat 240atgctctccg acgccgcgct gatggtgctc cacagaggca accgcgtgcg ggacatcacc 300aagcacttca gggacaccgc gcgcatgaag aaggggaccc ctgtggtcgg ggtcgtgaac 360aacgccgacg tcgggcgcct catcttctcc ggcgaggcgc tgacctacaa ggacatcgtc 420gtgtgcatgg acggggacac gatgccgggg ctcttcgcct acaaggccgc gaccaaggcc 480gggtactgcg ggggtgcggt gctcgccaag gacggcgccg acacgttcat cgtgggcacc 540cactccgccg gcggtaacgg cgtcggctac tgctcctgcg tgtcccggtc catgctgctg 600cggatgaagg cccacgttga ccccgagccg cagcacgag 63912213PRTArtificial SequenceFMDV - 3C protein (translation of SEQ ID NOs11 and 14) 12Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met Gly Asn Thr1 5 10 15Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val Ala Ile Cys Cys 20 25 30Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro Arg His Leu Phe 35 40 45Ala Glu Lys Tyr Asp Lys Ile Met Leu Asp Gly Arg Ala Met Thr Asp 50 55 60Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile Lys Val Lys Gly Gln Asp65 70 75 80Met Leu Ser Asp Ala Ala Leu Met Val Leu His Arg Gly Asn Arg Val 85 90 95Arg Asp Ile Thr Lys His Phe Arg Asp Thr Ala Arg Met Lys Lys Gly 100 105 110Thr Pro Val Val Gly Val Val Asn Asn Ala Asp Val Gly Arg Leu Ile 115 120 125Phe Ser Gly Glu Ala Leu Thr Tyr Lys Asp Ile Val Val Cys Met Asp 130 135 140Gly Asp Thr Met Pro Gly Leu Phe Ala Tyr Lys Ala Ala Thr Lys Ala145 150 155 160Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys Asp Gly Ala Asp Thr Phe 165 170 175Ile Val Gly Thr His Ser Ala Gly Gly Asn Gly Val Gly Tyr Cys Ser 180 185 190Cys Val Ser Arg Ser Met Leu Leu Arg Met Lys Ala His Val Asp Pro 195 200 205Glu Pro Gln His Glu 210132208DNAArtificial SequenceFMDV-P1 DNA optimized for mammalian expression 13atgggagctg ggcaatccag cccagcaacc ggctcgcaga accagtctgg caacactggc 60agcataatca acaactacta catgcaacag taccagaact ccatggacac acagttggga 120gacaatgcca tcagtggagg ctccaacgag ggctccacgg acacaacttc aacacacaca 180accaacactc aaaacaatga ctggttctcg aagctcgcca gttcagcttt taccggtctg 240ttcggtgcac tgctcgccga caagaagaca gaggaaacga cacttcttga ggaccgcatc 300ctcaccaccc gcaacgggca caccacctcg acgacccaat cgagtgtggg tgtcacacac 360gggtactcca cagaggagga ccacgttgct gggcccaaca catcgggcct ggagacgcga 420gtggtgcagg cagagagatt ctacaaaaag tacttgtttg actggacaac ggacaaggca 480tttggacacc tggaaaagct ggagctcccg tccgaccacc acggtgtctt tggacacttg 540gtggactcgt acgcctatat gagaaatggc tgggatgttg aggtgtccgc tgttggcaac 600cagttcaacg gcgggtgcct cctggtggcc atggtacctg aatggaagga atttgacaca 660cgggagaaat accaactcac ccttttcccg caccagttta ttagccccag aactaacatg 720actgcccaca tcacggtccc ctaccttggt gtgaacaggt atgatcagta caagaagcat 780aagccctgga cattggttgt catggtcgtg tcgccactta cggtcaacaa cactagtgcg 840gcacaaatca aggtctacgc caacatagct ccgacctatg ttcacgtggc cggtgaactc 900ccctcgaaag aggggatttt cccggttgca tgtgcggacg gttacggagg attggtgacg 960acagacccga agacagctga ccctgcttat ggcaaggtgt acaacccgcc taggactaac 1020taccctgggc gcttcaccaa cctgttggac gtggccgaag cgtgtcccac tttcctctgc 1080tttgacgacg ggaaaccgta cgtcaccacg cggacggatg acacccgact tttggccaag 1140tttgaccttt cccttgccgc aaaacatatg tccaacacat acctgtcagg gattgctcag 1200tactacacac agtactctgg caccatcaat ttgcatttca tgtttacagg ttccactgat 1260tcaaaggccc gatacatggt ggcctacatc ccacctgggg tggagacacc accggacaca 1320cctgaaaggg ctgcccactg cattcacgct gaatgggaca ctggactaaa ctccaaattc 1380actttctcaa tcccgtacgt atccgccgcg gattacgcgt acacagcgtc tgacacggca 1440gaaacaatca acgtacaggg atgggtctgc atctaccaaa ttacacacgg gaaggctgaa 1500aatgacacct tggtcgtgtc ggttagcgcc ggcaaagact ttgagttgcg cctcccgatt 1560gacccccgcc agcagaccac cgctaccggg gaatcagcag acccggtcac caccaccgtg 1620gagaactacg gcggtgagac acaaatccag agacgtcacc acacggacat tggtttcatc 1680atggacagat ttgtgaagat ccaaagcttg agcccaacac atgtcattga cctcatgcag 1740gctcaccaac acggtctggt gggtgccttg ctgcgtgcag ccacgtacta cttttctgac 1800ctggaaattg ttgtacggca cgaaggcaat ctgacctggg tgcccaacgg cgcccctgaa 1860tcagccctgt tgaacaccag caaccccact gcctacaaca aggcaccatt cacgagactc 1920gctctcccct acactgcgcc gcaccgtgtg ctggcaacag tgtacaacgg gacgagtaag 1980tatgctgtgg gtggttcagg cagaagaggc gacatggggt ctctcgcggc gcgagtcgtg 2040aaacagcttc ctgcttcatt taactacggt gcaatcaagg ccgacgccat ccacgaactt 2100ctcgtgcgca tgaaacgggc cgagctctac tgccccagac cgctgttggc aatagaggtg 2160tcttcgcaag acaggcacaa gcaaaagatc attgcaccag caaagcag 220814639DNAArtificial SequenceFMDV-3C DNA optimized for mammalian expression 14agtggtgccc caccgaccga cttgcaaaag atggtcatgg gcaacacaaa gcctgttgag 60ctcatccttg acgggaagac agtagccatc tgttgtgcta ctggagtgtt tggcactgct 120tacctcgtgc ctcgtcatct tttcgcagag aagtatgaca agatcatgct ggatggcaga 180gccatgacag acagtgacta cagagtgttt gagtttgaga ttaaagtaaa aggacaggac 240atgctctcag acgctgcgct catggtgctc caccgtggga accgcgtgag agatatcacg 300aaacactttc gtgatacagc aagaatgaag aaaggcaccc ccgtcgtcgg tgtggtcaac 360aacgccgacg ttgggagact gattttctct ggtgaggccc tcacctacaa ggatattgta 420gtgtgcatgg acggagacac catgcctggc ctctttgcct acaaagccgc caccaaggca 480ggctactgtg gaggagccgt tctcgccaag gacggggccg acactttcat cgtcggcact 540cactccgcag gaggcaatgg agttggatac tgctcatgcg tttccaggtc catgcttctc 600agaatgaagg cacacgttga ccctgaacca caacacgag 63915912DNAArtificial SequenceFMDV - VP0 DNA optimized for Lemna expression 15atgggcgccg ggcagtcctc gcctgcgacg gggagccaga accagtcggg caacaccggc 60tcgatcatca acaactacta catgcagcaa taccagaaca gcatggacac ccagctcggc 120gataacgcca tctccggcgg gtccaacgag ggctcgaccg acacgacctc cacccacacc 180acgaacaccc agaacaatga ctggttcagc aagctggcct ccagcgcctt cacgggcctg 240ttcggggcgc tgctcgcgga caagaagacc gaggagacca cgctgctcga ggaccggatc 300ctcaccacgc gcaacgggca cacgaccagc acgacccagt ccagcgtcgg ggtgacccac 360ggctactcca cggaggagga ccacgtcgcc gggccgaaca cgagcggcct cgagacccgc 420gtggtccagg cggagcgctt ctacaagaag tacctgttcg actggaccac ggacaaggcg 480ttcgggcacc tcgagaagct ggagctgccg agcgaccacc acggcgtctt cggccacctc 540gtggactcct acgcgtacat gagaaatggg tgggacgtcg aggtcagcgc cgtcggcaac 600cagttcaacg gcgggtgcct gctcgtggcg atggtcccgg agtggaagga gttcgacacc 660cgcgagaagt atcagctgac cctcttcccc caccagttca tcagcccgcg caccaacatg 720accgcccaca tcacggtgcc gtacctcggg gtgaaccgct acgaccagta caagaagcac 780aagccctgga ccctcgtggt catggtggtc tccccgctga ctgtgaacaa cacgtccgcc 840gcgcagatca aagtctacgc caacatcgcc cccacctacg tgcacgtcgc cggcgagctg 900ccctccaagg ag 91216912DNAArtificial SequenceFMDV-VP0 DNA optimized for mammalian expression 16atgggagctg ggcaatccag cccagcaacc ggctcgcaga accagtctgg caacactggc 60agcataatca acaactacta catgcaacag taccagaact ccatggacac acagttggga 120gacaatgcca tcagtggagg ctccaacgag ggctccacgg acacaacttc aacacacaca 180accaacactc aaaacaatga ctggttctcg aagctcgcca gttcagcttt taccggtctg 240ttcggtgcac tgctcgccga caagaagaca gaggaaacga cacttcttga ggaccgcatc 300ctcaccaccc gcaacgggca caccacctcg acgacccaat cgagtgtggg tgtcacacac 360gggtactcca cagaggagga ccacgttgct gggcccaaca catcgggcct ggagacgcga 420gtggtgcagg cagagagatt ctacaaaaag tacttgtttg actggacaac ggacaaggca 480tttggacacc tggaaaagct ggagctcccg tccgaccacc acggtgtctt tggacacttg 540gtggactcgt acgcctatat gagaaatggc tgggatgttg aggtgtccgc tgttggcaac 600cagttcaacg gcgggtgcct cctggtggcc atggtacctg aatggaagga atttgacaca 660cgggagaaat accaactcac ccttttcccg caccagttta ttagccccag aactaacatg 720actgcccaca tcacggtccc ctaccttggt gtgaacaggt atgatcagta caagaagcat 780aagccctgga cattggttgt catggtcgtg tcgccactta cggtcaacaa cactagtgcg 840gcacaaatca aggtctacgc caacatagct ccgacctatg ttcacgtggc cggtgaactc 900ccctcgaaag ag 91217304PRTArtificial SequenceFMDV - VP0 protein (translation of SEQ ID NOs15 and 16) 17Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser1 5 10 15Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu65 70 75 80Phe Gly Ala Leu Leu

Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90 95Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr 100 105 110Gln Ser Ser Val Gly Val Thr His Gly Tyr Ser Thr Glu Glu Asp His 115 120 125Val Ala Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala 130 135 140Glu Arg Phe Tyr Lys Lys Tyr Leu Phe Asp Trp Thr Thr Asp Lys Ala145 150 155 160Phe Gly His Leu Glu Lys Leu Glu Leu Pro Ser Asp His His Gly Val 165 170 175Phe Gly His Leu Val Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190Val Glu Val Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205Val Ala Met Val Pro Glu Trp Lys Glu Phe Asp Thr Arg Glu Lys Tyr 210 215 220Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn Met225 230 235 240Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln 245 250 255Tyr Lys Lys His Lys Pro Trp Thr Leu Val Val Met Val Val Ser Pro 260 265 270Leu Thr Val Asn Asn Thr Ser Ala Ala Gln Ile Lys Val Tyr Ala Asn 275 280 285Ile Ala Pro Thr Tyr Val His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 30018663DNAArtificial SequenceFMDV - VP3 DNA optimized for Lemna expression 18ggcatcttcc ccgtcgcctg cgccgacggg tacgggggcc tggtgaccac cgaccccaag 60acggccgacc cggcctacgg caaagtgtac aacccgccca ggacgaacta ccccggtcgc 120ttcaccaacc tcctggacgt ggcggaggcc tgcccgacct tcctgtgctt cgacgacggg 180aagccctacg tcaccacgcg caccgacgac acgcgcctgc tcgccaagtt cgacctcagc 240ctggccgcta agcacatgag caacacctac ctcagcggca tcgcccaata ctatacccaa 300tactcgggca ccatcaacct gcacttcatg ttcacgggca gcaccgacag caaggcccgg 360tacatggtgg cctacatccc gccgggcgtg gagacccctc ccgacacgcc cgagcgggct 420gcgcactgca tccacgccga gtgggacacc ggcctcaaca gcaagttcac gttcagcatc 480ccctacgtgt ccgccgcgga ttacgcttac accgcctcgg acacggccga gacgatcaac 540gtccagggct gggtctgcat ctaccagatc actcacggca aggccgagaa cgacaccctc 600gtcgtgagcg tctccgccgg gaaggacttc gagctgaggc tgcccatcga ccccaggcag 660cag 66319663DNAArtificial SequenceFMDV-VP3 DNA optimized for mammalian expression 19gggattttcc cggttgcatg tgcggacggt tacggaggat tggtgacgac agacccgaag 60acagctgacc ctgcttatgg caaggtgtac aacccgccta ggactaacta ccctgggcgc 120ttcaccaacc tgttggacgt ggccgaagcg tgtcccactt tcctctgctt tgacgacggg 180aaaccgtacg tcaccacgcg gacggatgac acccgacttt tggccaagtt tgacctttcc 240cttgccgcaa aacatatgtc caacacatac ctgtcaggga ttgctcagta ctacacacag 300tactctggca ccatcaattt gcatttcatg tttacaggtt ccactgattc aaaggcccga 360tacatggtgg cctacatccc acctggggtg gagacaccac cggacacacc tgaaagggct 420gcccactgca ttcacgctga atgggacact ggactaaact ccaaattcac tttctcaatc 480ccgtacgtat ccgccgcgga ttacgcgtac acagcgtctg acacggcaga aacaatcaac 540gtacagggat gggtctgcat ctaccaaatt acacacggga aggctgaaaa tgacaccttg 600gtcgtgtcgg ttagcgccgg caaagacttt gagttgcgcc tcccgattga cccccgccag 660cag 66320221PRTArtificial SequenceFMDV - VP3 protein (translation of SEQ ID NOs18 and 19) 20Gly Ile Phe Pro Val Ala Cys Ala Asp Gly Tyr Gly Gly Leu Val Thr1 5 10 15Thr Asp Pro Lys Thr Ala Asp Pro Ala Tyr Gly Lys Val Tyr Asn Pro 20 25 30Pro Arg Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala 35 40 45Glu Ala Cys Pro Thr Phe Leu Cys Phe Asp Asp Gly Lys Pro Tyr Val 50 55 60Thr Thr Arg Thr Asp Asp Thr Arg Leu Leu Ala Lys Phe Asp Leu Ser65 70 75 80Leu Ala Ala Lys His Met Ser Asn Thr Tyr Leu Ser Gly Ile Ala Gln 85 90 95Tyr Tyr Thr Gln Tyr Ser Gly Thr Ile Asn Leu His Phe Met Phe Thr 100 105 110Gly Ser Thr Asp Ser Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro 115 120 125Gly Val Glu Thr Pro Pro Asp Thr Pro Glu Arg Ala Ala His Cys Ile 130 135 140His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile145 150 155 160Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr Ala 165 170 175Glu Thr Ile Asn Val Gln Gly Trp Val Cys Ile Tyr Gln Ile Thr His 180 185 190Gly Lys Ala Glu Asn Asp Thr Leu Val Val Ser Val Ser Ala Gly Lys 195 200 205Asp Phe Glu Leu Arg Leu Pro Ile Asp Pro Arg Gln Gln 210 215 22021633DNAArtificial SequenceFMDV - VP1 DNA optimized for Lemna expression 21accacggcga ccggggagtc cgccgacccc gtgaccacga ccgtggagaa ctacggcggg 60gagactcaga tccagcggcg ccaccacacc gacatcggct tcatcatgga ccgcttcgtg 120aagatccagt ccctgtcgcc cacccacgtt atcgacctca tgcaggccca ccagcacggg 180ctcgtgggtg ccctcctgcg cgcggccacc tactacttca gcgacctcga gatcgttgtc 240cgccacgagg ggaacctcac ctgggtcccg aacggtgccc ccgagagcgc cctgctcaac 300acctccaacc ccacggcgta caacaaggcc cccttcacgc gcctcgccct gccttacacc 360gcgccccacc gcgtgctggc cacggtgtac aacgggacct ccaagtacgc cgtgggcggg 420agcggccgca gaggcgacat ggggagcctc gccgctaggg tcgtgaagca gctcccggcc 480tccttcaact acggcgccat caaggccgac gccatccacg aactcctggt ccgcatgaag 540cgcgccgaac tctactgccc ccggcccctc ctggccatcg aggtctcctc gcaggaccgc 600cacaagcaga agatcatcgc cccggccaag cag 63322633DNAArtificial SequenceFMDV-VP1 DNA optimized for mammalian expression 22accaccgcta ccggggaatc agcagacccg gtcaccacca ccgtggagaa ctacggcggt 60gagacacaaa tccagagacg tcaccacacg gacattggtt tcatcatgga cagatttgtg 120aagatccaaa gcttgagccc aacacatgtc attgacctca tgcaggctca ccaacacggt 180ctggtgggtg ccttgctgcg tgcagccacg tactactttt ctgacctgga aattgttgta 240cggcacgaag gcaatctgac ctgggtgccc aacggcgccc ctgaatcagc cctgttgaac 300accagcaacc ccactgccta caacaaggca ccattcacga gactcgctct cccctacact 360gcgccgcacc gtgtgctggc aacagtgtac aacgggacga gtaagtatgc tgtgggtggt 420tcaggcagaa gaggcgacat ggggtctctc gcggcgcgag tcgtgaaaca gcttcctgct 480tcatttaact acggtgcaat caaggccgac gccatccacg aacttctcgt gcgcatgaaa 540cgggccgagc tctactgccc cagaccgctg ttggcaatag aggtgtcttc gcaagacagg 600cacaagcaaa agatcattgc accagcaaag cag 63323211PRTArtificial SequenceFMDV - VP1 protein (translation of SEQ ID NOs21 and 22) 23Thr Thr Ala Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu1 5 10 15Asn Tyr Gly Gly Glu Thr Gln Ile Gln Arg Arg His His Thr Asp Ile 20 25 30Gly Phe Ile Met Asp Arg Phe Val Lys Ile Gln Ser Leu Ser Pro Thr 35 40 45His Val Ile Asp Leu Met Gln Ala His Gln His Gly Leu Val Gly Ala 50 55 60Leu Leu Arg Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val65 70 75 80Arg His Glu Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Ser 85 90 95Ala Leu Leu Asn Thr Ser Asn Pro Thr Ala Tyr Asn Lys Ala Pro Phe 100 105 110Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr 115 120 125Val Tyr Asn Gly Thr Ser Lys Tyr Ala Val Gly Gly Ser Gly Arg Arg 130 135 140Gly Asp Met Gly Ser Leu Ala Ala Arg Val Val Lys Gln Leu Pro Ala145 150 155 160Ser Phe Asn Tyr Gly Ala Ile Lys Ala Asp Ala Ile His Glu Leu Leu 165 170 175Val Arg Met Lys Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala 180 185 190Ile Glu Val Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro 195 200 205Ala Lys Gln 21024654DNAArtificial SequenceFMDV - VP2 DNA optimized for Lemna expression 24gacaagaaga ccgaggagac cacgctgctc gaggaccgga tcctcaccac gcgcaacggg 60cacacgacca gcacgaccca gtccagcgtc ggggtgaccc acggctactc cacggaggag 120gaccacgtcg ccgggccgaa cacgagcggc ctcgagaccc gcgtggtcca ggcggagcgc 180ttctacaaga agtacctgtt cgactggacc acggacaagg cgttcgggca cctcgagaag 240ctggagctgc cgagcgacca ccacggcgtc ttcggccacc tcgtggactc ctacgcgtac 300atgagaaatg ggtgggacgt cgaggtcagc gccgtcggca accagttcaa cggcgggtgc 360ctgctcgtgg cgatggtccc ggagtggaag gagttcgaca cccgcgagaa gtatcagctg 420accctcttcc cccaccagtt catcagcccg cgcaccaaca tgaccgccca catcacggtg 480ccgtacctcg gggtgaaccg ctacgaccag tacaagaagc acaagccctg gaccctcgtg 540gtcatggtgg tctccccgct gactgtgaac aacacgtccg ccgcgcagat caaagtctac 600gccaacatcg cccccaccta cgtgcacgtc gccggcgagc tgccctccaa ggag 65425654DNAArtificial SequenceFMDV-VP2 DNA optimized for mammalian expression 25gacaagaaga cagaggaaac gacacttctt gaggaccgca tcctcaccac ccgcaacggg 60cacaccacct cgacgaccca atcgagtgtg ggtgtcacac acgggtactc cacagaggag 120gaccacgttg ctgggcccaa cacatcgggc ctggagacgc gagtggtgca ggcagagaga 180ttctacaaaa agtacttgtt tgactggaca acggacaagg catttggaca cctggaaaag 240ctggagctcc cgtccgacca ccacggtgtc tttggacact tggtggactc gtacgcctat 300atgagaaatg gctgggatgt tgaggtgtcc gctgttggca accagttcaa cggcgggtgc 360ctcctggtgg ccatggtacc tgaatggaag gaatttgaca cacgggagaa ataccaactc 420acccttttcc cgcaccagtt tattagcccc agaactaaca tgactgccca catcacggtc 480ccctaccttg gtgtgaacag gtatgatcag tacaagaagc ataagccctg gacattggtt 540gtcatggtcg tgtcgccact tacggtcaac aacactagtg cggcacaaat caaggtctac 600gccaacatag ctccgaccta tgttcacgtg gccggtgaac tcccctcgaa agag 65426218PRTArtificial SequenceFMDV - VP2 protein (translation of SEQ ID NOs24 and 25) 26Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu Thr1 5 10 15Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly Val 20 25 30Thr His Gly Tyr Ser Thr Glu Glu Asp His Val Ala Gly Pro Asn Thr 35 40 45Ser Gly Leu Glu Thr Arg Val Val Gln Ala Glu Arg Phe Tyr Lys Lys 50 55 60Tyr Leu Phe Asp Trp Thr Thr Asp Lys Ala Phe Gly His Leu Glu Lys65 70 75 80Leu Glu Leu Pro Ser Asp His His Gly Val Phe Gly His Leu Val Asp 85 90 95Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Val Glu Val Ser Ala Val 100 105 110Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Met Val Pro Glu 115 120 125Trp Lys Glu Phe Asp Thr Arg Glu Lys Tyr Gln Leu Thr Leu Phe Pro 130 135 140His Gln Phe Ile Ser Pro Arg Thr Asn Met Thr Ala His Ile Thr Val145 150 155 160Pro Tyr Leu Gly Val Asn Arg Tyr Asp Gln Tyr Lys Lys His Lys Pro 165 170 175Trp Thr Leu Val Val Met Val Val Ser Pro Leu Thr Val Asn Asn Thr 180 185 190Ser Ala Ala Gln Ile Lys Val Tyr Ala Asn Ile Ala Pro Thr Tyr Val 195 200 205His Val Ala Gly Glu Leu Pro Ser Lys Glu 210 21527258DNAArtificial SequenceFMDV - VP4 DNA optimized for Lemna expression 27atgggcgccg ggcagtcctc gcctgcgacg gggagccaga accagtcggg caacaccggc 60tcgatcatca acaactacta catgcagcaa taccagaaca gcatggacac ccagctcggc 120gataacgcca tctccggcgg gtccaacgag ggctcgaccg acacgacctc cacccacacc 180acgaacaccc agaacaatga ctggttcagc aagctggcct ccagcgcctt cacgggcctg 240ttcggggcgc tgctcgcg 25828258DNAArtificial SequenceFMDV-VP4 DNA optimized for mammalian expression 28atgggagctg ggcaatccag cccagcaacc ggctcgcaga accagtctgg caacactggc 60agcataatca acaactacta catgcaacag taccagaact ccatggacac acagttggga 120gacaatgcca tcagtggagg ctccaacgag ggctccacgg acacaacttc aacacacaca 180accaacactc aaaacaatga ctggttctcg aagctcgcca gttcagcttt taccggtctg 240ttcggtgcac tgctcgcc 2582986PRTArtificial SequenceFMDV-VP4 protein (translation of SEQ ID NOs27 and 28) 29Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser1 5 10 15Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser 35 40 45Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu65 70 75 80Phe Gly Ala Leu Leu Ala 853016478DNAArtificial SequenceMERF01 plasmid 30gtaccgggcc cccccctcga cggatcaagt gcaaaggtcc gccttgtttc tcctctgtct 60cttgatctga ctaatcttgg tttatgattc gttgagtaat tttggggaaa gctagcttcg 120tccacagttt ttttttcgat gaacagtgcc gcagtggcgc tgatcttgta tgctatcctg 180caatcgtggt gaacttattt cttttatatc cttcactccc atgaaaaggc tagtaatctt 240tctcgatgta acatcgtcca gcactgctat taccgtgtgg tccatccgac agtctggctg 300aacacatcat acgatattga gcaaagatcg atctatcttc cctgttcttt aatgaaagac 360gtcattttca tcagtatgat ctaagaatgt tgcaacttgc aaggaggcgt ttctttcttt 420gaatttaact aactcgttga gtggccctgt ttctcggacg taaggccttt gctgctccac 480acatgtccat tcgaatttta ccgtgtttag caagggcgaa aagtttgcat cttgatgatt 540tagcttgact atgcgattgc tttcctggac ccgtgcagct gcggacggat cccccgctcg 600aggtcgacgg tatcgataag cttgatcaga tctgatcgaa ttcaccatgg gggccgggca 660gtcgtccccg gccacggggt cccagaacca gtccggcaac accggcagca tcatcaacaa 720ctactacatg cagcagtacc agaactccat ggacacccaa ctgggggaca acgccacgag 780cggcggcagc aatgagggct ccaccgacac cacgagcacc cacacgacca acacccagaa 840caacgactgg ttcagcaagc tcgcgtccag cgccttctcc gggctcttcg gggcgctcct 900ggccgacaag aagaccgagg agaccacgct gctcgaggac cgcatcctca ccacgcggaa 960cggccacacg acctccacca cgcagtcgag cgtcggggtc acttacggtt acgccacggc 1020cgaggacttc gtgagcggcc ccaacacctc cggcctcgag actcgggtcg cccaggccga 1080gcgcttcttc aagacgcacc tgttcgactg ggtgacctcc gaccccttcg gccgctgcca 1140cctcttggaa ctgcccaccg accacaaggg cgtgtacggg tcgttgacgg acagctacgc 1200gtacatgagg aacgggtggg acgtggaggt caccgccgtc ggcaaccagt tcaacggggg 1260ctgcctcctg gtggccatgg tccccgaact ctgctcgatc cagaagcggg agctgtacca 1320gctgaccctc ttcccgcacc agttcatcaa cccgcgcacc aacatgaccg cgcacatcac 1380cgtccccttc gtgggcgtga accgctacga ccagtacaaa gtccacaagc cctggacctt 1440ggtggtcatg gtcgtcgccc ccctcaccgt caactcggag ggggcgcccc agatcaaagt 1500ctacgccaac atcgccccca cgaacgtgca cgttgccggc gagttcccga gcaaggaggg 1560catcttcccg gttgcgtgct ccgacggcta cgggggcctc gtgacgaccg acccgaagac 1620cgccgaccct gcctacggca aggtgttcaa cccgcccaga aacatgctcc ctgggagatt 1680cacgaacttc ctcgacgtgg ccgaggcctg ccccacgttc ctgcacttcg agggggacgt 1740cccttacgtc accacgaaga ctgactcgga tcgcgtgctg gcccagttcg acctgtccct 1800cgccgctaag cacatgtcca acaccttcct cgccggcctc gcgcaatact acacccagta 1860cagcggcacc atcaacctcc acttcatgtt caccggcccc accgacgcca aggcccgcta 1920catgatcgct tacgccccgc ccggcatgga gcccccgaag acccccgagg cggccgcgca 1980ctgcatccac gccgagtggg acacggggct caactccaag ttcacgttct ccatccccta 2040cctcagcgcc gcggactacg cgtacaccgc cagcgacacc gcggagacca cgaacgtgca 2100gggctgggtg tgcctgttcc agatcaccca cgggaaggcc gacggcgacg ccctggtcgt 2160gctggcctcg gcggggaagg acttcgaact caggctgccg gtggacgccc gcacccagac 2220gactagcgcc ggcgagtccg cggacccggt gaccgcgacc gtcgagaact acgggggcga 2280gacccaagtg cagcgcagac agcacacgga cgtgtccttc atcctggatc gcttcgtgaa 2340agtgaccccg aaggaccaga tcaacgtgct ggacctgatg cagacgccgg cgcacaccct 2400cgtgggggcc ctcctgcgca ccgccacgta ctacttcgcg gacctcgagg tggcggtgaa 2460gcacgagggc aacctgacgt gggtgcccaa cggtgcgccg gaggccgcgc tcgacaacac 2520cacgaacccc accgcgtacc acaaggcccc cctgacccgg ctcgccctcc cctacaccgc 2580cccccacagg gtcctcgcca cggtctacaa cggcaacagc aagtacgggg acgggaccgt 2640cgccaacgtg cgcggggacc tccaagtgct cgcccagaag gccgcgcgcg ccctccccac 2700tagcttcaac tacggtgcca tcaaggccac gagggtgacg gagctgctct accgcatgaa 2760gcgcgccgag acctactgcc cccgccccct gctcgcgatc caccccgacc aggccagaca 2820caagcagaag atcgtcgccc ctgtcaagca gctcctgaac ttcgacctcc tgaagctggc 2880cggcgacgtg gagagcaatc cgggcccctt cttcttcgcg gacgtgcgga gcaacttctc 2940caagctggtg gacaccatca accagatgca ggaggacatg agcacgaagc acgggccgga 3000cttcaaccgg ctggtcagcg ccttcgagga gctggccacc ggggtcaagg ccatccgcac 3060cggtctcgac gaggccaagc cctggtacaa gctcatcaag ctgctctcgc ggctgtcctg 3120catggcggcc gtcgccgctc gctcgaagga ccctgtgctc gtcgccatca tgctggccga 3180caccggcctg gagatcctgg actccacctt cgtggtcaag aagatctcgg actcgctgtc 3240cagcctcttc cacgttccgg ctccggtgtt ctccttcggt gccccgatcc tcctcgccgg 3300cctggtcaag gtggcgtcca gcttcttccg ctccaccccg gaggacctcg agcgcgcgga 3360gaagcagtcc ggtgcgcctc ccaccgacct gcagaagatg gtgatgggca acaccaagcc 3420cgtcgagttg atcctcgacg ggaagaccgt ggcgatctgc tgcgccaccg gcgtgttcgg 3480caccgcctac ctcgtcccga gacacctgtt cgccgagaag tatgacaaga tcatgctgga 3540cgggcgggcc atgaccgact cggactaccg ggtcttcgag ttcgagatca aagtgaaggg 3600ccaggatatg ctctccgacg ccgcgctgat ggtgctccac agaggcaacc gcgtgcggga 3660catcaccaag cacttcaggg acaccgcgcg catgaagaag gggacccctg tggtcggggt

3720cgtgaacaac gccgacgtcg ggcgcctcat cttctccggc gaggcgctga cctacaagga 3780catcgtcgtg tgcatggacg gggacacgat gccggggctc ttcgcctaca aggccgcgac 3840caaggccggg tactgcgggg gtgcggtgct cgccaaggac ggcgccgaca cgttcatcgt 3900gggcacccac tccgccggcg gtaacggcgt cggctactgc tcctgcgtgt cccggtccat 3960gctgctgcgg atgaaggccc acgttgaccc cgagccgcag cacgagtaag agctcgaatt 4020tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 4080ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 4140aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 4200aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 4260catctatgtt actagatcgg gaattaattc agatcggctg agtggctcct tcaacgttgc 4320ggttctgtca gttccaaacg taaaacggct tgtcccgcgt catcggcggg ggtcataacg 4380tgactccctt aattctccgc tcatgatcag attgtcgttt cccgccttca gtttaaacta 4440tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat 4500aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc 4560caaccacagg gttccccaga tctggcgccg gccagcgaga cgagcaagat tggccgccgc 4620ccgaaacgat ccgacagcgc gcccagcaca ggtgcgcagg caaattgcac caacgcatac 4680agcgccagca gaatgccata gtgggcggtg acgtcgttcg agtgaaccag atcgcgcagg 4740aggcccggca gcaccggcat aatcaggccg atgccgacag cgtcgagcgc gacagtgctc 4800agaattacga tcaggggtat gttgggtttc acgtctggcc tccggaccag cctccgctgg 4860tccgattgaa cgcgcggatt ctttatcact gataagttgg tggacatatt atgtttatca 4920gtgataaagt gtcaagcatg acaaagttgc agccgaatac agtgatccgt gccgccctgg 4980acctgttgaa cgaggtcggc gtagacggtc tgacgacacg caaactggcg gaacggttgg 5040gggttcagca gccggcgctt tactggcact tcaggaacaa gcgggcgctg ctcgacgcac 5100tggccgaagc catgctggcg gagaatcata cgcattcggt gccgagagcc gacgacgact 5160ggcgctcatt tctgatcggg aatgcccgca gcttcaggca ggcgctgctc gcctaccgcg 5220atggcgcgcg catccatgcc ggcacgcgac cgggcgcacc gcagatggaa acggccgacg 5280cgcagcttcg cttcctctgc gaggcgggtt tttcggccgg ggacgccgtc aatgcgctga 5340tgacaatcag ctacttcact gttggggccg tgcttgagga gcaggccggc gacagcgatg 5400ccggcgagcg cggcggcacc gttgaacagg ctccgctctc gccgctgttg cgggccgcga 5460tagacgcctt cgacgaagcc ggtccggacg cagcgttcga gcagggactc gcggtgattg 5520tcgatggatt ggcgaaaagg aggctcgttg tcaggaacgt tgaaggaccg agaaagggtg 5580acgattgatc aggaccgctg ccggagcgca acccactcac tacagcagag ccatgtagac 5640aacatcccct ccccctttcc accgcgtcag acgcccgtag cagcccgcta cgggcttttt 5700catgccctgc cctagcgtcc aagcctcacg gccgcgctcg gcctctctgg cggccttctg 5760gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 5820ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 5880aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5940ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6000gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6060cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6120gggaagcgtg gcgcttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt 6180cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact 6240ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga 6300gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg 6360ctctgcgagg ctggccggct accgccggcg taacagatga gggcaagcgg atggctgatg 6420aaaccaagcc aaccaggaag ggcagcccac ctatcaaggt gtactgcctt ccagacgaac 6480gaagagcgat tgaggaaaag gcggcggcgg ccggcatgag cctgtcggcc tacctgctgg 6540ccgtcggcca gggctacaaa atcacgggcg tcgtggacta tgagcacgtc cgcgagctgg 6600cccgcatcaa tggcgacctg ggccgcctgg gcggcctgct gaaactctgg ctcaccgacg 6660acccgcgcac ggcgcggttc ggtgatgcca cgatcctcgc cctgctggcg aagatcgaag 6720agaagcagga cgagcttggc aaggtcatga tgggcgtggt ccgcccgagg gcagagccat 6780gactttttta gccgctaaaa cggccggggg gtgcgcgtga ttgccaagca cgtccccatg 6840cgctccatca agaagagcga cttcgcggag ctggtgaagt acatcaccga cgagcaaggc 6900aagaccgagc gcctttgcga cgctcaccgg gctggttgcc ctcgccgctg ggctggcggc 6960cgtctatggc cctgcaaacg cgccagaaac gccgtcgaag ccgtgtgcga gacaccgcgg 7020ccgccggcgt tgtggatacc tcgcggaaaa cttggccctc actgacagat gaggggcgga 7080cgttgacact tgaggggccg actcacccgg cgcggcgttg acagatgagg ggcaggctcg 7140atttcggccg gcgacgtgga gctggccagc ctcgcaaatc ggcgaaaacg cctgatttta 7200cgcgagtttc ccacagatga tgtggacaag cctggggata agtgccctgc ggtattgaca 7260cttgaggggc gcgactactg acagatgagg ggcgcgatcc ttgacacttg aggggcagag 7320tgctgacaga tgaggggcgc acctattgac atttgagggg ctgtccacag gcagaaaatc 7380cagcatttgc aagggtttcc gcccgttttt cggccaccgc taacctgtct tttaacctgc 7440ttttaaacca atatttataa accttgtttt taaccagggc tgcgccctgt gcgcgtgacc 7500gcgcacgccg aaggggggtg cccccccttc tcgaaccctc ccggcccgct aacgcgggcc 7560tcccatcccc ccaggggctg cgcccctcgg ccgcgaacgg cctcacccca aaaatggcag 7620cgctggcagt ccttgccatt gccgggatcg gggcagtaac gggatgggcg atcagcccga 7680gcgcgacgcc cggaagcatt gacgtgccgc aggtgctggc atcgacattc agcgaccagg 7740tgccgggcag tgagggcggc ggcctgggtg gcggcctgcc cttcacttcg gccgtcgggg 7800cattcacgga cttcatggcg gggccggcaa tttttacctt gggcattctt ggcatagtgg 7860tcgcgggtgc cgtgctcgtg ttcgggggtg cgataaaccc agcgaaccat ttgaggtgat 7920aggtaagatt ataccgaggt atgaaaacga gaattggacc tttacagaat tactctatga 7980agcgccatat ttaaaaagct accaagacga agaggatgaa gaggatgagg aggcagattg 8040ccttgaatat attgacaata ctgataagat aatatatctt ttatatagaa gatatcgccg 8100tatgtaagga tttcaggggg caaggcatag gcagcgcgct tatcaatata tctatagaat 8160gggcaaagca taaaaacttg catggactaa tgcttgaaac ccaggacaat aaccttatag 8220cttgtaaatt ctatcataat tgggtaatga ctccaactta ttgatagtgt tttatgttca 8280gataatgccc gatgactttg tcatgcagct ccaccgattt tgagaacgac agcgacttcc 8340gtcccagccg tgccaggtgc tgcctcagat tcaggttatg ccgctcaatt cgctgcgtat 8400atcgcttgct gattacgtgc agctttccct tcaggcggga ttcatacagc ggccagccat 8460ccgtcatcca tatcaccacg tcaaagggtg acagcaggct cataagacgc cccagcgtcg 8520ccatagtgcg ttcaccgaat acgtgcgcaa caaccgtctt ccggagactg tcatacgcgt 8580aaaacagcca gcgctggcgc gatttagccc cgacatagcc ccactgttcg tccatttccg 8640cgcagacgat gacgtcactg cccggctgta tgcgcgaggt taccgactgc ggcctgagtt 8700ttttaagtga cgtaaaatcg tgttgaggcc aacgcccata atgcgggctg ttgcccggca 8760tccaacgcca ttcatggcca tatcaatgat tttctggtgc gtaccgggtt gagaagcggt 8820gtaagtgaac tgcagttgcc atgttttacg gcagtgagag cagagatagc gctgatgtcc 8880ggcggtgctt ttgccgttac gcaccacccc gtcagtagct gaacaggagg gacagctgat 8940agacacagaa gccactggag cacctcaaaa acaccatcat acactaaatc agtaagttgg 9000cagcatcacc cataattgtg gtttcaaaat cggctccgtc gatactatgt tatacgccaa 9060ctttgaaaac aactttgaaa aagctgtttt ctggtattta aggttttaga atgcaaggaa 9120cagtgaattg gagttcgtct tgttataatt agcttcttgg ggtatcttta aatactgtag 9180aaaagaggaa ggaaataata aatggctaaa atgagaatat caccggaatt gaaaaaactg 9240atcgaaaaat accgctgcgt aaaagatacg gaaggaatgt ctcctgctaa ggtatataag 9300ctggtgggag aaaatgaaaa cctatattta aaaatgacgg acagccggta taaagggacc 9360acctatgatg tggaacggga aaaggacatg atgctatggc tggaaggaaa gctgcctgtt 9420ccaaaggtcc tgcactttga acggcatgat ggctggagca atctgctcat gagtgaggcc 9480gatggcgtcc tttgctcgga agagtatgaa gatgaacaaa gccctgaaaa gattatcgag 9540ctgtatgcgg agtgcatcag gctctttcac tccatcgaca tatcggattg tccctatacg 9600aatagcttag acagccgctt agccgaattg gattacttac tgaataacga tctggccgat 9660gtggattgcg aaaactggga agaagacact ccatttaaag atccgcgcga gctgtatgat 9720tttttaaaga cggaaaagcc cgaagaggaa cttgtctttt cccacggcga cctgggagac 9780agcaacatct ttgtgaaaga tggcaaagta agtggcttta ttgatcttgg gagaagcggc 9840agggcggaca agtggtatga cattgccttc tgcgtccggt cgatcaggga ggatatcggg 9900gaagaacagt atgtcgagct attttttgac ttactgggga tcaagcctga ttgggagaaa 9960ataaaatatt atattttact ggatgaattg ttttagtacc tagatgtggc gcaacgatgc 10020cggcgacaag caggagcgca ccgacttctt ccgcatcaag tgttttggct ctcaggccga 10080ggcccacggc aagtatttgg gcaaggggtc gctggtattc gtgcagggca agattcggaa 10140taccaagtac gagaaggacg gccagacggt ctacgggacc gacttcattg ccgataaggt 10200ggattatctg gacaccaagg caccaggcgg gtcaaatcag gaataagggc acattgcccc 10260ggcgtgagtc ggggcaatcc cgcaaggagg gtgaatgaat cggacgtttg accggaaggc 10320atacaggcaa gaactgatcg acgcggggtt ttccgccgag gatgccgaaa ccatcgcaag 10380ccgcaccgtc atgcgtgcgc cccgcgaaac cttccagtcc gtcggctcga tggtccagca 10440agctacggcc aagatcgagc gcgacagcgt gcaactggct ccccctgccc tgcccgcgcc 10500atcggccgcc gtggagcgtt cgcgtcgtct cgaacaggag gcggcaggtt tggcgaagtc 10560gatgaccatc gacacgcgag gaactatgac gaccaagaag cgaaaaaccg ccggcgagga 10620cctggcaaaa caggtcagcg aggccaagca ggccgcgttg ctgaaacaca cgaagcagca 10680gatcaaggaa atgcagcttt ccttgttcga tattgcgccg tggccggaca cgatgcgagc 10740gatgccaaac gacacggccc gctctgccct gttcaccacg cgcaacaaga aaatcccgcg 10800cgaggcgctg caaaacaagg tcattttcca cgtcaacaag gacgtgaaga tcacctacac 10860cggcgtcgag ctgcgggccg acgatgacga actggtgtgg cagcaggtgt tggagtacgc 10920gaagcgcacc cctatcggcg agccgatcac cttcacgttc tacgagcttt gccaggacct 10980gggctggtcg atcaatggcc ggtattacac gaaggccgag gaatgcctgt cgcgcctaca 11040ggcgacggcg atgggcttca cgtccgaccg cgttgggcac ctggaatcgg tgtcgctgct 11100gcaccgcttc cgcgtcctgg accgtggcaa gaaaacgtcc cgttgccagg tcctgatcga 11160cgaggaaatc gtcgtgctgt ttgctggcga ccactacacg aaattcatat gggagaagta 11220ccgcaagctg tcgccgacgg cccgacggat gttcgactat ttcagctcgc accgggagcc 11280gtacccgctc aagctggaaa ccttccgcct catgtgcgga tcggattcca cccgcgtgaa 11340gaagtggcgc gagcaggtcg gcgaagcctg cgaagagttg cgaggcagcg gcctggtgga 11400acacgcctgg gtcaatgatg acctggtgca ttgcaaacgc tagggccttg tggggtcagt 11460tccggctggg ggttcagcag ccagcgcttt actggcattt caggaacaag cgggcactgc 11520tcgacgcact tgcttcgctc agtatcgctc gggacgcacg gcgcgctcta cgaactgccg 11580ataaacagag gattaaaatt gacaattgtg attaaggctc agattcgacg gcttggagcg 11640gccgacgtgc aggatttccg cgagatccga ttgtcggccc tgaagaaagc tccagagatg 11700ttcgggtccg tttacgagca cgaggagaaa aagcccatgg aggcgttcgc tgaacggttg 11760cgagatgccg tggcattcgg cgcctacatc gacggcgaga tcattgggct gtcggtcttc 11820aaacaggagg acggccccaa ggacgctcac aaggcgcatc tgtccggcgt tttcgtggag 11880cccgaacagc gaggccgagg ggtcgccggt atgctgctgc gggcgttgcc ggcgggttta 11940ttgctcgtga tgatcgtccg acagattcca acgggaatct ggtggatgcg catcttcatc 12000ctcggcgcac ttaatatttc gctattctgg agcttgttgt ttatttcggt ctaccgcctg 12060ccgggcgggg tcgcggcgac ggtaggcgct gtgcagccgc tgatggtcgt gttcatctct 12120gccgctctgc taggtagccc gatacgattg atggcggtcc tgggggctat ttgcggaact 12180gcgggcgtgg cgctgttggt gttgacacca aacgcagcgc tagatcctgt cggcgtcgca 12240gcgggcctgg cgggggcggt ttccatggcg ttcggaaccg tgctgacccg caagtggcaa 12300cctcccgtgc ctctgctcac ctttaccgcc tggcaactgg cggccggagg acttctgctc 12360gttccagtag ctttagtgtt tgatccgcca atcccgatgc ctacaggaac caatgttctc 12420ggcctggcgt ggctcggcct gatcggagcg ggtttaacct acttcctttg gttccggggg 12480atctcgcgac tcgaacctac agttgtttcc ttactgggct ttctcagccc cagatctggg 12540gtcgatcagc cggggatgca tcaggccgac agtcggaact tcgggtcccc gacctgtacc 12600attcggtgag caatggatag gggagttgat atcgtcaacg ttcacttcta aagaaatagc 12660gccactcagc ttcctcagcg gctttatcca gcgatttcct attatgtcgg catagttctc 12720aagatcgaca gcctgtcacg gttaagcgag aaatgaataa gaaggctgat aattcggatc 12780tctgcgaggg agatgatatt tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac 12840atgctaccct ccgcgagatc atccgtgttt caaacccggc agcttagttg ccgttcttcc 12900gaatagcatc ggtaacatga gcaaagtctg ccgccttaca acggctctcc cgctgacgcc 12960gtcccggact gatgggctgc ctgtatcgag tggtgatttt gtgccgagct gccggtcggg 13020gagctgttgg ctggctggtg gcaggatata ttgtggtgta aacaaattga cgcttagaca 13080acttaataac acattgcgga cgtttttaat gtactggggt ggtttttctt ttcaccagtg 13140agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 13200ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttccg aaatcggcaa 13260aatcccttat aaatcaaaag aatagcccga gatagggttg agtgttgttc cagtttggaa 13320caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 13380gggcgatggc ccactacgtg aaccatcacc caaatcaagt tttttggggt cgaggtgccg 13440taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 13500ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcca ttcaggctgc 13560gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag 13620ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt 13680gtaaaacgac ggccagtgaa ttgccatctt gaaagaaata tagtttaaat atttattgat 13740aaaataagtc aggtattata gtccaagcaa aaacataatt tattgatgca aagtttaaat 13800tcagaaatat ttcaataact gattatatca gctggtacat tgccgtagat gaaagactga 13860gtgcgatatt atgtgtaata cataaattga tgatatagct agcttagctc atcgggggat 13920ccttaatcga ctctagctag aacgaattgt taggtggcgg tacttgggtc gatatcaaag 13980tgcatcactt cttcccgtat gcccaacttt gtatagagag ccactgcggg atcgtcaccg 14040taatctgctt gcacgtagat cacataagca ccaagcgcgt tggcctcatg cttgaggaga 14100ttgatgagcg cggtggcaat gccctgcctc cggtgctcgc cggagactgc gagatcatag 14160atatagatct cactacgcgg ctgctcaaac ctgggcagaa cgtaagccgc gagagcgcca 14220acaaccgctt cttggtcgaa ggcagcaagc gcgatgaatg tcttactacg gagcaagttc 14280ccgaggtaat cggagtccgg ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga 14340ccgaaaagat caagagcagc ccgcatggat ttgacttggt cagggccgag cctacatgtg 14400cgaatgatgc ccatacttga gccacctaac tttgttttag ggcgactgcc ctgctgcgta 14460acatcgttgc tgctgcgtac catggagatc tggattgaga gtgaatatga gactctaatt 14520ggataccgag gggaatttat ggaagtcagt ggagcatttt tgacaagaaa tatttgctag 14580ctgatagtga ccttaggcga cttttgaacg cgcaataatg gtttctgacg tatgtgctta 14640gctcattaaa ctccagaaac ccgcggctga gtggctcctt caacgttgcg gttctgtcag 14700ttccaaacgt aaaacggctt gtcccgcgtc atcggcgggg gtcataacgt gactccctta 14760attctccgct catgatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat 14820ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg 14880ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc 14940tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt 15000catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca 15060gcccttgcgc cctgagtgct tgcggcagcg tgaagctctg gacatcatgt tggatatgaa 15120acaactatta tttatctaca tgttttagat gttatctgat tatttttata ccgtagtctt 15180ctattgatga ggagtctaag gctatagaat tatatatcta aatgattaat atatatatta 15240ttaataatta acaataatta atatattata atttatatat atatatttta tattattata 15300ataatattct tacaaatata attattatat tcgacggtat cgataagctc gggatccctg 15360aaagcgacgt tggatgttaa catctacaaa ttgccttttc ttatcgacca tgtacgtaag 15420cgcttacgtt tttggtggac ccttgaggaa actggtagct gttgtgggcc tgtggtctca 15480agatggatca ttaatttcca ccttcaccta cgatgggggg catcgcaccg gtgagtaata 15540ttgtacggct aagagcgaat ttggcctgta ggatccctga aagcgacgtt ggatgttaac 15600atctacaaat tgccttttct tatcgaccat gtacgtaagc gcttacgttt ttggtggacc 15660cttgaggaaa ctggtagctg ttgtgggcct gtggtctcaa gatggatcat taatttccac 15720cttcacctac gatggggggc atcgcaccgg tgagtaatat tgtacggcta agagcgaatt 15780tggcctgtag gatccctgaa agcgacgttg gatgttaaca tctacaaatt gccttttctt 15840atcgaccatg tacgtaagcg cttacgtttt tggtggaccc ttgaggaaac tggtagctgt 15900tgtgggcctg tggtctcaag atggatcatt aatttccacc ttcacctacg atggggggca 15960tcgcaccggt gagtaatatt gtacggctaa gagcgaattt ggcctgtagg atccgcgagc 16020tggtcaatcc cattgctttt gaagcagctc aacattgatc tctttctcga tcgagggaga 16080tttttcaaat cagtgcgcaa gacgtgacgt aagtatccga gtcagttttt atttttctac 16140taatttggtc gtttatttcg gcgtgtagga catggcaacc gggcctgaat ttcgcgggta 16200ttctgtttct attccaactt tttcttgatc cgcagccatt aacgactttt gaatagatac 16260gctgacacgc caagcctcgc tagtcaaaag tgtaccaaac aacgctttac agcaagaacg 16320gaatgcgcgt gacgctcgcg gtgacgccat ttcgcctttt cagaaatgga taaatagcct 16380tgcttcctat tatatcttcc cccaaattaa ttaagaaact cccgaggtga gcaaggatcc 16440ggagtcgagc gcgaagaaga gaaagaggga aagcgcgg 164783116821DNAArtificial SequenceMERF02 plasmid 31gtaccgggcc cccccctcga cggatcaagt gcaaaggtcc gccttgtttc tcctctgtct 60cttgatctga ctaatcttgg tttatgattc gttgagtaat tttggggaaa gctagcttcg 120tccacagttt ttttttcgat gaacagtgcc gcagtggcgc tgatcttgta tgctatcctg 180caatcgtggt gaacttattt cttttatatc cttcactccc atgaaaaggc tagtaatctt 240tctcgatgta acatcgtcca gcactgctat taccgtgtgg tccatccgac agtctggctg 300aacacatcat acgatattga gcaaagatcg atctatcttc cctgttcttt aatgaaagac 360gtcattttca tcagtatgat ctaagaatgt tgcaacttgc aaggaggcgt ttctttcttt 420gaatttaact aactcgttga gtggccctgt ttctcggacg taaggccttt gctgctccac 480acatgtccat tcgaatttta ccgtgtttag caagggcgaa aagtttgcat cttgatgatt 540tagcttgact atgcgattgc tttcctggac ccgtgcagct gcggacggat cccccgctcg 600aggtcgacgg tatcgataag cttgatcaga tctgatcgaa ttcaccatgg gggccgggca 660gtcgtccccg gccacggggt cccagaacca gtccggcaac accggcagca tcatcaacaa 720ctactacatg cagcagtacc agaactccat ggacacccaa ctgggggaca acgccacgag 780cggcggcagc aatgagggct ccaccgacac cacgagcacc cacacgacca acacccagaa 840caacgactgg ttcagcaagc tcgcgtccag cgccttctcc gggctcttcg gggcgctcct 900ggccgacaag aagaccgagg agaccacgct gctcgaggac cgcatcctca ccacgcggaa 960cggccacacg acctccacca cgcagtcgag cgtcggggtc acttacggtt acgccacggc 1020cgaggacttc gtgagcggcc ccaacacctc cggcctcgag actcgggtcg cccaggccga 1080gcgcttcttc aagacgcacc tgttcgactg ggtgacctcc gaccccttcg gccgctgcca 1140cctcttggaa ctgcccaccg accacaaggg cgtgtacggg tcgttgacgg acagctacgc 1200gtacatgagg aacgggtggg acgtggaggt caccgccgtc ggcaaccagt tcaacggggg 1260ctgcctcctg gtggccatgg tccccgaact ctgctcgatc cagaagcggg agctgtacca 1320gctgaccctc ttcccgcacc agttcatcaa cccgcgcacc aacatgaccg cgcacatcac 1380cgtccccttc gtgggcgtga accgctacga ccagtacaaa gtccacaagc cctggacctt 1440ggtggtcatg gtcgtcgccc ccctcaccgt caactcggag ggggcgcccc agatcaaagt 1500ctacgccaac atcgccccca cgaacgtgca cgttgccggc gagttcccga gcaaggaggg 1560catcttcccg gttgcgtgct ccgacggcta cgggggcctc gtgacgaccg acccgaagac 1620cgccgaccct gcctacggca aggtgttcaa cccgcccaga aacatgctcc ctgggagatt 1680cacgaacttc ctcgacgtgg ccgaggcctg ccccacgttc ctgcacttcg agggggacgt 1740cccttacgtc accacgaaga ctgactcgga tcgcgtgctg gcccagttcg acctgtccct 1800cgccgctaag cacatgtcca acaccttcct cgccggcctc gcgcaatact acacccagta 1860cagcggcacc atcaacctcc acttcatgtt caccggcccc accgacgcca aggcccgcta 1920catgatcgct tacgccccgc ccggcatgga gcccccgaag acccccgagg cggccgcgca 1980ctgcatccac gccgagtggg acacggggct caactccaag ttcacgttct ccatccccta 2040cctcagcgcc gcggactacg cgtacaccgc cagcgacacc gcggagacca cgaacgtgca 2100gggctgggtg tgcctgttcc agatcaccca cgggaaggcc gacggcgacg ccctggtcgt 2160gctggcctcg gcggggaagg acttcgaact caggctgccg gtggacgccc gcacccagac 2220gactagcgcc ggcgagtccg

cggacccggt gaccgcgacc gtcgagaact acgggggcga 2280gacccaagtg cagcgcagac agcacacgga cgtgtccttc atcctggatc gcttcgtgaa 2340agtgaccccg aaggaccaga tcaacgtgct ggacctgatg cagacgccgg cgcacaccct 2400cgtgggggcc ctcctgcgca ccgccacgta ctacttcgcg gacctcgagg tggcggtgaa 2460gcacgagggc aacctgacgt gggtgcccaa cggtgcgccg gaggccgcgc tcgacaacac 2520cacgaacccc accgcgtacc acaaggcccc cctgacccgg ctcgccctcc cctacaccgc 2580cccccacagg gtcctcgcca cggtctacaa cggcaacagc aagtacgggg acgggaccgt 2640cgccaacgtg cgcggggacc tccaagtgct cgcccagaag gccgcgcgcg ccctccccac 2700tagcttcaac tacggtgcca tcaaggccac gagggtgacg gagctgctct accgcatgaa 2760gcgcgccgag acctactgcc cccgccccct gctcgcgatc caccccgacc aggccagaca 2820caagcagaag atcgtcgccc ctgtcaagca gctcctgaac ttcgacctcc tgaagctggc 2880cggcgacgtg gagagcaatc cgggcccctt cttcttcgcg gacgtgcgga gcaacttctc 2940caagctggtg gacaccatca accagatgca ggaggacatg agcacgaagc acgggccgga 3000cttcaaccgg ctggtcagcg ccttcgagga gctggccacc ggggtcaagg ccatccgcac 3060cggtctcgac gaggccaagc cctggtacaa gctcatcaag ctgctctcgc ggctgtcctg 3120catggcggcc gtcgccgctc gctcgaagga ccctgtgctc gtcgccatca tgctggccga 3180caccggcctg gagatcctgg actccacctt cgtggtcaag aagatctcgg actcgctgtc 3240cagcctcttc cacgttccgg ctccggtgtt ctccttcggt gccccgatcc tcctcgccgg 3300cctggtcaag gtggcgtcca gcttcttccg ctccaccccg gaggacctcg agcgcgcgga 3360gaagcagtcc ggtgcgcctc ccaccgacct gcagaagatg gtgatgggca acaccaagcc 3420cgtcgagttg atcctcgacg ggaagaccgt ggcgatctgc tgcgccaccg gcgtgttcgg 3480caccgcctac ctcgtcccga gacacctgtt cgccgagaag tatgacaaga tcatgctgga 3540cgggcgggcc atgaccgact cggactaccg ggtcttcgag ttcgagatca aagtgaaggg 3600ccaggatatg ctctccgacg ccgcgctgat ggtgctccac agaggcaacc gcgtgcggga 3660catcaccaag cacttcaggg acaccgcgcg catgaagaag gggacccctg tggtcggggt 3720cgtgaacaac gccgacgtcg ggcgcctcat cttctccggc gaggcgctga cctacaagga 3780catcgtcgtg tgcatggacg gggacacgat gccggggctc ttcgcctaca aggccgcgac 3840caaggccggg tactgcgggg gtgcggtgct cgccaaggac ggcgccgaca cgttcatcgt 3900gggcacccac tccgccggcg gtaacggcgt cggctactgc tcctgcgtgt cccggtccat 3960gctgctgcgg atgaaggccc acgttgaccc cgagccgcag cacgagtaag agctcgaatt 4020tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 4080ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 4140aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 4200aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 4260catctatgtt actagatcgg gaattaattc agatcggctg agtggctcct tcaacgttgc 4320ggttctgtca gttccaaacg taaaacggct tgtcccgcgt catcggcggg ggtcataacg 4380tgactccctt aattctccgc tcatgatcag attgtcgttt cccgccttca gtttaaacta 4440tcagtgtttg acaggatata ttggcgggta aacctaagag aaaagagcgt ttattagaat 4500aatcggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc 4560caaccacagg gttccccaga tctggcgccg gccagcgaga cgagcaagat tggccgccgc 4620ccgaaacgat ccgacagcgc gcccagcaca ggtgcgcagg caaattgcac caacgcatac 4680agcgccagca gaatgccata gtgggcggtg acgtcgttcg agtgaaccag atcgcgcagg 4740aggcccggca gcaccggcat aatcaggccg atgccgacag cgtcgagcgc gacagtgctc 4800agaattacga tcaggggtat gttgggtttc acgtctggcc tccggaccag cctccgctgg 4860tccgattgaa cgcgcggatt ctttatcact gataagttgg tggacatatt atgtttatca 4920gtgataaagt gtcaagcatg acaaagttgc agccgaatac agtgatccgt gccgccctgg 4980acctgttgaa cgaggtcggc gtagacggtc tgacgacacg caaactggcg gaacggttgg 5040gggttcagca gccggcgctt tactggcact tcaggaacaa gcgggcgctg ctcgacgcac 5100tggccgaagc catgctggcg gagaatcata cgcattcggt gccgagagcc gacgacgact 5160ggcgctcatt tctgatcggg aatgcccgca gcttcaggca ggcgctgctc gcctaccgcg 5220atggcgcgcg catccatgcc ggcacgcgac cgggcgcacc gcagatggaa acggccgacg 5280cgcagcttcg cttcctctgc gaggcgggtt tttcggccgg ggacgccgtc aatgcgctga 5340tgacaatcag ctacttcact gttggggccg tgcttgagga gcaggccggc gacagcgatg 5400ccggcgagcg cggcggcacc gttgaacagg ctccgctctc gccgctgttg cgggccgcga 5460tagacgcctt cgacgaagcc ggtccggacg cagcgttcga gcagggactc gcggtgattg 5520tcgatggatt ggcgaaaagg aggctcgttg tcaggaacgt tgaaggaccg agaaagggtg 5580acgattgatc aggaccgctg ccggagcgca acccactcac tacagcagag ccatgtagac 5640aacatcccct ccccctttcc accgcgtcag acgcccgtag cagcccgcta cgggcttttt 5700catgccctgc cctagcgtcc aagcctcacg gccgcgctcg gcctctctgg cggccttctg 5760gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 5820ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 5880aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 5940ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6000gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6060cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6120gggaagcgtg gcgcttttcc gctgcataac cctgcttcgg ggtcattata gcgatttttt 6180cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact 6240ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga 6300gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg 6360ctctgcgagg ctggccggct accgccggcg taacagatga gggcaagcgg atggctgatg 6420aaaccaagcc aaccaggaag ggcagcccac ctatcaaggt gtactgcctt ccagacgaac 6480gaagagcgat tgaggaaaag gcggcggcgg ccggcatgag cctgtcggcc tacctgctgg 6540ccgtcggcca gggctacaaa atcacgggcg tcgtggacta tgagcacgtc cgcgagctgg 6600cccgcatcaa tggcgacctg ggccgcctgg gcggcctgct gaaactctgg ctcaccgacg 6660acccgcgcac ggcgcggttc ggtgatgcca cgatcctcgc cctgctggcg aagatcgaag 6720agaagcagga cgagcttggc aaggtcatga tgggcgtggt ccgcccgagg gcagagccat 6780gactttttta gccgctaaaa cggccggggg gtgcgcgtga ttgccaagca cgtccccatg 6840cgctccatca agaagagcga cttcgcggag ctggtgaagt acatcaccga cgagcaaggc 6900aagaccgagc gcctttgcga cgctcaccgg gctggttgcc ctcgccgctg ggctggcggc 6960cgtctatggc cctgcaaacg cgccagaaac gccgtcgaag ccgtgtgcga gacaccgcgg 7020ccgccggcgt tgtggatacc tcgcggaaaa cttggccctc actgacagat gaggggcgga 7080cgttgacact tgaggggccg actcacccgg cgcggcgttg acagatgagg ggcaggctcg 7140atttcggccg gcgacgtgga gctggccagc ctcgcaaatc ggcgaaaacg cctgatttta 7200cgcgagtttc ccacagatga tgtggacaag cctggggata agtgccctgc ggtattgaca 7260cttgaggggc gcgactactg acagatgagg ggcgcgatcc ttgacacttg aggggcagag 7320tgctgacaga tgaggggcgc acctattgac atttgagggg ctgtccacag gcagaaaatc 7380cagcatttgc aagggtttcc gcccgttttt cggccaccgc taacctgtct tttaacctgc 7440ttttaaacca atatttataa accttgtttt taaccagggc tgcgccctgt gcgcgtgacc 7500gcgcacgccg aaggggggtg cccccccttc tcgaaccctc ccggcccgct aacgcgggcc 7560tcccatcccc ccaggggctg cgcccctcgg ccgcgaacgg cctcacccca aaaatggcag 7620cgctggcagt ccttgccatt gccgggatcg gggcagtaac gggatgggcg atcagcccga 7680gcgcgacgcc cggaagcatt gacgtgccgc aggtgctggc atcgacattc agcgaccagg 7740tgccgggcag tgagggcggc ggcctgggtg gcggcctgcc cttcacttcg gccgtcgggg 7800cattcacgga cttcatggcg gggccggcaa tttttacctt gggcattctt ggcatagtgg 7860tcgcgggtgc cgtgctcgtg ttcgggggtg cgataaaccc agcgaaccat ttgaggtgat 7920aggtaagatt ataccgaggt atgaaaacga gaattggacc tttacagaat tactctatga 7980agcgccatat ttaaaaagct accaagacga agaggatgaa gaggatgagg aggcagattg 8040ccttgaatat attgacaata ctgataagat aatatatctt ttatatagaa gatatcgccg 8100tatgtaagga tttcaggggg caaggcatag gcagcgcgct tatcaatata tctatagaat 8160gggcaaagca taaaaacttg catggactaa tgcttgaaac ccaggacaat aaccttatag 8220cttgtaaatt ctatcataat tgggtaatga ctccaactta ttgatagtgt tttatgttca 8280gataatgccc gatgactttg tcatgcagct ccaccgattt tgagaacgac agcgacttcc 8340gtcccagccg tgccaggtgc tgcctcagat tcaggttatg ccgctcaatt cgctgcgtat 8400atcgcttgct gattacgtgc agctttccct tcaggcggga ttcatacagc ggccagccat 8460ccgtcatcca tatcaccacg tcaaagggtg acagcaggct cataagacgc cccagcgtcg 8520ccatagtgcg ttcaccgaat acgtgcgcaa caaccgtctt ccggagactg tcatacgcgt 8580aaaacagcca gcgctggcgc gatttagccc cgacatagcc ccactgttcg tccatttccg 8640cgcagacgat gacgtcactg cccggctgta tgcgcgagct gctgccagtg gcgataagtc 8700gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 8760aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 8820cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 8880tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 8940ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 9000atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 9060cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 9120ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 9180gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac 9240gcatctgtgc ggtatttcac accgcacgac tgcggcctga gttttttaag tgacgtaaaa 9300tcgtgttgag gccaacgccc ataatgcggg ctgttgcccg gcatccaacg ccattcatgg 9360ccatatcaat gattttctgg tgcgtaccgg gttgagaagc ggtgtaagtg aactgcagtt 9420gccatgtttt acggcagtga gagcagagat agcgctgatg tccggcggtg cttttgccgt 9480tacgcaccac cccgtcagta gctgaacagg agggacagct gatagacaca gaagccactg 9540gagcacctca aaaacaccat catacactaa atcagtaagt tggcagcatc acccataatt 9600gtggtttcaa aatcggctcc gtcgatacta tgttatacgc caactttgaa aacaactttg 9660aaaaagctgt tttctggtat ttaaggtttt agaatgcaag gaacagtgaa ttggagttcg 9720tcttgttata attagcttct tggggtatct ttaaatactg tagaaaagag gaaggaaata 9780ataaatggct aaaatgagaa tatcaccgga attgaaaaaa ctgatcgaaa aataccgctg 9840cgtaaaagat acggaaggaa tgtctcctgc taaggtatat aagctggtgg gagaaaatga 9900aaacctatat ttaaaaatga cggacagccg gtataaaggg accacctatg atgtggaacg 9960ggaaaaggac atgatgctat ggctggaagg aaagctgcct gttccaaagg tcctgcactt 10020tgaacggcat gatggctgga gcaatctgct catgagtgag gccgatggcg tcctttgctc 10080ggaagagtat gaagatgaac aaagccctga aaagattatc gagctgtatg cggagtgcat 10140caggctcttt cactccatcg acatatcgga ttgtccctat acgaatagct tagacagccg 10200cttagccgaa ttggattact tactgaataa cgatctggcc gatgtggatt gcgaaaactg 10260ggaagaagac actccattta aagatccgcg cgagctgtat gattttttaa agacggaaaa 10320gcccgaagag gaacttgtct tttcccacgg cgacctggga gacagcaaca tctttgtgaa 10380agatggcaaa gtaagtggct ttattgatct tgggagaagc ggcagggcgg acaagtggta 10440tgacattgcc ttctgcgtcc ggtcgatcag ggaggatatc ggggaagaac agtatgtcga 10500gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat attatatttt 10560actggatgaa ttgttttagt acctagatgt ggcgcaacga tgccggcgac aagcaggagc 10620gcaccgactt cttccgcatc aagtgttttg gctctcaggc cgaggcccac ggcaagtatt 10680tgggcaaggg gtcgctggta ttcgtgcagg gcaagattcg gaataccaag tacgagaagg 10740acggccagac ggtctacggg accgacttca ttgccgataa ggtggattat ctggacacca 10800aggcaccagg cgggtcaaat caggaataag ggcacattgc cccggcgtga gtcggggcaa 10860tcccgcaagg agggtgaatg aatcggacgt ttgaccggaa ggcatacagg caagaactga 10920tcgacgcggg gttttccgcc gaggatgccg aaaccatcgc aagccgcacc gtcatgcgtg 10980cgccccgcga aaccttccag tccgtcggct cgatggtcca gcaagctacg gccaagatcg 11040agcgcgacag cgtgcaactg gctccccctg ccctgcccgc gccatcggcc gccgtggagc 11100gttcgcgtcg tctcgaacag gaggcggcag gtttggcgaa gtcgatgacc atcgacacgc 11160gaggaactat gacgaccaag aagcgaaaaa ccgccggcga ggacctggca aaacaggtca 11220gcgaggccaa gcaggccgcg ttgctgaaac acacgaagca gcagatcaag gaaatgcagc 11280tttccttgtt cgatattgcg ccgtggccgg acacgatgcg agcgatgcca aacgacacgg 11340cccgctctgc cctgttcacc acgcgcaaca agaaaatccc gcgcgaggcg ctgcaaaaca 11400aggtcatttt ccacgtcaac aaggacgtga agatcaccta caccggcgtc gagctgcggg 11460ccgacgatga cgaactggtg tggcagcagg tgttggagta cgcgaagcgc acccctatcg 11520gcgagccgat caccttcacg ttctacgagc tttgccagga cctgggctgg tcgatcaatg 11580gccggtatta cacgaaggcc gaggaatgcc tgtcgcgcct acaggcgacg gcgatgggct 11640tcacgtccga ccgcgttggg cacctggaat cggtgtcgct gctgcaccgc ttccgcgtcc 11700tggaccgtgg caagaaaacg tcccgttgcc aggtcctgat cgacgaggaa atcgtcgtgc 11760tgtttgctgg cgaccactac acgaaattca tatgggagaa gtaccgcaag ctgtcgccga 11820cggcccgacg gatgttcgac tatttcagct cgcaccggga gccgtacccg ctcaagctgg 11880aaaccttccg cctcatgtgc ggatcggatt ccacccgcgt gaagaagtgg cgcgagcagg 11940tcggcgaagc ctgcgaagag ttgcgaggca gcggcctggt ggaacacgcc tgggtcaatg 12000atgacctggt gcattgcaaa cgctagggcc ttgtggggtc agttccggct gggggttcag 12060cagccagcgc tttactggca tttcaggaac aagcgggcac tgctcgacgc acttgcttcg 12120ctcagtatcg ctcgggacgc acggcgcgct ctacgaactg ccgataaaca gaggattaaa 12180attgacaatt gtgattaagg ctcagattcg acggcttgga gcggccgacg tgcaggattt 12240ccgcgagatc cgattgtcgg ccctgaagaa agctccagag atgttcgggt ccgtttacga 12300gcacgaggag aaaaagccca tggaggcgtt cgctgaacgg ttgcgagatg ccgtggcatt 12360cggcgcctac atcgacggcg agatcattgg gctgtcggtc ttcaaacagg aggacggccc 12420caaggacgct cacaaggcgc atctgtccgg cgttttcgtg gagcccgaac agcgaggccg 12480aggggtcgcc ggtatgctgc tgcgggcgtt gccggcgggt ttattgctcg tgatgatcgt 12540ccgacagatt ccaacgggaa tctggtggat gcgcatcttc atcctcggcg cacttaatat 12600ttcgctattc tggagcttgt tgtttatttc ggtctaccgc ctgccgggcg gggtcgcggc 12660gacggtaggc gctgtgcagc cgctgatggt cgtgttcatc tctgccgctc tgctaggtag 12720cccgatacga ttgatggcgg tcctgggggc tatttgcgga actgcgggcg tggcgctgtt 12780ggtgttgaca ccaaacgcag cgctagatcc tgtcggcgtc gcagcgggcc tggcgggggc 12840ggtttccatg gcgttcggaa ccgtgctgac ccgcaagtgg caacctcccg tgcctctgct 12900cacctttacc gcctggcaac tggcggccgg aggacttctg ctcgttccag tagctttagt 12960gtttgatccg ccaatcccga tgcctacagg aaccaatgtt ctcggcctgg cgtggctcgg 13020cctgatcgga gcgggtttaa cctacttcct ttggttccgg gggatctcgc gactcgaacc 13080tacagttgtt tccttactgg gctttctcag ccccagatct ggggtcgatc agccggggat 13140gcatcaggcc gacagtcgga acttcgggtc cccgacctgt accattcggt gagcaatgga 13200taggggagtt gatatcgtca acgttcactt ctaaagaaat agcgccactc agcttcctca 13260gcggctttat ccagcgattt cctattatgt cggcatagtt ctcaagatcg acagcctgtc 13320acggttaagc gagaaatgaa taagaaggct gataattcgg atctctgcga gggagatgat 13380atttgatcac aggcagcaac gctctgtcat cgttacaatc aacatgctac cctccgcgag 13440atcatccgtg tttcaaaccc ggcagcttag ttgccgttct tccgaatagc atcggtaaca 13500tgagcaaagt ctgccgcctt acaacggctc tcccgctgac gccgtcccgg actgatgggc 13560tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg 13620gtggcaggat atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc 13680ggacgttttt aatgtactgg ggtggttttt cttttcacca gtgagacggg caacagctga 13740ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 13800agcaggcgaa aatcctgttt gatggtggtt ccgaaatcgg caaaatccct tataaatcaa 13860aagaatagcc cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 13920agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 13980gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 14040accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 14100aggaagggaa gaaagcgaaa ggagcgggcg ccattcaggc tgcgcaactg ttgggaaggg 14160cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg 14220cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt 14280gaattaattc ccatcttgaa agaaatatag tttaaatatt tattgataaa ataacaagtc 14340aggtattata gtccaagcaa aaacataaat ttattgatgc aagtttaaat tcagaaatat 14400ttcaataact gattatatca gctggtacat tgccgtagat gaaagactga gtgcgatatt 14460atgtgtaata cataaattga tgatatagct agcttagctc atcgggggat ccgtcgaagc 14520tagcttgggt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga 14580atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc 14640ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg 14700gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc 14760atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa 14820cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc 14880ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca 14940ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc 15000ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca 15060gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc 15120cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt 15180cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca 15240gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga 15300acctgcgtgc aatccatctt gttcaatcca agctcccatg ggccctcgac tagagtcgag 15360atctggattg agagtgaata tgagactcta attggatacc gaggggaatt tatggaacgt 15420cagtggagca tttttgacaa gaaatatttg ctagctgata gtgaccttag gcgacttttg 15480aacgcgcaat aatggtttct gacgtatgtg cttagctcat taaactccag aaacccgcgg 15540ctgagtggct ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg 15600cgtcatcggc gggggtcata acgtgactcc cttaattctc cgctcatgat cttgatcccc 15660tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa 15720ccttaccaga gggcgcccca gctggcaatt ccggttcgct tgctgtccat aaaaccgccc 15780agtctagcta tcgccatgta agcccactgc aagctacctg ctttctcttt gcgcttgcgt 15840tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg 15900gactggcttt ctacgtgttc cgcttccttt agcagccctt gcgccctgag tgcttgcggc 15960agcgtgaagc ttctgccatg catcattcat catatttagg tcatgaaatg atgcccttta 16020gtagctatga taggaaaaca aattctgtcg cgcaccattc atcatatttg gggccatctt 16080cattcttgag agggagggtc ctctttgagg aaggataatt tatcttggtt tcagagaaaa 16140tacttgaagt gggtgccaac ttggttgagc agtgaaacta gacataaaga ttacatgatg 16200ttcttggtgg tcgtaatatg tcaaatagat ggtatgctaa aaaacagtta aaactagatg 16260agaagattgt gaaagactga tttgccagag aaaatctagt agatatttat tggagttggc 16320ctagcatgtg gggcagtgca tgcttgaatg ccccctgatc atgggtagat gtcagccaca 16380aggagcccaa taaaccttgg gctttgatac catgttaaat aaaaaatata tatgcctcat 16440atatatgatc tataaactcc taaattgcat ctaaacactc gatggaagaa gggtgaaatc 16500agtttatcga tttaactaaa ttaatttatt ttgggggttt ttttggcttt taaaatgggt 16560ttgaagataa ctctctctct ctctcccccc gcccatgcaa agaggtcggc aattttagat 16620aaagacgtcc attttttcga cgcgtgtcgt tacgtggcga aacgtcgtgg aaggacgagt 16680ctttgagggc acgcgtcaga ctatcgtggt catcctctgc ctacttatat ccctctggtc 16740ttcttcttct taattaagaa actcccgagg tgagcaagga tccggagtcg agcgcgaaga 16800agagaaagag ggaaagcgcg g 168213217675DNAArtificial SequenceMERF03 plasmid 32cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 60gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 120catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 180cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 240tatgttacta gatcgggtgt acatctagaa gctctggaca tcatgttgga tatgaaacaa 300ctattattta tctacatgtt ttagatgtta tctgattatt tttataccgt agtcttctat 360tgatgaggag tctaaggcta tagaattata tatctaaatg

attaatatat atattattaa 420taattaacaa taattaatat attataattt atatatatat attttatatt attataataa 480tattcttaca aatataatta ttatattcga cggtatcgat aagctcgggc ccaaatcgta 540cgggccggcc gtaagcttat cgatgaagga agtggaggag agaggacgcc atggtagcag 600aggaaggtct ggcttgatct cccgacgatt cctctctcat cagtgaaaca agagaataag 660aggcatcgcg attcttggaa ggtacagagg gaagttgatc aaagagaggc tccgggaaga 720agcagatggc ggggaagacg aaacatggcg cctgacaaca taggctatca taggataatc 780cactctccct ctgtctttct ctctgtttct ttctctctct ctttatctct ctctctctct 840cgaacatttc acacattttt gggcctgttc tttggcgtag ctagcccttt cttggtccat 900attttgaggc caaggccaat catgcgcagc cacgttgcat ggcgggagga ccccatctat 960ccattccgtc agttcctggc ttttgggaca atctgaacag tacataaacc acgggctcgg 1020gcttgggccc gccgaaaagc ccgtccgtac aattttctga cgtacaatat taatttccca 1080gaaaagaaaa ttcattaaaa aataactatg tcacccacga actcgtgatc tagatataag 1140gcaggcaact ttcccgtgac agtcggactt gtggggttcc cttgacggcg ccgccgtcta 1200ctgacggcgc ttgaatgacg tcattattac tatatttaat atatccgaga ataaatggct 1260ccctggatcc cccgtgaatg gtcacgtcat ccacgcgggc tttgacttcg cctaggtcgc 1320cacgttgggg cctcataacg tgcaagctga cgtggctatc attgcctcga tggcgatctt 1380gaacgaacca cataaccaat ctgagacggc gacgaggatt ctgtgttttc ctcggggatc 1440tctggccgtc cgatgaacta acacgcacta tttcaaaaag ggggttaaac acgattgtta 1500ggtttctttg agtcttccac atctccgctc cacgatcttg tacctcttct tgcgacgatc 1560tactcgccct cgaggtcgac accatgtccg gtgcgcctcc caccgacctg cagaagatgg 1620tgatgggcaa caccaagccc gtcgagttga tcctcgacgg gaagaccgtg gcgatctgct 1680gcgccaccgg cgtgttcggc accgcctacc tcgtcccgag acacctgttc gccgagaagt 1740atgacaagat catgctggac gggcgggcca tgaccgactc ggactaccgg gtcttcgagt 1800tcgagatcaa agtgaagggc caggatatgc tctccgacgc cgcgctgatg gtgctccaca 1860gaggcaaccg cgtgcgggac atcaccaagc acttcaggga caccgcgcgc atgaagaagg 1920ggacccctgt ggtcggggtc gtgaacaacg ccgacgtcgg gcgcctcatc ttctccggcg 1980aggcgctgac ctacaaggac atcgtcgtgt gcatggacgg ggacacgatg ccggggctct 2040tcgcctacaa ggccgcgacc aaggccgggt actgcggggg tgcggtgctc gccaaggacg 2100gcgccgacac gttcatcgtg ggcacccact ccgccggcgg taacggcgtc ggctactgct 2160cctgcgtgtc ccggtccatg ctgctgcgga tgaaggccca cgttgacccc gagccgcagc 2220acgagtaaaa gcttatcgat gtgcacgatc gttcaaacat ttggcaataa agtttcttaa 2280gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta 2340agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta 2400gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg 2460ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggtggacaac tcgtcacgta 2520ctagtggcgc gcccacgtga tttaaatcgt acgggccggc cgtatactct agtggatccc 2580ccaattcaga tcggctgagt ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa 2640aacggcttgt cccgcgtcat cggcgggggt cataacgtga ctcccttaat tctccgctca 2700tgatcagatt gtcgtttccc gccttcagtt taaactatca gtgtttgaca ggatatattg 2760gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg 2820aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt ccccagatct 2880ggcgccggcc agcgagacga gcaagattgg ccgccgcccg aaacgatccg acagcgcgcc 2940cagcacaggt gcgcaggcaa attgcaccaa cgcatacagc gccagcagaa tgccatagtg 3000ggcggtgacg tcgttcgagt gaaccagatc gcgcaggagg cccggcagca ccggcataat 3060caggccgatg ccgacagcgt cgagcgcgac agtgctcaga attacgatca ggggtatgtt 3120gggtttcacg tctggcctcc ggaccagcct ccgctggtcc gattgaacgc gcggattctt 3180tatcactgat aagttggtgg acatattatg tttatcagtg ataaagtgtc aagcatgaca 3240aagttgcagc cgaatacagt gatccgtgcc gccctggacc tgttgaacga ggtcggcgta 3300gacggtctga cgacacgcaa actggcggaa cggttggggg ttcagcagcc ggcgctttac 3360tggcacttca ggaacaagcg ggcgctgctc gacgcactgg ccgaagccat gctggcggag 3420aatcatacgc attcggtgcc gagagccgac gacgactggc gctcatttct gatcgggaat 3480gcccgcagct tcaggcaggc gctgctcgcc taccgcgatg gcgcgcgcat ccatgccggc 3540acgcgaccgg gcgcaccgca gatggaaacg gccgacgcgc agcttcgctt cctctgcgag 3600gcgggttttt cggccgggga cgccgtcaat gcgctgatga caatcagcta cttcactgtt 3660ggggccgtgc ttgaggagca ggccggcgac agcgatgccg gcgagcgcgg cggcaccgtt 3720gaacaggctc cgctctcgcc gctgttgcgg gccgcgatag acgccttcga cgaagccggt 3780ccggacgcag cgttcgagca gggactcgcg gtgattgtcg atggattggc gaaaaggagg 3840ctcgttgtca ggaacgttga aggaccgaga aagggtgacg attgatcagg accgctgccg 3900gagcgcaacc cactcactac agcagagcca tgtagacaac atcccctccc cctttccacc 3960gcgtcagacg cccgtagcag cccgctacgg gctttttcat gccctgccct agcgtccaag 4020cctcacggcc gcgctcggcc tctctggcgg ccttctggcg ctcttccgct tcctcgctca 4080ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4140taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4200agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4260cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4320tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4380tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg cttttccgct 4440gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca 4500cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt 4560cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc 4620ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccggctacc 4680gccggcgtaa cagatgaggg caagcggatg gctgatgaaa ccaagccaac caggaagggc 4740agcccaccta tcaaggtgta ctgccttcca gacgaacgaa gagcgattga ggaaaaggcg 4800gcggcggccg gcatgagcct gtcggcctac ctgctggccg tcggccaggg ctacaaaatc 4860acgggcgtcg tggactatga gcacgtccgc gagctggccc gcatcaatgg cgacctgggc 4920cgcctgggcg gcctgctgaa actctggctc accgacgacc cgcgcacggc gcggttcggt 4980gatgccacga tcctcgccct gctggcgaag atcgaagaga agcaggacga gcttggcaag 5040gtcatgatgg gcgtggtccg cccgagggca gagccatgac ttttttagcc gctaaaacgg 5100ccggggggtg cgcgtgattg ccaagcacgt ccccatgcgc tccatcaaga agagcgactt 5160cgcggagctg gtgaagtaca tcaccgacga gcaaggcaag accgagcgcc tttgcgacgc 5220tcaccgggct ggttgccctc gccgctgggc tggcggccgt ctatggccct gcaaacgcgc 5280cagaaacgcc gtcgaagccg tgtgcgagac accgcggccg ccggcgttgt ggatacctcg 5340cggaaaactt ggccctcact gacagatgag gggcggacgt tgacacttga ggggccgact 5400cacccggcgc ggcgttgaca gatgaggggc aggctcgatt tcggccggcg acgtggagct 5460ggccagcctc gcaaatcggc gaaaacgcct gattttacgc gagtttccca cagatgatgt 5520ggacaagcct ggggataagt gccctgcggt attgacactt gaggggcgcg actactgaca 5580gatgaggggc gcgatccttg acacttgagg ggcagagtgc tgacagatga ggggcgcacc 5640tattgacatt tgaggggctg tccacaggca gaaaatccag catttgcaag ggtttccgcc 5700cgtttttcgg ccaccgctaa cctgtctttt aacctgcttt taaaccaata tttataaacc 5760ttgtttttaa ccagggctgc gccctgtgcg cgtgaccgcg cacgccgaag gggggtgccc 5820ccccttctcg aaccctcccg gcccgctaac gcgggcctcc catcccccca ggggctgcgc 5880ccctcggccg cgaacggcct caccccaaaa atggcagcgc tggcagtcct tgccattgcc 5940gggatcgggg cagtaacggg atgggcgatc agcccgagcg cgacgcccgg aagcattgac 6000gtgccgcagg tgctggcatc gacattcagc gaccaggtgc cgggcagtga gggcggcggc 6060ctgggtggcg gcctgccctt cacttcggcc gtcggggcat tcacggactt catggcgggg 6120ccggcaattt ttaccttggg cattcttggc atagtggtcg cgggtgccgt gctcgtgttc 6180gggggtgcga taaacccagc gaaccatttg aggtgatagg taagattata ccgaggtatg 6240aaaacgagaa ttggaccttt acagaattac tctatgaagc gccatattta aaaagctacc 6300aagacgaaga ggatgaagag gatgaggagg cagattgcct tgaatatatt gacaatactg 6360ataagataat atatctttta tatagaagat atcgccgtat gtaaggattt cagggggcaa 6420ggcataggca gcgcgcttat caatatatct atagaatggg caaagcataa aaacttgcat 6480ggactaatgc ttgaaaccca ggacaataac cttatagctt gtaaattcta tcataattgg 6540gtaatgactc caacttattg atagtgtttt atgttcagat aatgcccgat gactttgtca 6600tgcagctcca ccgattttga gaacgacagc gacttccgtc ccagccgtgc caggtgctgc 6660ctcagattca ggttatgccg ctcaattcgc tgcgtatatc gcttgctgat tacgtgcagc 6720tttcccttca ggcgggattc atacagcggc cagccatccg tcatccatat caccacgtca 6780aagggtgaca gcaggctcat aagacgcccc agcgtcgcca tagtgcgttc accgaatacg 6840tgcgcaacaa ccgtcttccg gagactgtca tacgcgtaaa acagccagcg ctggcgcgat 6900ttagccccga catagcccca ctgttcgtcc atttccgcgc agacgatgac gtcactgccc 6960ggctgtatgc gcgaggttac cgactgcggc ctgagttttt taagtgacgt aaaatcgtgt 7020tgaggccaac gcccataatg cgggctgttg cccggcatcc aacgccattc atggccatat 7080caatgatttt ctggtgcgta ccgggttgag aagcggtgta agtgaactgc agttgccatg 7140ttttacggca gtgagagcag agatagcgct gatgtccggc ggtgcttttg ccgttacgca 7200ccaccccgtc agtagctgaa caggagggac agctgataga cacagaagcc actggagcac 7260ctcaaaaaca ccatcataca ctaaatcagt aagttggcag catcacccat aattgtggtt 7320tcaaaatcgg ctccgtcgat actatgttat acgccaactt tgaaaacaac tttgaaaaag 7380ctgttttctg gtatttaagg ttttagaatg caaggaacag tgaattggag ttcgtcttgt 7440tataattagc ttcttggggt atctttaaat actgtagaaa agaggaagga aataataaat 7500ggctaaaatg agaatatcac cggaattgaa aaaactgatc gaaaaatacc gctgcgtaaa 7560agatacggaa ggaatgtctc ctgctaaggt atataagctg gtgggagaaa atgaaaacct 7620atatttaaaa atgacggaca gccggtataa agggaccacc tatgatgtgg aacgggaaaa 7680ggacatgatg ctatggctgg aaggaaagct gcctgttcca aaggtcctgc actttgaacg 7740gcatgatggc tggagcaatc tgctcatgag tgaggccgat ggcgtccttt gctcggaaga 7800gtatgaagat gaacaaagcc ctgaaaagat tatcgagctg tatgcggagt gcatcaggct 7860ctttcactcc atcgacatat cggattgtcc ctatacgaat agcttagaca gccgcttagc 7920cgaattggat tacttactga ataacgatct ggccgatgtg gattgcgaaa actgggaaga 7980agacactcca tttaaagatc cgcgcgagct gtatgatttt ttaaagacgg aaaagcccga 8040agaggaactt gtcttttccc acggcgacct gggagacagc aacatctttg tgaaagatgg 8100caaagtaagt ggctttattg atcttgggag aagcggcagg gcggacaagt ggtatgacat 8160tgccttctgc gtccggtcga tcagggagga tatcggggaa gaacagtatg tcgagctatt 8220ttttgactta ctggggatca agcctgattg ggagaaaata aaatattata ttttactgga 8280tgaattgttt tagtacctag atgtggcgca acgatgccgg cgacaagcag gagcgcaccg 8340acttcttccg catcaagtgt tttggctctc aggccgaggc ccacggcaag tatttgggca 8400aggggtcgct ggtattcgtg cagggcaaga ttcggaatac caagtacgag aaggacggcc 8460agacggtcta cgggaccgac ttcattgccg ataaggtgga ttatctggac accaaggcac 8520caggcgggtc aaatcaggaa taagggcaca ttgccccggc gtgagtcggg gcaatcccgc 8580aaggagggtg aatgaatcgg acgtttgacc ggaaggcata caggcaagaa ctgatcgacg 8640cggggttttc cgccgaggat gccgaaacca tcgcaagccg caccgtcatg cgtgcgcccc 8700gcgaaacctt ccagtccgtc ggctcgatgg tccagcaagc tacggccaag atcgagcgcg 8760acagcgtgca actggctccc cctgccctgc ccgcgccatc ggccgccgtg gagcgttcgc 8820gtcgtctcga acaggaggcg gcaggtttgg cgaagtcgat gaccatcgac acgcgaggaa 8880ctatgacgac caagaagcga aaaaccgccg gcgaggacct ggcaaaacag gtcagcgagg 8940ccaagcaggc cgcgttgctg aaacacacga agcagcagat caaggaaatg cagctttcct 9000tgttcgatat tgcgccgtgg ccggacacga tgcgagcgat gccaaacgac acggcccgct 9060ctgccctgtt caccacgcgc aacaagaaaa tcccgcgcga ggcgctgcaa aacaaggtca 9120ttttccacgt caacaaggac gtgaagatca cctacaccgg cgtcgagctg cgggccgacg 9180atgacgaact ggtgtggcag caggtgttgg agtacgcgaa gcgcacccct atcggcgagc 9240cgatcacctt cacgttctac gagctttgcc aggacctggg ctggtcgatc aatggccggt 9300attacacgaa ggccgaggaa tgcctgtcgc gcctacaggc gacggcgatg ggcttcacgt 9360ccgaccgcgt tgggcacctg gaatcggtgt cgctgctgca ccgcttccgc gtcctggacc 9420gtggcaagaa aacgtcccgt tgccaggtcc tgatcgacga ggaaatcgtc gtgctgtttg 9480ctggcgacca ctacacgaaa ttcatatggg agaagtaccg caagctgtcg ccgacggccc 9540gacggatgtt cgactatttc agctcgcacc gggagccgta cccgctcaag ctggaaacct 9600tccgcctcat gtgcggatcg gattccaccc gcgtgaagaa gtggcgcgag caggtcggcg 9660aagcctgcga agagttgcga ggcagcggcc tggtggaaca cgcctgggtc aatgatgacc 9720tggtgcattg caaacgctag ggccttgtgg ggtcagttcc ggctgggggt tcagcagcca 9780gcgctttact ggcatttcag gaacaagcgg gcactgctcg acgcacttgc ttcgctcagt 9840atcgctcggg acgcacggcg cgctctacga actgccgata aacagaggat taaaattgac 9900aattgtgatt aaggctcaga ttcgacggct tggagcggcc gacgtgcagg atttccgcga 9960gatccgattg tcggccctga agaaagctcc agagatgttc gggtccgttt acgagcacga 10020ggagaaaaag cccatggagg cgttcgctga acggttgcga gatgccgtgg cattcggcgc 10080ctacatcgac ggcgagatca ttgggctgtc ggtcttcaaa caggaggacg gccccaagga 10140cgctcacaag gcgcatctgt ccggcgtttt cgtggagccc gaacagcgag gccgaggggt 10200cgccggtatg ctgctgcggg cgttgccggc gggtttattg ctcgtgatga tcgtccgaca 10260gattccaacg ggaatctggt ggatgcgcat cttcatcctc ggcgcactta atatttcgct 10320attctggagc ttgttgttta tttcggtcta ccgcctgccg ggcggggtcg cggcgacggt 10380aggcgctgtg cagccgctga tggtcgtgtt catctctgcc gctctgctag gtagcccgat 10440acgattgatg gcggtcctgg gggctatttg cggaactgcg ggcgtggcgc tgttggtgtt 10500gacaccaaac gcagcgctag atcctgtcgg cgtcgcagcg ggcctggcgg gggcggtttc 10560catggcgttc ggaaccgtgc tgacccgcaa gtggcaacct cccgtgcctc tgctcacctt 10620taccgcctgg caactggcgg ccggaggact tctgctcgtt ccagtagctt tagtgtttga 10680tccgccaatc ccgatgccta caggaaccaa tgttctcggc ctggcgtggc tcggcctgat 10740cggagcgggt ttaacctact tcctttggtt ccgggggatc tcgcgactcg aacctacagt 10800tgtttcctta ctgggctttc tcagccccag atctggggtc gatcagccgg ggatgcatca 10860ggccgacagt cggaacttcg ggtccccgac ctgtaccatt cggtgagcaa tggatagggg 10920agttgatatc gtcaacgttc acttctaaag aaatagcgcc actcagcttc ctcagcggct 10980ttatccagcg atttcctatt atgtcggcat agttctcaag atcgacagcc tgtcacggtt 11040aagcgagaaa tgaataagaa ggctgataat tcggatctct gcgagggaga tgatatttga 11100tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg cgagatcatc 11160cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt aacatgagca 11220aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat gggctgcctg 11280tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg gctggtggca 11340ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca ttgcggacgt 11400ttttaatgta ctggggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 11460ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg 11520cgaaaatcct gtttgatggt ggttccgaaa tcggcaaaat cccttataaa tcaaaagaat 11580agcccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 11640tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 11700catcacccaa atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 11760aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 11820ggaagaaagc gaaaggagcg ggcgccattc aggctgcgca actgttggga agggcgatcg 11880gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 11940agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg 12000ccatcttgaa agaaatatag tttaaatatt tattgataaa ataagtcagg tattatagtc 12060caagcaaaaa cataatttat tgatgcaaag tttaaattca gaaatatttc aataactgat 12120tatatcagct ggtacattgc cgtagatgaa agactgagtg cgatattatg tgtaatacat 12180aaattgatga tatagctagc ttagctcatc gggggatcct taatcgactc tagctagaac 12240gaattgttag gtggcggtac ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc 12300caactttgta tagagagcca ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac 12360ataagcacca agcgcgttgg cctcatgctt gaggagattg atgagcgcgg tggcaatgcc 12420ctgcctccgg tgctcgccgg agactgcgag atcatagata tagatctcac tacgcggctg 12480ctcaaacctg ggcagaacgt aagccgcgag agcgccaaca accgcttctt ggtcgaaggc 12540agcaagcgcg atgaatgtct tactacggag caagttcccg aggtaatcgg agtccggctg 12600atgttgggag taggtggcta cgtctccgaa ctcacgaccg aaaagatcaa gagcagcccg 12660catggatttg acttggtcag ggccgagcct acatgtgcga atgatgccca tacttgagcc 12720acctaacttt gttttagggc gactgccctg ctgcgtaaca tcgttgctgc tgcgtaccat 12780ggagatctgg attgagagtg aatatgagac tctaattgga taccgagggg aatttatgga 12840agtcagtgga gcatttttga caagaaatat ttgctagctg atagtgacct taggcgactt 12900ttgaacgcgc aataatggtt tctgacgtat gtgcttagct cattaaactc cagaaacccg 12960cggctgagtg gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc 13020ccgcgtcatc ggcgggggtc ataacgtgac tcccttaatt ctccgctcat gatcttgatc 13080ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc 13140caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg 13200cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg 13260cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct 13320gcggactggc tttctacgtg ttccgcttcc tttagcagcc cttgcgccct gagtgcttgc 13380ggcagcgtga agctctggac atcatgttgg atatgaaaca actattattt atctacatgt 13440tttagatgtt atctgattat ttttataccg tagtcttcta ttgatgagga gtctaaggct 13500atagaattat atatctaaat gattaatata tatattatta ataattaaca ataattaata 13560tattataatt tatatatata tattttatat tattataata atattcttac aaatataatt 13620attatattcg acggtatcga taagctcggg atccctgaaa gcgacgttgg atgttaacat 13680ctacaaattg ccttttctta tcgaccatgt acgtaagcgc ttacgttttt ggtggaccct 13740tgaggaaact ggtagctgtt gtgggcctgt ggtctcaaga tggatcatta atttccacct 13800tcacctacga tggggggcat cgcaccggtg agtaatattg tacggctaag agcgaatttg 13860gcctgtagga tccctgaaag cgacgttgga tgttaacatc tacaaattgc cttttcttat 13920cgaccatgta cgtaagcgct tacgtttttg gtggaccctt gaggaaactg gtagctgttg 13980tgggcctgtg gtctcaagat ggatcattaa tttccacctt cacctacgat ggggggcatc 14040gcaccggtga gtaatattgt acggctaaga gcgaatttgg cctgtaggat ccctgaaagc 14100gacgttggat gttaacatct acaaattgcc ttttcttatc gaccatgtac gtaagcgctt 14160acgtttttgg tggacccttg aggaaactgg tagctgttgt gggcctgtgg tctcaagatg 14220gatcattaat ttccaccttc acctacgatg gggggcatcg caccggtgag taatattgta 14280cggctaagag cgaatttggc ctgtaggatc cgcgagctgg tcaatcccat tgcttttgaa 14340gcagctcaac attgatctct ttctcgatcg agggagattt ttcaaatcag tgcgcaagac 14400gtgacgtaag tatccgagtc agtttttatt tttctactaa tttggtcgtt tatttcggcg 14460tgtaggacat ggcaaccggg cctgaatttc gcgggtattc tgtttctatt ccaacttttt 14520cttgatccgc agccattaac gacttttgaa tagatacgct gacacgccaa gcctcgctag 14580tcaaaagtgt accaaacaac gctttacagc aagaacggaa tgcgcgtgac gctcgcggtg 14640acgccatttc gccttttcag aaatggataa atagccttgc ttcctattat atcttccccc 14700aaattaatta agaaactccc gaggtgagca aggatccgga gtcgagcgcg aagaagagaa 14760agagggaaag cgcgggtacc gggccccccc ctcgacggat caagtgcaaa ggtccgcctt 14820gtttctcctc tgtctcttga tctgactaat cttggtttat gattcgttga gtaattttgg 14880ggaaagctag cttcgtccac agtttttttt tcgatgaaca gtgccgcagt ggcgctgatc 14940ttgtatgcta tcctgcaatc gtggtgaact tatttctttt atatccttca ctcccatgaa 15000aaggctagta atctttctcg atgtaacatc gtccagcact gctattaccg tgtggtccat 15060ccgacagtct ggctgaacac atcatacgat attgagcaaa gatcgatcta tcttccctgt 15120tctttaatga aagacgtcat tttcatcagt atgatctaag aatgttgcaa cttgcaagga 15180ggcgtttctt tctttgaatt taactaactc gttgagtggc cctgtttctc ggacgtaagg 15240cctttgctgc tccacacatg tccattcgaa ttttaccgtg tttagcaagg gcgaaaagtt 15300tgcatcttga tgatttagct tgactatgcg attgctttcc tggacccgtg cagctgcgga 15360cggatccccc gctcgaagct agcttgatca gatctgatcg aattcaccat gggggccggg 15420cagtcgtccc cggccacggg gtcccagaac cagtccggca

acaccggcag catcatcaac 15480aactactaca tgcagcagta ccagaactcc atggacaccc aactggggga caacgccacg 15540agcggcggca gcaatgaggg ctccaccgac accacgagca cccacacgac caacacccag 15600aacaacgact ggttcagcaa gctcgcgtcc agcgccttct ccgggctctt cggggcgctc 15660ctggccgaca agaagaccga ggagaccacg ctgctcgagg accgcatcct caccacgcgg 15720aacggccaca cgacctccac cacgcagtcg agcgtcgggg tcacttacgg ttacgccacg 15780gccgaggact tcgtgagcgg ccccaacacc tccggcctcg agactcgggt cgcccaggcc 15840gagcgcttct tcaagacgca cctgttcgac tgggtgacct ccgacccctt cggccgctgc 15900cacctcttgg aactgcccac cgaccacaag ggcgtgtacg ggtcgttgac ggacagctac 15960gcgtacatga ggaacgggtg ggacgtggag gtcaccgccg tcggcaacca gttcaacggg 16020ggctgcctcc tggtggccat ggtccccgaa ctctgctcga tccagaagcg ggagctgtac 16080cagctgaccc tcttcccgca ccagttcatc aacccgcgca ccaacatgac cgcgcacatc 16140accgtcccct tcgtgggcgt gaaccgctac gaccagtaca aagtccacaa gccctggacc 16200ttggtggtca tggtcgtcgc ccccctcacc gtcaactcgg agggggcgcc ccagatcaaa 16260gtctacgcca acatcgcccc cacgaacgtg cacgttgccg gcgagttccc gagcaaggag 16320ggcatcttcc cggttgcgtg ctccgacggc tacgggggcc tcgtgacgac cgacccgaag 16380accgccgacc ctgcctacgg caaggtgttc aacccgccca gaaacatgct ccctgggaga 16440ttcacgaact tcctcgacgt ggccgaggcc tgccccacgt tcctgcactt cgagggggac 16500gtcccttacg tcaccacgaa gactgactcg gatcgcgtgc tggcccagtt cgacctgtcc 16560ctcgccgcta agcacatgtc caacaccttc ctcgccggcc tcgcgcaata ctacacccag 16620tacagcggca ccatcaacct ccacttcatg ttcaccggcc ccaccgacgc caaggcccgc 16680tacatgatcg cttacgcccc gcccggcatg gagcccccga agacccccga ggcggccgcg 16740cactgcatcc acgccgagtg ggacacgggg ctcaactcca agttcacgtt ctccatcccc 16800tacctcagcg ccgcggacta cgcgtacacc gccagcgaca ccgcggagac cacgaacgtg 16860cagggctggg tgtgcctgtt ccagatcacc cacgggaagg ccgacggcga cgccctggtc 16920gtgctggcct cggcggggaa ggacttcgaa ctcaggctgc cggtggacgc ccgcacccag 16980acgactagcg ccggcgagtc cgcggacccg gtgaccgcga ccgtcgagaa ctacgggggc 17040gagacccaag tgcagcgcag acagcacacg gacgtgtcct tcatcctgga tcgcttcgtg 17100aaagtgaccc cgaaggacca gatcaacgtg ctggacctga tgcagacgcc ggcgcacacc 17160ctcgtggggg ccctcctgcg caccgccacg tactacttcg cggacctcga ggtggcggtg 17220aagcacgagg gcaacctgac gtgggtgccc aacggtgcgc cggaggccgc gctcgacaac 17280accacgaacc ccaccgcgta ccacaaggcc cccctgaccc ggctcgccct cccctacacc 17340gccccccaca gggtcctcgc cacggtctac aacggcaaca gcaagtacgg ggacgggacc 17400gtcgccaacg tgcgcgggga cctccaagtg ctcgcccaga aggccgcgcg cgccctcccc 17460actagcttca actacggtgc catcaaggcc acgagggtga cggagctgct ctaccgcatg 17520aagcgcgccg agacctactg cccccgcccc ctgctcgcga tccaccccga ccaggccaga 17580cacaagcaga agatcgtcgc ccctgtcaag cagctcctga acttcgacct cctgaagctg 17640gccggcgacg tggagagcaa tccgggctaa gagct 176753318089DNAArtificial SequenceMERF04 plasmid 33cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 60gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 120catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 180cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 240tatgttacta gatcgggtgt acatctagaa gctctggaca tcatgttgga tatgaaacaa 300ctattattta tctacatgtt ttagatgtta tctgattatt tttataccgt agtcttctat 360tgatgaggag tctaaggcta tagaattata tatctaaatg attaatatat atattattaa 420taattaacaa taattaatat attataattt atatatatat attttatatt attataataa 480tattcttaca aatataatta ttatattcga cggtatcgat aagctcgggc ccagcttctg 540ccatgcatca ttcatcatat ttaggtcatg aaatgatgcc ctttagtagc tatgatagga 600aaacaaattc tgtcgcgcac cattcatcat atttggggcc atcttcattc ttgagaggga 660gggtcctctt tgaggaagga taatttatct tggtttcaga gaaaatactt gaagtgggtg 720ccaacttggt tgagcagtga aactagacat aaagattaca tgatgttctt ggtggtcgta 780atatgtcaaa tagatggtat gctaaaaaac agttaaaact agatgagaag attgtgaaag 840actgatttgc cagagaaaat ctagtagata tttattggag ttggcctagc atgtggggca 900gtgcatgctt gaatgccccc tgatcatggg tagatgtcag ccacaaggag cccaataaac 960cttgggcttt gataccatgt taaataaaaa atatatatgc ctcatatata tgatctataa 1020actcctaaat tgcatctaaa cactcgatgg aagaagggtg aaatcagttt atcgatttaa 1080ctaaattaat ttattttggg ggtttttttg gcttttaaaa tgggtttgaa gataactctc 1140tctctctctc cccccgccca tgcaaagagg tcggcaattt tagataaaga cgtccatttt 1200ttcgacgcgt gtcgttacgt ggcgaaacgt cgtggaagga cgagtctttg agggcacgcg 1260tcagactatc gtggtcatcc tctgcctact tatatccctc tggtcttctt cttcttaatt 1320aagaaactcc cgaggtgagc aaggatccgg agtcgagcgc gaagaagaga aagagggaaa 1380gcgcgggtac cgggcccccc cctcgacgga tcaagtgcaa aggtccgcct tgtttctcct 1440ctgtctcttg atctgactaa tcttggttta tgattcgttg agtaattttg gggaaagcta 1500gcttcgtcca cagttttttt ttcgatgaac agtgccgcag tggcgctgat cttgtatgct 1560atcctgcaat cgtggtgaac ttatttcttt tatatccttc actcccatga aaaggctagt 1620aatctttctc gatgtaacat cgtccagcac tgctattacc gtgtggtcca tccgacagtc 1680tggctgaaca catcatacga tattgagcaa agatcgatct atcttccctg ttctttaatg 1740aaagacgtca ttttcatcag tatgatctaa gaatgttgca acttgcaagg aggcgtttct 1800ttctttgaat ttaactaact cgttgagtgg ccctgtttct cggacgtaag gcctttgctg 1860ctccacacat gtccattcga attttaccgt gtttagcaag ggcgaaaagt ttgcatcttg 1920atgatttagc ttgactatgc gattgctttc ctggacccgt gcagctgcgg acggatcccc 1980cgctcgaggt cgacaccatg tccggtgcgc ctcccaccga cctgcagaag atggtgatgg 2040gcaacaccaa gcccgtcgag ttgatcctcg acgggaagac cgtggcgatc tgctgcgcca 2100ccggcgtgtt cggcaccgcc tacctcgtcc cgagacacct gttcgccgag aagtatgaca 2160agatcatgct ggacgggcgg gccatgaccg actcggacta ccgggtcttc gagttcgaga 2220tcaaagtgaa gggccaggat atgctctccg acgccgcgct gatggtgctc cacagaggca 2280accgcgtgcg ggacatcacc aagcacttca gggacaccgc gcgcatgaag aaggggaccc 2340ctgtggtcgg ggtcgtgaac aacgccgacg tcgggcgcct catcttctcc ggcgaggcgc 2400tgacctacaa ggacatcgtc gtgtgcatgg acggggacac gatgccgggg ctcttcgcct 2460acaaggccgc gaccaaggcc gggtactgcg ggggtgcggt gctcgccaag gacggcgccg 2520acacgttcat cgtgggcacc cactccgccg gcggtaacgg cgtcggctac tgctcctgcg 2580tgtcccggtc catgctgctg cggatgaagg cccacgttga ccccgagccg cagcacgagt 2640aaaagcttat cgatgtgcac gatcgttcaa acatttggca ataaagtttc ttaagattga 2700atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg 2760taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc 2820cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat 2880tatcgcgcgc ggtgtcatct atgttactag atcgggtgga caactcgtca cgtactagtg 2940gcgcgcccac gtgatttaaa tcgtacgggc cggccgtata ctctagtgga tcccccaatt 3000cagatcggct gagtggctcc ttcaacgttg cggttctgtc agttccaaac gtaaaacggc 3060ttgtcccgcg tcatcggcgg gggtcataac gtgactccct taattctccg ctcatgatca 3120gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt 3180aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg cgtgaaaagg 3240tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttccccag atctggcgcc 3300ggccagcgag acgagcaaga ttggccgccg cccgaaacga tccgacagcg cgcccagcac 3360aggtgcgcag gcaaattgca ccaacgcata cagcgccagc agaatgccat agtgggcggt 3420gacgtcgttc gagtgaacca gatcgcgcag gaggcccggc agcaccggca taatcaggcc 3480gatgccgaca gcgtcgagcg cgacagtgct cagaattacg atcaggggta tgttgggttt 3540cacgtctggc ctccggacca gcctccgctg gtccgattga acgcgcggat tctttatcac 3600tgataagttg gtggacatat tatgtttatc agtgataaag tgtcaagcat gacaaagttg 3660cagccgaata cagtgatccg tgccgccctg gacctgttga acgaggtcgg cgtagacggt 3720ctgacgacac gcaaactggc ggaacggttg ggggttcagc agccggcgct ttactggcac 3780ttcaggaaca agcgggcgct gctcgacgca ctggccgaag ccatgctggc ggagaatcat 3840acgcattcgg tgccgagagc cgacgacgac tggcgctcat ttctgatcgg gaatgcccgc 3900agcttcaggc aggcgctgct cgcctaccgc gatggcgcgc gcatccatgc cggcacgcga 3960ccgggcgcac cgcagatgga aacggccgac gcgcagcttc gcttcctctg cgaggcgggt 4020ttttcggccg gggacgccgt caatgcgctg atgacaatca gctacttcac tgttggggcc 4080gtgcttgagg agcaggccgg cgacagcgat gccggcgagc gcggcggcac cgttgaacag 4140gctccgctct cgccgctgtt gcgggccgcg atagacgcct tcgacgaagc cggtccggac 4200gcagcgttcg agcagggact cgcggtgatt gtcgatggat tggcgaaaag gaggctcgtt 4260gtcaggaacg ttgaaggacc gagaaagggt gacgattgat caggaccgct gccggagcgc 4320aacccactca ctacagcaga gccatgtaga caacatcccc tccccctttc caccgcgtca 4380gacgcccgta gcagcccgct acgggctttt tcatgccctg ccctagcgtc caagcctcac 4440ggccgcgctc ggcctctctg gcggccttct ggcgctcttc cgcttcctcg ctcactgact 4500cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4560ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4620aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4680acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4740gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4800ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgcttttc cgctgcataa 4860ccctgcttcg gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat 4920acaggatttt gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg 4980ggcaggatag gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat 5040tcgcacctgg cggtgctcaa cgggaatcct gctctgcgag gctggccggc taccgccggc 5100gtaacagatg agggcaagcg gatggctgat gaaaccaagc caaccaggaa gggcagccca 5160cctatcaagg tgtactgcct tccagacgaa cgaagagcga ttgaggaaaa ggcggcggcg 5220gccggcatga gcctgtcggc ctacctgctg gccgtcggcc agggctacaa aatcacgggc 5280gtcgtggact atgagcacgt ccgcgagctg gcccgcatca atggcgacct gggccgcctg 5340ggcggcctgc tgaaactctg gctcaccgac gacccgcgca cggcgcggtt cggtgatgcc 5400acgatcctcg ccctgctggc gaagatcgaa gagaagcagg acgagcttgg caaggtcatg 5460atgggcgtgg tccgcccgag ggcagagcca tgactttttt agccgctaaa acggccgggg 5520ggtgcgcgtg attgccaagc acgtccccat gcgctccatc aagaagagcg acttcgcgga 5580gctggtgaag tacatcaccg acgagcaagg caagaccgag cgcctttgcg acgctcaccg 5640ggctggttgc cctcgccgct gggctggcgg ccgtctatgg ccctgcaaac gcgccagaaa 5700cgccgtcgaa gccgtgtgcg agacaccgcg gccgccggcg ttgtggatac ctcgcggaaa 5760acttggccct cactgacaga tgaggggcgg acgttgacac ttgaggggcc gactcacccg 5820gcgcggcgtt gacagatgag gggcaggctc gatttcggcc ggcgacgtgg agctggccag 5880cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt cccacagatg atgtggacaa 5940gcctggggat aagtgccctg cggtattgac acttgagggg cgcgactact gacagatgag 6000gggcgcgatc cttgacactt gaggggcaga gtgctgacag atgaggggcg cacctattga 6060catttgaggg gctgtccaca ggcagaaaat ccagcatttg caagggtttc cgcccgtttt 6120tcggccaccg ctaacctgtc ttttaacctg cttttaaacc aatatttata aaccttgttt 6180ttaaccaggg ctgcgccctg tgcgcgtgac cgcgcacgcc gaaggggggt gccccccctt 6240ctcgaaccct cccggcccgc taacgcgggc ctcccatccc cccaggggct gcgcccctcg 6300gccgcgaacg gcctcacccc aaaaatggca gcgctggcag tccttgccat tgccgggatc 6360ggggcagtaa cgggatgggc gatcagcccg agcgcgacgc ccggaagcat tgacgtgccg 6420caggtgctgg catcgacatt cagcgaccag gtgccgggca gtgagggcgg cggcctgggt 6480ggcggcctgc ccttcacttc ggccgtcggg gcattcacgg acttcatggc ggggccggca 6540atttttacct tgggcattct tggcatagtg gtcgcgggtg ccgtgctcgt gttcgggggt 6600gcgataaacc cagcgaacca tttgaggtga taggtaagat tataccgagg tatgaaaacg 6660agaattggac ctttacagaa ttactctatg aagcgccata tttaaaaagc taccaagacg 6720aagaggatga agaggatgag gaggcagatt gccttgaata tattgacaat actgataaga 6780taatatatct tttatataga agatatcgcc gtatgtaagg atttcagggg gcaaggcata 6840ggcagcgcgc ttatcaatat atctatagaa tgggcaaagc ataaaaactt gcatggacta 6900atgcttgaaa cccaggacaa taaccttata gcttgtaaat tctatcataa ttgggtaatg 6960actccaactt attgatagtg ttttatgttc agataatgcc cgatgacttt gtcatgcagc 7020tccaccgatt ttgagaacga cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga 7080ttcaggttat gccgctcaat tcgctgcgta tatcgcttgc tgattacgtg cagctttccc 7140ttcaggcggg attcatacag cggccagcca tccgtcatcc atatcaccac gtcaaagggt 7200gacagcaggc tcataagacg ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca 7260acaaccgtct tccggagact gtcatacgcg taaaacagcc agcgctggcg cgatttagcc 7320ccgacatagc cccactgttc gtccatttcc gcgcagacga tgacgtcact gcccggctgt 7380atgcgcgagg ttaccgactg cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc 7440caacgcccat aatgcgggct gttgcccggc atccaacgcc attcatggcc atatcaatga 7500ttttctggtg cgtaccgggt tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac 7560ggcagtgaga gcagagatag cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc 7620cgtcagtagc tgaacaggag ggacagctga tagacacaga agccactgga gcacctcaaa 7680aacaccatca tacactaaat cagtaagttg gcagcatcac ccataattgt ggtttcaaaa 7740tcggctccgt cgatactatg ttatacgcca actttgaaaa caactttgaa aaagctgttt 7800tctggtattt aaggttttag aatgcaagga acagtgaatt ggagttcgtc ttgttataat 7860tagcttcttg gggtatcttt aaatactgta gaaaagagga aggaaataat aaatggctaa 7920aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac 7980ggaaggaatg tctcctgcta aggtatataa gctggtggga gaaaatgaaa acctatattt 8040aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat 8100gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg aacggcatga 8160tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga 8220agatgaacaa agccctgaaa agattatcga gctgtatgcg gagtgcatca ggctctttca 8280ctccatcgac atatcggatt gtccctatac gaatagctta gacagccgct tagccgaatt 8340ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg aagaagacac 8400tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga 8460acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt 8520aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt 8580ctgcgtccgg tcgatcaggg aggatatcgg ggaagaacag tatgtcgagc tattttttga 8640cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt 8700gttttagtac ctagatgtgg cgcaacgatg ccggcgacaa gcaggagcgc accgacttct 8760tccgcatcaa gtgttttggc tctcaggccg aggcccacgg caagtatttg ggcaaggggt 8820cgctggtatt cgtgcagggc aagattcgga ataccaagta cgagaaggac ggccagacgg 8880tctacgggac cgacttcatt gccgataagg tggattatct ggacaccaag gcaccaggcg 8940ggtcaaatca ggaataaggg cacattgccc cggcgtgagt cggggcaatc ccgcaaggag 9000ggtgaatgaa tcggacgttt gaccggaagg catacaggca agaactgatc gacgcggggt 9060tttccgccga ggatgccgaa accatcgcaa gccgcaccgt catgcgtgcg ccccgcgaaa 9120ccttccagtc cgtcggctcg atggtccagc aagctacggc caagatcgag cgcgacagcg 9180tgcaactggc tccccctgcc ctgcccgcgc catcggccgc cgtggagcgt tcgcgtcgtc 9240tcgaacagga ggcggcaggt ttggcgaagt cgatgaccat cgacacgcga ggaactatga 9300cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa acaggtcagc gaggccaagc 9360aggccgcgtt gctgaaacac acgaagcagc agatcaagga aatgcagctt tccttgttcg 9420atattgcgcc gtggccggac acgatgcgag cgatgccaaa cgacacggcc cgctctgccc 9480tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct gcaaaacaag gtcattttcc 9540acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga gctgcgggcc gacgatgacg 9600aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac ccctatcggc gagccgatca 9660ccttcacgtt ctacgagctt tgccaggacc tgggctggtc gatcaatggc cggtattaca 9720cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc gatgggcttc acgtccgacc 9780gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt ccgcgtcctg gaccgtggca 9840agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat cgtcgtgctg tttgctggcg 9900accactacac gaaattcata tgggagaagt accgcaagct gtcgccgacg gcccgacgga 9960tgttcgacta tttcagctcg caccgggagc cgtacccgct caagctggaa accttccgcc 10020tcatgtgcgg atcggattcc acccgcgtga agaagtggcg cgagcaggtc ggcgaagcct 10080gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg ggtcaatgat gacctggtgc 10140attgcaaacg ctagggcctt gtggggtcag ttccggctgg gggttcagca gccagcgctt 10200tactggcatt tcaggaacaa gcgggcactg ctcgacgcac ttgcttcgct cagtatcgct 10260cgggacgcac ggcgcgctct acgaactgcc gataaacaga ggattaaaat tgacaattgt 10320gattaaggct cagattcgac ggcttggagc ggccgacgtg caggatttcc gcgagatccg 10380attgtcggcc ctgaagaaag ctccagagat gttcgggtcc gtttacgagc acgaggagaa 10440aaagcccatg gaggcgttcg ctgaacggtt gcgagatgcc gtggcattcg gcgcctacat 10500cgacggcgag atcattgggc tgtcggtctt caaacaggag gacggcccca aggacgctca 10560caaggcgcat ctgtccggcg ttttcgtgga gcccgaacag cgaggccgag gggtcgccgg 10620tatgctgctg cgggcgttgc cggcgggttt attgctcgtg atgatcgtcc gacagattcc 10680aacgggaatc tggtggatgc gcatcttcat cctcggcgca cttaatattt cgctattctg 10740gagcttgttg tttatttcgg tctaccgcct gccgggcggg gtcgcggcga cggtaggcgc 10800tgtgcagccg ctgatggtcg tgttcatctc tgccgctctg ctaggtagcc cgatacgatt 10860gatggcggtc ctgggggcta tttgcggaac tgcgggcgtg gcgctgttgg tgttgacacc 10920aaacgcagcg ctagatcctg tcggcgtcgc agcgggcctg gcgggggcgg tttccatggc 10980gttcggaacc gtgctgaccc gcaagtggca acctcccgtg cctctgctca cctttaccgc 11040ctggcaactg gcggccggag gacttctgct cgttccagta gctttagtgt ttgatccgcc 11100aatcccgatg cctacaggaa ccaatgttct cggcctggcg tggctcggcc tgatcggagc 11160gggtttaacc tacttccttt ggttccgggg gatctcgcga ctcgaaccta cagttgtttc 11220cttactgggc tttctcagcc ccagatctgg ggtcgatcag ccggggatgc atcaggccga 11280cagtcggaac ttcgggtccc cgacctgtac cattcggtga gcaatggata ggggagttga 11340tatcgtcaac gttcacttct aaagaaatag cgccactcag cttcctcagc ggctttatcc 11400agcgatttcc tattatgtcg gcatagttct caagatcgac agcctgtcac ggttaagcga 11460gaaatgaata agaaggctga taattcggat ctctgcgagg gagatgatat ttgatcacag 11520gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt 11580tcaaacccgg cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct 11640gccgccttac aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga 11700gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat 11760attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa 11820tgtactgggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc 11880gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa 11940tcctgtttga tggtggttcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg 12000agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 12060ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 12120ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 12180gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 12240aagcgaaagg agcgggcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 12300gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 12360gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgccatct 12420tgaaagaaat atagtttaaa tatttattga taaaataagt caggtattat agtccaagca 12480aaaacataat ttattgatgc aaagtttaaa ttcagaaata tttcaataac tgattatatc 12540agctggtaca ttgccgtaga tgaaagactg agtgcgatat tatgtgtaat acataaattg 12600atgatatagc tagcttagct catcggggga tccttaatcg actctagcta gaacgaattg 12660ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact tcttcccgta tgcccaactt 12720tgtatagaga gccactgcgg gatcgtcacc gtaatctgct tgcacgtaga tcacataagc

12780accaagcgcg ttggcctcat gcttgaggag attgatgagc gcggtggcaa tgccctgcct 12840ccggtgctcg ccggagactg cgagatcata gatatagatc tcactacgcg gctgctcaaa 12900cctgggcaga acgtaagccg cgagagcgcc aacaaccgct tcttggtcga aggcagcaag 12960cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa tcggagtccg gctgatgttg 13020ggagtaggtg gctacgtctc cgaactcacg accgaaaaga tcaagagcag cccgcatgga 13080tttgacttgg tcagggccga gcctacatgt gcgaatgatg cccatacttg agccacctaa 13140ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctgcgta ccatggagat 13200ctggattgag agtgaatatg agactctaat tggataccga ggggaattta tggaagtcag 13260tggagcattt ttgacaagaa atatttgcta gctgatagtg accttaggcg acttttgaac 13320gcgcaataat ggtttctgac gtatgtgctt agctcattaa actccagaaa cccgcggctg 13380agtggctcct tcaacgttgc ggttctgtca gttccaaacg taaaacggct tgtcccgcgt 13440catcggcggg ggtcataacg tgactccctt aattctccgc tcatgatctt gatcccctgc 13500gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct 13560taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa accgcccagt 13620ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt 13680cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac 13740tggctttcta cgtgttccgc ttcctttagc agcccttgcg ccctgagtgc ttgcggcagc 13800gtgaagctct ggacatcatg ttggatatga aacaactatt atttatctac atgttttaga 13860tgttatctga ttatttttat accgtagtct tctattgatg aggagtctaa ggctatagaa 13920ttatatatct aaatgattaa tatatatatt attaataatt aacaataatt aatatattat 13980aatttatata tatatatttt atattattat aataatattc ttacaaatat aattattata 14040ttcgacggta tcgataagct cgggatccct gaaagcgacg ttggatgtta acatctacaa 14100attgcctttt cttatcgacc atgtacgtaa gcgcttacgt ttttggtgga cccttgagga 14160aactggtagc tgttgtgggc ctgtggtctc aagatggatc attaatttcc accttcacct 14220acgatggggg gcatcgcacc ggtgagtaat attgtacggc taagagcgaa tttggcctgt 14280aggatccctg aaagcgacgt tggatgttaa catctacaaa ttgccttttc ttatcgacca 14340tgtacgtaag cgcttacgtt tttggtggac ccttgaggaa actggtagct gttgtgggcc 14400tgtggtctca agatggatca ttaatttcca ccttcaccta cgatgggggg catcgcaccg 14460gtgagtaata ttgtacggct aagagcgaat ttggcctgta ggatccctga aagcgacgtt 14520ggatgttaac atctacaaat tgccttttct tatcgaccat gtacgtaagc gcttacgttt 14580ttggtggacc cttgaggaaa ctggtagctg ttgtgggcct gtggtctcaa gatggatcat 14640taatttccac cttcacctac gatggggggc atcgcaccgg tgagtaatat tgtacggcta 14700agagcgaatt tggcctgtag gatccgcgag ctggtcaatc ccattgcttt tgaagcagct 14760caacattgat ctctttctcg atcgagggag atttttcaaa tcagtgcgca agacgtgacg 14820taagtatccg agtcagtttt tatttttcta ctaatttggt cgtttatttc ggcgtgtagg 14880acatggcaac cgggcctgaa tttcgcgggt attctgtttc tattccaact ttttcttgat 14940ccgcagccat taacgacttt tgaatagata cgctgacacg ccaagcctcg ctagtcaaaa 15000gtgtaccaaa caacgcttta cagcaagaac ggaatgcgcg tgacgctcgc ggtgacgcca 15060tttcgccttt tcagaaatgg ataaatagcc ttgcttccta ttatatcttc ccccaaatta 15120attaagaaac tcccgaggtg agcaaggatc cggagtcgag cgcgaagaag agaaagaggg 15180aaagcgcggg taccgggccc ccccctcgac ggatcaagtg caaaggtccg ccttgtttct 15240cctctgtctc ttgatctgac taatcttggt ttatgattcg ttgagtaatt ttggggaaag 15300ctagcttcgt ccacagtttt tttttcgatg aacagtgccg cagtggcgct gatcttgtat 15360gctatcctgc aatcgtggtg aacttatttc ttttatatcc ttcactccca tgaaaaggct 15420agtaatcttt ctcgatgtaa catcgtccag cactgctatt accgtgtggt ccatccgaca 15480gtctggctga acacatcata cgatattgag caaagatcga tctatcttcc ctgttcttta 15540atgaaagacg tcattttcat cagtatgatc taagaatgtt gcaacttgca aggaggcgtt 15600tctttctttg aatttaacta actcgttgag tggccctgtt tctcggacgt aaggcctttg 15660ctgctccaca catgtccatt cgaattttac cgtgtttagc aagggcgaaa agtttgcatc 15720ttgatgattt agcttgacta tgcgattgct ttcctggacc cgtgcagctg cggacggatc 15780ccccgctcga agctagcttg atcagatctg atcgaattca ccatgggggc cgggcagtcg 15840tccccggcca cggggtccca gaaccagtcc ggcaacaccg gcagcatcat caacaactac 15900tacatgcagc agtaccagaa ctccatggac acccaactgg gggacaacgc cacgagcggc 15960ggcagcaatg agggctccac cgacaccacg agcacccaca cgaccaacac ccagaacaac 16020gactggttca gcaagctcgc gtccagcgcc ttctccgggc tcttcggggc gctcctggcc 16080gacaagaaga ccgaggagac cacgctgctc gaggaccgca tcctcaccac gcggaacggc 16140cacacgacct ccaccacgca gtcgagcgtc ggggtcactt acggttacgc cacggccgag 16200gacttcgtga gcggccccaa cacctccggc ctcgagactc gggtcgccca ggccgagcgc 16260ttcttcaaga cgcacctgtt cgactgggtg acctccgacc ccttcggccg ctgccacctc 16320ttggaactgc ccaccgacca caagggcgtg tacgggtcgt tgacggacag ctacgcgtac 16380atgaggaacg ggtgggacgt ggaggtcacc gccgtcggca accagttcaa cgggggctgc 16440ctcctggtgg ccatggtccc cgaactctgc tcgatccaga agcgggagct gtaccagctg 16500accctcttcc cgcaccagtt catcaacccg cgcaccaaca tgaccgcgca catcaccgtc 16560cccttcgtgg gcgtgaaccg ctacgaccag tacaaagtcc acaagccctg gaccttggtg 16620gtcatggtcg tcgcccccct caccgtcaac tcggaggggg cgccccagat caaagtctac 16680gccaacatcg cccccacgaa cgtgcacgtt gccggcgagt tcccgagcaa ggagggcatc 16740ttcccggttg cgtgctccga cggctacggg ggcctcgtga cgaccgaccc gaagaccgcc 16800gaccctgcct acggcaaggt gttcaacccg cccagaaaca tgctccctgg gagattcacg 16860aacttcctcg acgtggccga ggcctgcccc acgttcctgc acttcgaggg ggacgtccct 16920tacgtcacca cgaagactga ctcggatcgc gtgctggccc agttcgacct gtccctcgcc 16980gctaagcaca tgtccaacac cttcctcgcc ggcctcgcgc aatactacac ccagtacagc 17040ggcaccatca acctccactt catgttcacc ggccccaccg acgccaaggc ccgctacatg 17100atcgcttacg ccccgcccgg catggagccc ccgaagaccc ccgaggcggc cgcgcactgc 17160atccacgccg agtgggacac ggggctcaac tccaagttca cgttctccat cccctacctc 17220agcgccgcgg actacgcgta caccgccagc gacaccgcgg agaccacgaa cgtgcagggc 17280tgggtgtgcc tgttccagat cacccacggg aaggccgacg gcgacgccct ggtcgtgctg 17340gcctcggcgg ggaaggactt cgaactcagg ctgccggtgg acgcccgcac ccagacgact 17400agcgccggcg agtccgcgga cccggtgacc gcgaccgtcg agaactacgg gggcgagacc 17460caagtgcagc gcagacagca cacggacgtg tccttcatcc tggatcgctt cgtgaaagtg 17520accccgaagg accagatcaa cgtgctggac ctgatgcaga cgccggcgca caccctcgtg 17580ggggccctcc tgcgcaccgc cacgtactac ttcgcggacc tcgaggtggc ggtgaagcac 17640gagggcaacc tgacgtgggt gcccaacggt gcgccggagg ccgcgctcga caacaccacg 17700aaccccaccg cgtaccacaa ggcccccctg acccggctcg ccctccccta caccgccccc 17760cacagggtcc tcgccacggt ctacaacggc aacagcaagt acggggacgg gaccgtcgcc 17820aacgtgcgcg gggacctcca agtgctcgcc cagaaggccg cgcgcgccct ccccactagc 17880ttcaactacg gtgccatcaa ggccacgagg gtgacggagc tgctctaccg catgaagcgc 17940gccgagacct actgcccccg ccccctgctc gcgatccacc ccgaccaggc cagacacaag 18000cagaagatcg tcgcccctgt caagcagctc ctgaacttcg acctcctgaa gctggccggc 18060gacgtggaga gcaatccggg ctaagagct 180893418235DNAArtificial SequenceMERF05 plasmid 34cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 60gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 120catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 180cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 240tatgttacta gatcgggtgt acatctagaa gctctggaca tcatgttgga tatgaaacaa 300ctattattta tctacatgtt ttagatgtta tctgattatt tttataccgt agtcttctat 360tgatgaggag tctaaggcta tagaattata tatctaaatg attaatatat atattattaa 420taattaacaa taattaatat attataattt atatatatat attttatatt attataataa 480tattcttaca aatataatta ttatattcga cggtatcgat aagctcgggc ccaaatcgta 540cgggccggcc gtaagcttat cgatgaagga agtggaggag agaggacgcc atggtagcag 600aggaaggtct ggcttgatct cccgacgatt cctctctcat cagtgaaaca agagaataag 660aggcatcgcg attcttggaa ggtacagagg gaagttgatc aaagagaggc tccgggaaga 720agcagatggc ggggaagacg aaacatggcg cctgacaaca taggctatca taggataatc 780cactctccct ctgtctttct ctctgtttct ttctctctct ctttatctct ctctctctct 840cgaacatttc acacattttt gggcctgttc tttggcgtag ctagcccttt cttggtccat 900attttgaggc caaggccaat catgcgcagc cacgttgcat ggcgggagga ccccatctat 960ccattccgtc agttcctggc ttttgggaca atctgaacag tacataaacc acgggctcgg 1020gcttgggccc gccgaaaagc ccgtccgtac aattttctga cgtacaatat taatttccca 1080gaaaagaaaa ttcattaaaa aataactatg tcacccacga actcgtgatc tagatataag 1140gcaggcaact ttcccgtgac agtcggactt gtggggttcc cttgacggcg ccgccgtcta 1200ctgacggcgc ttgaatgacg tcattattac tatatttaat atatccgaga ataaatggct 1260ccctggatcc cccgtgaatg gtcacgtcat ccacgcgggc tttgacttcg cctaggtcgc 1320cacgttgggg cctcataacg tgcaagctga cgtggctatc attgcctcga tggcgatctt 1380gaacgaacca cataaccaat ctgagacggc gacgaggatt ctgtgttttc ctcggggatc 1440tctggccgtc cgatgaacta acacgcacta tttcaaaaag ggggttaaac acgattgtta 1500ggtttctttg agtcttccac atctccgctc cacgatcttg tacctcttct tgcgacgatc 1560tactcgccct cgaggtcgac accatgtccg gtgcgcctcc caccgacctg cagaagatgg 1620tgatgggcaa caccaagccc gtcgagttga tcctcgacgg gaagaccgtg gcgatctgct 1680gcgccaccgg cgtgttcggc accgcctacc tcgtcccgag acacctgttc gccgagaagt 1740atgacaagat catgctggac gggcgggcca tgaccgactc ggactaccgg gtcttcgagt 1800tcgagatcaa agtgaagggc caggatatgc tctccgacgc cgcgctgatg gtgctccaca 1860gaggcaaccg cgtgcgggac atcaccaagc acttcaggga caccgcgcgc atgaagaagg 1920ggacccctgt ggtcggggtc gtgaacaacg ccgacgtcgg gcgcctcatc ttctccggcg 1980aggcgctgac ctacaaggac atcgtcgtgt gcatggacgg ggacacgatg ccggggctct 2040tcgcctacaa ggccgcgacc aaggccgggt actgcggggg tgcggtgctc gccaaggacg 2100gcgccgacac gttcatcgtg ggcacccact ccgccggcgg taacggcgtc ggctactgct 2160cctgcgtgtc ccggtccatg ctgctgcgga tgaaggccca cgttgacccc gagccgcagc 2220acgagtaaaa gcttatcgat gtgcacgatc gttcaaacat ttggcaataa agtttcttaa 2280gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta 2340agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta 2400gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg 2460ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggtggacaac tcgtcacgta 2520ctagtggcgc gcccacgtga tttaaatcgt acgggccggc cgtatactct agtggatccc 2580ccaattcaga tcggctgagt ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa 2640aacggcttgt cccgcgtcat cggcgggggt cataacgtga ctcccttaat tctccgctca 2700tgatcagatt gtcgtttccc gccttcagtt taaactatca gtgtttgaca ggatatattg 2760gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta aaagggcgtg 2820aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt ccccagatct 2880ggcgccggcc agcgagacga gcaagattgg ccgccgcccg aaacgatccg acagcgcgcc 2940cagcacaggt gcgcaggcaa attgcaccaa cgcatacagc gccagcagaa tgccatagtg 3000ggcggtgacg tcgttcgagt gaaccagatc gcgcaggagg cccggcagca ccggcataat 3060caggccgatg ccgacagcgt cgagcgcgac agtgctcaga attacgatca ggggtatgtt 3120gggtttcacg tctggcctcc ggaccagcct ccgctggtcc gattgaacgc gcggattctt 3180tatcactgat aagttggtgg acatattatg tttatcagtg ataaagtgtc aagcatgaca 3240aagttgcagc cgaatacagt gatccgtgcc gccctggacc tgttgaacga ggtcggcgta 3300gacggtctga cgacacgcaa actggcggaa cggttggggg ttcagcagcc ggcgctttac 3360tggcacttca ggaacaagcg ggcgctgctc gacgcactgg ccgaagccat gctggcggag 3420aatcatacgc attcggtgcc gagagccgac gacgactggc gctcatttct gatcgggaat 3480gcccgcagct tcaggcaggc gctgctcgcc taccgcgatg gcgcgcgcat ccatgccggc 3540acgcgaccgg gcgcaccgca gatggaaacg gccgacgcgc agcttcgctt cctctgcgag 3600gcgggttttt cggccgggga cgccgtcaat gcgctgatga caatcagcta cttcactgtt 3660ggggccgtgc ttgaggagca ggccggcgac agcgatgccg gcgagcgcgg cggcaccgtt 3720gaacaggctc cgctctcgcc gctgttgcgg gccgcgatag acgccttcga cgaagccggt 3780ccggacgcag cgttcgagca gggactcgcg gtgattgtcg atggattggc gaaaaggagg 3840ctcgttgtca ggaacgttga aggaccgaga aagggtgacg attgatcagg accgctgccg 3900gagcgcaacc cactcactac agcagagcca tgtagacaac atcccctccc cctttccacc 3960gcgtcagacg cccgtagcag cccgctacgg gctttttcat gccctgccct agcgtccaag 4020cctcacggcc gcgctcggcc tctctggcgg ccttctggcg ctcttccgct tcctcgctca 4080ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4140taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4200agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4260cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4320tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4380tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg cttttccgct 4440gcataaccct gcttcggggt cattatagcg attttttcgg tatatccatc ctttttcgca 4500cgatatacag gattttgcca aagggttcgt gtagactttc cttggtgtat ccaacggcgt 4560cagccgggca ggataggtga agtaggccca cccgcgagcg ggtgttcctt cttcactgtc 4620ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccggctacc 4680gccggcgtaa cagatgaggg caagcggatg gctgatgaaa ccaagccaac caggaagggc 4740agcccaccta tcaaggtgta ctgccttcca gacgaacgaa gagcgattga ggaaaaggcg 4800gcggcggccg gcatgagcct gtcggcctac ctgctggccg tcggccaggg ctacaaaatc 4860acgggcgtcg tggactatga gcacgtccgc gagctggccc gcatcaatgg cgacctgggc 4920cgcctgggcg gcctgctgaa actctggctc accgacgacc cgcgcacggc gcggttcggt 4980gatgccacga tcctcgccct gctggcgaag atcgaagaga agcaggacga gcttggcaag 5040gtcatgatgg gcgtggtccg cccgagggca gagccatgac ttttttagcc gctaaaacgg 5100ccggggggtg cgcgtgattg ccaagcacgt ccccatgcgc tccatcaaga agagcgactt 5160cgcggagctg gtgaagtaca tcaccgacga gcaaggcaag accgagcgcc tttgcgacgc 5220tcaccgggct ggttgccctc gccgctgggc tggcggccgt ctatggccct gcaaacgcgc 5280cagaaacgcc gtcgaagccg tgtgcgagac accgcggccg ccggcgttgt ggatacctcg 5340cggaaaactt ggccctcact gacagatgag gggcggacgt tgacacttga ggggccgact 5400cacccggcgc ggcgttgaca gatgaggggc aggctcgatt tcggccggcg acgtggagct 5460ggccagcctc gcaaatcggc gaaaacgcct gattttacgc gagtttccca cagatgatgt 5520ggacaagcct ggggataagt gccctgcggt attgacactt gaggggcgcg actactgaca 5580gatgaggggc gcgatccttg acacttgagg ggcagagtgc tgacagatga ggggcgcacc 5640tattgacatt tgaggggctg tccacaggca gaaaatccag catttgcaag ggtttccgcc 5700cgtttttcgg ccaccgctaa cctgtctttt aacctgcttt taaaccaata tttataaacc 5760ttgtttttaa ccagggctgc gccctgtgcg cgtgaccgcg cacgccgaag gggggtgccc 5820ccccttctcg aaccctcccg gcccgctaac gcgggcctcc catcccccca ggggctgcgc 5880ccctcggccg cgaacggcct caccccaaaa atggcagcgc tggcagtcct tgccattgcc 5940gggatcgggg cagtaacggg atgggcgatc agcccgagcg cgacgcccgg aagcattgac 6000gtgccgcagg tgctggcatc gacattcagc gaccaggtgc cgggcagtga gggcggcggc 6060ctgggtggcg gcctgccctt cacttcggcc gtcggggcat tcacggactt catggcgggg 6120ccggcaattt ttaccttggg cattcttggc atagtggtcg cgggtgccgt gctcgtgttc 6180gggggtgcga taaacccagc gaaccatttg aggtgatagg taagattata ccgaggtatg 6240aaaacgagaa ttggaccttt acagaattac tctatgaagc gccatattta aaaagctacc 6300aagacgaaga ggatgaagag gatgaggagg cagattgcct tgaatatatt gacaatactg 6360ataagataat atatctttta tatagaagat atcgccgtat gtaaggattt cagggggcaa 6420ggcataggca gcgcgcttat caatatatct atagaatggg caaagcataa aaacttgcat 6480ggactaatgc ttgaaaccca ggacaataac cttatagctt gtaaattcta tcataattgg 6540gtaatgactc caacttattg atagtgtttt atgttcagat aatgcccgat gactttgtca 6600tgcagctcca ccgattttga gaacgacagc gacttccgtc ccagccgtgc caggtgctgc 6660ctcagattca ggttatgccg ctcaattcgc tgcgtatatc gcttgctgat tacgtgcagc 6720tttcccttca ggcgggattc atacagcggc cagccatccg tcatccatat caccacgtca 6780aagggtgaca gcaggctcat aagacgcccc agcgtcgcca tagtgcgttc accgaatacg 6840tgcgcaacaa ccgtcttccg gagactgtca tacgcgtaaa acagccagcg ctggcgcgat 6900ttagccccga catagcccca ctgttcgtcc atttccgcgc agacgatgac gtcactgccc 6960ggctgtatgc gcgaggttac cgactgcggc ctgagttttt taagtgacgt aaaatcgtgt 7020tgaggccaac gcccataatg cgggctgttg cccggcatcc aacgccattc atggccatat 7080caatgatttt ctggtgcgta ccgggttgag aagcggtgta agtgaactgc agttgccatg 7140ttttacggca gtgagagcag agatagcgct gatgtccggc ggtgcttttg ccgttacgca 7200ccaccccgtc agtagctgaa caggagggac agctgataga cacagaagcc actggagcac 7260ctcaaaaaca ccatcataca ctaaatcagt aagttggcag catcacccat aattgtggtt 7320tcaaaatcgg ctccgtcgat actatgttat acgccaactt tgaaaacaac tttgaaaaag 7380ctgttttctg gtatttaagg ttttagaatg caaggaacag tgaattggag ttcgtcttgt 7440tataattagc ttcttggggt atctttaaat actgtagaaa agaggaagga aataataaat 7500ggctaaaatg agaatatcac cggaattgaa aaaactgatc gaaaaatacc gctgcgtaaa 7560agatacggaa ggaatgtctc ctgctaaggt atataagctg gtgggagaaa atgaaaacct 7620atatttaaaa atgacggaca gccggtataa agggaccacc tatgatgtgg aacgggaaaa 7680ggacatgatg ctatggctgg aaggaaagct gcctgttcca aaggtcctgc actttgaacg 7740gcatgatggc tggagcaatc tgctcatgag tgaggccgat ggcgtccttt gctcggaaga 7800gtatgaagat gaacaaagcc ctgaaaagat tatcgagctg tatgcggagt gcatcaggct 7860ctttcactcc atcgacatat cggattgtcc ctatacgaat agcttagaca gccgcttagc 7920cgaattggat tacttactga ataacgatct ggccgatgtg gattgcgaaa actgggaaga 7980agacactcca tttaaagatc cgcgcgagct gtatgatttt ttaaagacgg aaaagcccga 8040agaggaactt gtcttttccc acggcgacct gggagacagc aacatctttg tgaaagatgg 8100caaagtaagt ggctttattg atcttgggag aagcggcagg gcggacaagt ggtatgacat 8160tgccttctgc gtccggtcga tcagggagga tatcggggaa gaacagtatg tcgagctatt 8220ttttgactta ctggggatca agcctgattg ggagaaaata aaatattata ttttactgga 8280tgaattgttt tagtacctag atgtggcgca acgatgccgg cgacaagcag gagcgcaccg 8340acttcttccg catcaagtgt tttggctctc aggccgaggc ccacggcaag tatttgggca 8400aggggtcgct ggtattcgtg cagggcaaga ttcggaatac caagtacgag aaggacggcc 8460agacggtcta cgggaccgac ttcattgccg ataaggtgga ttatctggac accaaggcac 8520caggcgggtc aaatcaggaa taagggcaca ttgccccggc gtgagtcggg gcaatcccgc 8580aaggagggtg aatgaatcgg acgtttgacc ggaaggcata caggcaagaa ctgatcgacg 8640cggggttttc cgccgaggat gccgaaacca tcgcaagccg caccgtcatg cgtgcgcccc 8700gcgaaacctt ccagtccgtc ggctcgatgg tccagcaagc tacggccaag atcgagcgcg 8760acagcgtgca actggctccc cctgccctgc ccgcgccatc ggccgccgtg gagcgttcgc 8820gtcgtctcga acaggaggcg gcaggtttgg cgaagtcgat gaccatcgac acgcgaggaa 8880ctatgacgac caagaagcga aaaaccgccg gcgaggacct ggcaaaacag gtcagcgagg 8940ccaagcaggc cgcgttgctg aaacacacga agcagcagat caaggaaatg cagctttcct 9000tgttcgatat tgcgccgtgg ccggacacga tgcgagcgat gccaaacgac acggcccgct 9060ctgccctgtt caccacgcgc aacaagaaaa tcccgcgcga ggcgctgcaa aacaaggtca 9120ttttccacgt caacaaggac gtgaagatca cctacaccgg cgtcgagctg cgggccgacg 9180atgacgaact ggtgtggcag caggtgttgg agtacgcgaa gcgcacccct atcggcgagc 9240cgatcacctt cacgttctac gagctttgcc aggacctggg ctggtcgatc aatggccggt 9300attacacgaa ggccgaggaa tgcctgtcgc gcctacaggc gacggcgatg ggcttcacgt 9360ccgaccgcgt tgggcacctg gaatcggtgt cgctgctgca ccgcttccgc gtcctggacc 9420gtggcaagaa aacgtcccgt tgccaggtcc tgatcgacga ggaaatcgtc gtgctgtttg 9480ctggcgacca ctacacgaaa ttcatatggg agaagtaccg caagctgtcg ccgacggccc 9540gacggatgtt cgactatttc agctcgcacc gggagccgta cccgctcaag ctggaaacct 9600tccgcctcat gtgcggatcg gattccaccc gcgtgaagaa gtggcgcgag caggtcggcg 9660aagcctgcga agagttgcga

ggcagcggcc tggtggaaca cgcctgggtc aatgatgacc 9720tggtgcattg caaacgctag ggccttgtgg ggtcagttcc ggctgggggt tcagcagcca 9780gcgctttact ggcatttcag gaacaagcgg gcactgctcg acgcacttgc ttcgctcagt 9840atcgctcggg acgcacggcg cgctctacga actgccgata aacagaggat taaaattgac 9900aattgtgatt aaggctcaga ttcgacggct tggagcggcc gacgtgcagg atttccgcga 9960gatccgattg tcggccctga agaaagctcc agagatgttc gggtccgttt acgagcacga 10020ggagaaaaag cccatggagg cgttcgctga acggttgcga gatgccgtgg cattcggcgc 10080ctacatcgac ggcgagatca ttgggctgtc ggtcttcaaa caggaggacg gccccaagga 10140cgctcacaag gcgcatctgt ccggcgtttt cgtggagccc gaacagcgag gccgaggggt 10200cgccggtatg ctgctgcggg cgttgccggc gggtttattg ctcgtgatga tcgtccgaca 10260gattccaacg ggaatctggt ggatgcgcat cttcatcctc ggcgcactta atatttcgct 10320attctggagc ttgttgttta tttcggtcta ccgcctgccg ggcggggtcg cggcgacggt 10380aggcgctgtg cagccgctga tggtcgtgtt catctctgcc gctctgctag gtagcccgat 10440acgattgatg gcggtcctgg gggctatttg cggaactgcg ggcgtggcgc tgttggtgtt 10500gacaccaaac gcagcgctag atcctgtcgg cgtcgcagcg ggcctggcgg gggcggtttc 10560catggcgttc ggaaccgtgc tgacccgcaa gtggcaacct cccgtgcctc tgctcacctt 10620taccgcctgg caactggcgg ccggaggact tctgctcgtt ccagtagctt tagtgtttga 10680tccgccaatc ccgatgccta caggaaccaa tgttctcggc ctggcgtggc tcggcctgat 10740cggagcgggt ttaacctact tcctttggtt ccgggggatc tcgcgactcg aacctacagt 10800tgtttcctta ctgggctttc tcagccccag atctggggtc gatcagccgg ggatgcatca 10860ggccgacagt cggaacttcg ggtccccgac ctgtaccatt cggtgagcaa tggatagggg 10920agttgatatc gtcaacgttc acttctaaag aaatagcgcc actcagcttc ctcagcggct 10980ttatccagcg atttcctatt atgtcggcat agttctcaag atcgacagcc tgtcacggtt 11040aagcgagaaa tgaataagaa ggctgataat tcggatctct gcgagggaga tgatatttga 11100tcacaggcag caacgctctg tcatcgttac aatcaacatg ctaccctccg cgagatcatc 11160cgtgtttcaa acccggcagc ttagttgccg ttcttccgaa tagcatcggt aacatgagca 11220aagtctgccg ccttacaacg gctctcccgc tgacgccgtc ccggactgat gggctgcctg 11280tatcgagtgg tgattttgtg ccgagctgcc ggtcggggag ctgttggctg gctggtggca 11340ggatatattg tggtgtaaac aaattgacgc ttagacaact taataacaca ttgcggacgt 11400ttttaatgta ctggggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 11460ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg 11520cgaaaatcct gtttgatggt ggttccgaaa tcggcaaaat cccttataaa tcaaaagaat 11580agcccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 11640tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 11700catcacccaa atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 11760aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 11820ggaagaaagc gaaaggagcg ggcgccattc aggctgcgca actgttggga agggcgatcg 11880gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 11940agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaatta 12000attcccatct tgaaagaaat atagtttaaa tatttattga taaaataaca agtcaggtat 12060tatagtccaa gcaaaaacat aaatttattg atgcaagttt aaattcagaa atatttcaat 12120aactgattat atcagctggt acattgccgt agatgaaaga ctgagtgcga tattatgtgt 12180aatacataaa ttgatgatat agctagctta gctcatcggg ggatccgtcg aagctagctt 12240gggtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg 12300agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc 12360aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca 12420gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc 12480atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc 12540ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc 12600catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc 12660cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg 12720agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct 12780tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca 12840cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac 12900aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat 12960tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc 13020gtgcaatcca tcttgttcaa tccaagctcc catgggccct cgactagagt cgagatctgg 13080attgagagtg aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg 13140agcatttttg acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg 13200caataatggt ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctgagt 13260ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat 13320cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcttgat cccctgcgcc 13380atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc ccaaccttac 13440cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc gcccagtcta 13500gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt gcgttttccc 13560ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc tgcggactgg 13620ctttctacgt gttccgcttc ctttagcagc ccttgcgccc tgagtgcttg cggcagcgtg 13680aagctctgga catcatgttg gatatgaaac aactattatt tatctacatg ttttagatgt 13740tatctgatta tttttatacc gtagtcttct attgatgagg agtctaaggc tatagaatta 13800tatatctaaa tgattaatat atatattatt aataattaac aataattaat atattataat 13860ttatatatat atattttata ttattataat aatattctta caaatataat tattatattc 13920gacggtatcg atgatggaca gataatgaga tgaattagaa aaaaaaaatt cgtgttgtaa 13980gatagaatac ttgctatcta ctgatgaatg cagttcagtt ttcctcacga tcttaaagat 14040cgcgcactat cctcagcttc actctggaaa ttttgattct cttcttctgc tcagcagcct 14100cgactctgtc tagggtttcg tacaatcgga cgccattcta catgaatcga gcacagggaa 14160tgaagacaat taggagatcc tcgatgtcct ccgacttact tgcatgactt gacggggaag 14220atctcgagca gggaagcgac gcctctccgg aggactcgcc tcgccgagag gacctcctcc 14280gcgacacgga ccatggcctc cacggggtag aagctggccc tgttctttat tctcttgagg 14340atcatcggcc gaagcctccg caaatccatc cccgaggagt agaatctcgc ctgcaggaag 14400catctgtcga gatcctcgcc gaggcggcgg agatacctcg ccggcgccgc catggcgccg 14460gggacggagc accaccacgg agaagaagaa ccctaaccca aggcattaac gaagttgcgc 14520agattataca aaagccctca aatatctttc attttctatt tcactgatac attttcatta 14580ttgtatatga gtgtttattt aaattattcc gtattagaaa agcacctcca gaacccgaca 14640aaatagggtg acgtcatcat ggtgtcatga ccgcccaaca gccgcagatt taaaatcggt 14700ggatgagtgc ggccacgcca cgaaagcgat gggccttcgt cgatgccgtg agaatccatc 14760tgacataaag taaacggcgc cgtcagtatt gacggcgtat gacacgtgga aagaagctat 14820tggttcacgc atcggtggtt ccgctagcct ccgtccaccg ctagtactat aaatacggtc 14880ccgaggcctc ctcaccactc gcacatatcc tctttgtttt cctctccgtg aaagaagcga 14940ggaagcgcgt cgtctctccc aaggtaagga gcagatctct ttgatcgttt ttgttcttct 15000tttgttttgt tttttttttc tgcggatctt cggttgcatc atgccttggc tgtttttatt 15060agtttaggat atcctcgttt ggatctgagc cgatcatata tgttaaaggt tgtgttcgat 15120ctctttgttc attttcgcat gaaaaggatg tatccttttg atgtgaggcg atcttctatg 15180gttaagactt tgttcggtct attgatcatt tctgttcttc gtttttgagt ttttttctgc 15240ggatatcgca tcatccctag gtttttgctt tggttaggat gcatcctttg gatttgagcc 15300gatctccctt ggttaaggct gtgtctgttg cagaggagaa agtctgtcga ggtccttatg 15360caggctttgt ccagatgcgc gtgctctctc atgctatgaa tttatgtttt gagaactcct 15420cccggttttt ctagatccgg atttgaagta ttcattgcgg ttccccttcg gttttatgta 15480tttctcgagt tgatttggtc catgatcgtg ttctgtccag atctctcttg atatggatga 15540gatattcgtt acctctttca aacatcggtg gatgttcttt ttagtcttgg ctcaccttta 15600tctagaaatt aattttcggt ttgaaacccc tgcttgttaa ggtgatgtat tccttcttta 15660tagatttcgg tgtgttattt cttaacggtg atctgtccga tccatgtgtt gcacctcttg 15720ttttctgtgt aatcctctgt gaattataat tatgttttga aaacgtactt aagtaagggg 15780catgttcccc gtttaaaact tttgttctat caatttgtgg ttaatagatc ctgatttgtg 15840gtcgccttat tctgtcttta atcgtggatt ttatttatct tgagcgcgtc cttttctttt 15900aaaatcatgt gtttaacctt tcagtcgtca tatgttccat caggtcgacg tacaagggcg 15960aattcaccat gggggccggg cagtcgtccc cggccacggg gtcccagaac cagtccggca 16020acaccggcag catcatcaac aactactaca tgcagcagta ccagaactcc atggacaccc 16080aactggggga caacgccacg agcggcggca gcaatgaggg ctccaccgac accacgagca 16140cccacacgac caacacccag aacaacgact ggttcagcaa gctcgcgtcc agcgccttct 16200ccgggctctt cggggcgctc ctggccgaca agaagaccga ggagaccacg ctgctcgagg 16260accgcatcct caccacgcgg aacggccaca cgacctccac cacgcagtcg agcgtcgggg 16320tcacttacgg ttacgccacg gccgaggact tcgtgagcgg ccccaacacc tccggcctcg 16380agactcgggt cgcccaggcc gagcgcttct tcaagacgca cctgttcgac tgggtgacct 16440ccgacccctt cggccgctgc cacctcttgg aactgcccac cgaccacaag ggcgtgtacg 16500ggtcgttgac ggacagctac gcgtacatga ggaacgggtg ggacgtggag gtcaccgccg 16560tcggcaacca gttcaacggg ggctgcctcc tggtggccat ggtccccgaa ctctgctcga 16620tccagaagcg ggagctgtac cagctgaccc tcttcccgca ccagttcatc aacccgcgca 16680ccaacatgac cgcgcacatc accgtcccct tcgtgggcgt gaaccgctac gaccagtaca 16740aagtccacaa gccctggacc ttggtggtca tggtcgtcgc ccccctcacc gtcaactcgg 16800agggggcgcc ccagatcaaa gtctacgcca acatcgcccc cacgaacgtg cacgttgccg 16860gcgagttccc gagcaaggag ggcatcttcc cggttgcgtg ctccgacggc tacgggggcc 16920tcgtgacgac cgacccgaag accgccgacc ctgcctacgg caaggtgttc aacccgccca 16980gaaacatgct ccctgggaga ttcacgaact tcctcgacgt ggccgaggcc tgccccacgt 17040tcctgcactt cgagggggac gtcccttacg tcaccacgaa gactgactcg gatcgcgtgc 17100tggcccagtt cgacctgtcc ctcgccgcta agcacatgtc caacaccttc ctcgccggcc 17160tcgcgcaata ctacacccag tacagcggca ccatcaacct ccacttcatg ttcaccggcc 17220ccaccgacgc caaggcccgc tacatgatcg cttacgcccc gcccggcatg gagcccccga 17280agacccccga ggcggccgcg cactgcatcc acgccgagtg ggacacgggg ctcaactcca 17340agttcacgtt ctccatcccc tacctcagcg ccgcggacta cgcgtacacc gccagcgaca 17400ccgcggagac cacgaacgtg cagggctggg tgtgcctgtt ccagatcacc cacgggaagg 17460ccgacggcga cgccctggtc gtgctggcct cggcggggaa ggacttcgaa ctcaggctgc 17520cggtggacgc ccgcacccag acgactagcg ccggcgagtc cgcggacccg gtgaccgcga 17580ccgtcgagaa ctacgggggc gagacccaag tgcagcgcag acagcacacg gacgtgtcct 17640tcatcctgga tcgcttcgtg aaagtgaccc cgaaggacca gatcaacgtg ctggacctga 17700tgcagacgcc ggcgcacacc ctcgtggggg ccctcctgcg caccgccacg tactacttcg 17760cggacctcga ggtggcggtg aagcacgagg gcaacctgac gtgggtgccc aacggtgcgc 17820cggaggccgc gctcgacaac accacgaacc ccaccgcgta ccacaaggcc cccctgaccc 17880ggctcgccct cccctacacc gccccccaca gggtcctcgc cacggtctac aacggcaaca 17940gcaagtacgg ggacgggacc gtcgccaacg tgcgcgggga cctccaagtg ctcgcccaga 18000aggccgcgcg cgccctcccc actagcttca actacggtgc catcaaggcc acgagggtga 18060cggagctgct ctaccgcatg aagcgcgccg agacctactg cccccgcccc ctgctcgcga 18120tccaccccga ccaggccaga cacaagcaga agatcgtcgc ccctgtcaag cagctcctga 18180acttcgacct cctgaagctg gccggcgacg tggagagcaa tccgggctaa gagct 182353518649DNAArtificial SequenceMERF06 plasmid 35cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 60gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 120catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 180cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 240tatgttacta gatcgggtgt acatctagaa gctctggaca tcatgttgga tatgaaacaa 300ctattattta tctacatgtt ttagatgtta tctgattatt tttataccgt agtcttctat 360tgatgaggag tctaaggcta tagaattata tatctaaatg attaatatat atattattaa 420taattaacaa taattaatat attataattt atatatatat attttatatt attataataa 480tattcttaca aatataatta ttatattcga cggtatcgat aagctcgggc ccagcttctg 540ccatgcatca ttcatcatat ttaggtcatg aaatgatgcc ctttagtagc tatgatagga 600aaacaaattc tgtcgcgcac cattcatcat atttggggcc atcttcattc ttgagaggga 660gggtcctctt tgaggaagga taatttatct tggtttcaga gaaaatactt gaagtgggtg 720ccaacttggt tgagcagtga aactagacat aaagattaca tgatgttctt ggtggtcgta 780atatgtcaaa tagatggtat gctaaaaaac agttaaaact agatgagaag attgtgaaag 840actgatttgc cagagaaaat ctagtagata tttattggag ttggcctagc atgtggggca 900gtgcatgctt gaatgccccc tgatcatggg tagatgtcag ccacaaggag cccaataaac 960cttgggcttt gataccatgt taaataaaaa atatatatgc ctcatatata tgatctataa 1020actcctaaat tgcatctaaa cactcgatgg aagaagggtg aaatcagttt atcgatttaa 1080ctaaattaat ttattttggg ggtttttttg gcttttaaaa tgggtttgaa gataactctc 1140tctctctctc cccccgccca tgcaaagagg tcggcaattt tagataaaga cgtccatttt 1200ttcgacgcgt gtcgttacgt ggcgaaacgt cgtggaagga cgagtctttg agggcacgcg 1260tcagactatc gtggtcatcc tctgcctact tatatccctc tggtcttctt cttcttaatt 1320aagaaactcc cgaggtgagc aaggatccgg agtcgagcgc gaagaagaga aagagggaaa 1380gcgcgggtac cgggcccccc cctcgacgga tcaagtgcaa aggtccgcct tgtttctcct 1440ctgtctcttg atctgactaa tcttggttta tgattcgttg agtaattttg gggaaagcta 1500gcttcgtcca cagttttttt ttcgatgaac agtgccgcag tggcgctgat cttgtatgct 1560atcctgcaat cgtggtgaac ttatttcttt tatatccttc actcccatga aaaggctagt 1620aatctttctc gatgtaacat cgtccagcac tgctattacc gtgtggtcca tccgacagtc 1680tggctgaaca catcatacga tattgagcaa agatcgatct atcttccctg ttctttaatg 1740aaagacgtca ttttcatcag tatgatctaa gaatgttgca acttgcaagg aggcgtttct 1800ttctttgaat ttaactaact cgttgagtgg ccctgtttct cggacgtaag gcctttgctg 1860ctccacacat gtccattcga attttaccgt gtttagcaag ggcgaaaagt ttgcatcttg 1920atgatttagc ttgactatgc gattgctttc ctggacccgt gcagctgcgg acggatcccc 1980cgctcgaggt cgacaccatg tccggtgcgc ctcccaccga cctgcagaag atggtgatgg 2040gcaacaccaa gcccgtcgag ttgatcctcg acgggaagac cgtggcgatc tgctgcgcca 2100ccggcgtgtt cggcaccgcc tacctcgtcc cgagacacct gttcgccgag aagtatgaca 2160agatcatgct ggacgggcgg gccatgaccg actcggacta ccgggtcttc gagttcgaga 2220tcaaagtgaa gggccaggat atgctctccg acgccgcgct gatggtgctc cacagaggca 2280accgcgtgcg ggacatcacc aagcacttca gggacaccgc gcgcatgaag aaggggaccc 2340ctgtggtcgg ggtcgtgaac aacgccgacg tcgggcgcct catcttctcc ggcgaggcgc 2400tgacctacaa ggacatcgtc gtgtgcatgg acggggacac gatgccgggg ctcttcgcct 2460acaaggccgc gaccaaggcc gggtactgcg ggggtgcggt gctcgccaag gacggcgccg 2520acacgttcat cgtgggcacc cactccgccg gcggtaacgg cgtcggctac tgctcctgcg 2580tgtcccggtc catgctgctg cggatgaagg cccacgttga ccccgagccg cagcacgagt 2640aaaagcttat cgatgtgcac gatcgttcaa acatttggca ataaagtttc ttaagattga 2700atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg 2760taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc 2820cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat 2880tatcgcgcgc ggtgtcatct atgttactag atcgggtgga caactcgtca cgtactagtg 2940gcgcgcccac gtgatttaaa tcgtacgggc cggccgtata ctctagtgga tcccccaatt 3000cagatcggct gagtggctcc ttcaacgttg cggttctgtc agttccaaac gtaaaacggc 3060ttgtcccgcg tcatcggcgg gggtcataac gtgactccct taattctccg ctcatgatca 3120gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt 3180aaacctaaga gaaaagagcg tttattagaa taatcggata tttaaaaggg cgtgaaaagg 3240tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttccccag atctggcgcc 3300ggccagcgag acgagcaaga ttggccgccg cccgaaacga tccgacagcg cgcccagcac 3360aggtgcgcag gcaaattgca ccaacgcata cagcgccagc agaatgccat agtgggcggt 3420gacgtcgttc gagtgaacca gatcgcgcag gaggcccggc agcaccggca taatcaggcc 3480gatgccgaca gcgtcgagcg cgacagtgct cagaattacg atcaggggta tgttgggttt 3540cacgtctggc ctccggacca gcctccgctg gtccgattga acgcgcggat tctttatcac 3600tgataagttg gtggacatat tatgtttatc agtgataaag tgtcaagcat gacaaagttg 3660cagccgaata cagtgatccg tgccgccctg gacctgttga acgaggtcgg cgtagacggt 3720ctgacgacac gcaaactggc ggaacggttg ggggttcagc agccggcgct ttactggcac 3780ttcaggaaca agcgggcgct gctcgacgca ctggccgaag ccatgctggc ggagaatcat 3840acgcattcgg tgccgagagc cgacgacgac tggcgctcat ttctgatcgg gaatgcccgc 3900agcttcaggc aggcgctgct cgcctaccgc gatggcgcgc gcatccatgc cggcacgcga 3960ccgggcgcac cgcagatgga aacggccgac gcgcagcttc gcttcctctg cgaggcgggt 4020ttttcggccg gggacgccgt caatgcgctg atgacaatca gctacttcac tgttggggcc 4080gtgcttgagg agcaggccgg cgacagcgat gccggcgagc gcggcggcac cgttgaacag 4140gctccgctct cgccgctgtt gcgggccgcg atagacgcct tcgacgaagc cggtccggac 4200gcagcgttcg agcagggact cgcggtgatt gtcgatggat tggcgaaaag gaggctcgtt 4260gtcaggaacg ttgaaggacc gagaaagggt gacgattgat caggaccgct gccggagcgc 4320aacccactca ctacagcaga gccatgtaga caacatcccc tccccctttc caccgcgtca 4380gacgcccgta gcagcccgct acgggctttt tcatgccctg ccctagcgtc caagcctcac 4440ggccgcgctc ggcctctctg gcggccttct ggcgctcttc cgcttcctcg ctcactgact 4500cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4560ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4620aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4680acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4740gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4800ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgcttttc cgctgcataa 4860ccctgcttcg gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat 4920acaggatttt gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg 4980ggcaggatag gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat 5040tcgcacctgg cggtgctcaa cgggaatcct gctctgcgag gctggccggc taccgccggc 5100gtaacagatg agggcaagcg gatggctgat gaaaccaagc caaccaggaa gggcagccca 5160cctatcaagg tgtactgcct tccagacgaa cgaagagcga ttgaggaaaa ggcggcggcg 5220gccggcatga gcctgtcggc ctacctgctg gccgtcggcc agggctacaa aatcacgggc 5280gtcgtggact atgagcacgt ccgcgagctg gcccgcatca atggcgacct gggccgcctg 5340ggcggcctgc tgaaactctg gctcaccgac gacccgcgca cggcgcggtt cggtgatgcc 5400acgatcctcg ccctgctggc gaagatcgaa gagaagcagg acgagcttgg caaggtcatg 5460atgggcgtgg tccgcccgag ggcagagcca tgactttttt agccgctaaa acggccgggg 5520ggtgcgcgtg attgccaagc acgtccccat gcgctccatc aagaagagcg acttcgcgga 5580gctggtgaag tacatcaccg acgagcaagg caagaccgag cgcctttgcg acgctcaccg 5640ggctggttgc cctcgccgct gggctggcgg ccgtctatgg ccctgcaaac gcgccagaaa 5700cgccgtcgaa gccgtgtgcg agacaccgcg gccgccggcg ttgtggatac ctcgcggaaa 5760acttggccct cactgacaga tgaggggcgg acgttgacac ttgaggggcc gactcacccg 5820gcgcggcgtt gacagatgag gggcaggctc gatttcggcc ggcgacgtgg agctggccag 5880cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt cccacagatg atgtggacaa 5940gcctggggat aagtgccctg cggtattgac acttgagggg cgcgactact gacagatgag 6000gggcgcgatc cttgacactt gaggggcaga gtgctgacag atgaggggcg cacctattga 6060catttgaggg gctgtccaca ggcagaaaat ccagcatttg caagggtttc cgcccgtttt 6120tcggccaccg ctaacctgtc ttttaacctg cttttaaacc aatatttata aaccttgttt 6180ttaaccaggg ctgcgccctg tgcgcgtgac cgcgcacgcc gaaggggggt gccccccctt 6240ctcgaaccct cccggcccgc taacgcgggc ctcccatccc cccaggggct gcgcccctcg 6300gccgcgaacg gcctcacccc aaaaatggca gcgctggcag tccttgccat tgccgggatc 6360ggggcagtaa cgggatgggc gatcagcccg agcgcgacgc ccggaagcat tgacgtgccg 6420caggtgctgg catcgacatt cagcgaccag gtgccgggca

gtgagggcgg cggcctgggt 6480ggcggcctgc ccttcacttc ggccgtcggg gcattcacgg acttcatggc ggggccggca 6540atttttacct tgggcattct tggcatagtg gtcgcgggtg ccgtgctcgt gttcgggggt 6600gcgataaacc cagcgaacca tttgaggtga taggtaagat tataccgagg tatgaaaacg 6660agaattggac ctttacagaa ttactctatg aagcgccata tttaaaaagc taccaagacg 6720aagaggatga agaggatgag gaggcagatt gccttgaata tattgacaat actgataaga 6780taatatatct tttatataga agatatcgcc gtatgtaagg atttcagggg gcaaggcata 6840ggcagcgcgc ttatcaatat atctatagaa tgggcaaagc ataaaaactt gcatggacta 6900atgcttgaaa cccaggacaa taaccttata gcttgtaaat tctatcataa ttgggtaatg 6960actccaactt attgatagtg ttttatgttc agataatgcc cgatgacttt gtcatgcagc 7020tccaccgatt ttgagaacga cagcgacttc cgtcccagcc gtgccaggtg ctgcctcaga 7080ttcaggttat gccgctcaat tcgctgcgta tatcgcttgc tgattacgtg cagctttccc 7140ttcaggcggg attcatacag cggccagcca tccgtcatcc atatcaccac gtcaaagggt 7200gacagcaggc tcataagacg ccccagcgtc gccatagtgc gttcaccgaa tacgtgcgca 7260acaaccgtct tccggagact gtcatacgcg taaaacagcc agcgctggcg cgatttagcc 7320ccgacatagc cccactgttc gtccatttcc gcgcagacga tgacgtcact gcccggctgt 7380atgcgcgagg ttaccgactg cggcctgagt tttttaagtg acgtaaaatc gtgttgaggc 7440caacgcccat aatgcgggct gttgcccggc atccaacgcc attcatggcc atatcaatga 7500ttttctggtg cgtaccgggt tgagaagcgg tgtaagtgaa ctgcagttgc catgttttac 7560ggcagtgaga gcagagatag cgctgatgtc cggcggtgct tttgccgtta cgcaccaccc 7620cgtcagtagc tgaacaggag ggacagctga tagacacaga agccactgga gcacctcaaa 7680aacaccatca tacactaaat cagtaagttg gcagcatcac ccataattgt ggtttcaaaa 7740tcggctccgt cgatactatg ttatacgcca actttgaaaa caactttgaa aaagctgttt 7800tctggtattt aaggttttag aatgcaagga acagtgaatt ggagttcgtc ttgttataat 7860tagcttcttg gggtatcttt aaatactgta gaaaagagga aggaaataat aaatggctaa 7920aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac 7980ggaaggaatg tctcctgcta aggtatataa gctggtggga gaaaatgaaa acctatattt 8040aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat 8100gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg aacggcatga 8160tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga 8220agatgaacaa agccctgaaa agattatcga gctgtatgcg gagtgcatca ggctctttca 8280ctccatcgac atatcggatt gtccctatac gaatagctta gacagccgct tagccgaatt 8340ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg aagaagacac 8400tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga 8460acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt 8520aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt 8580ctgcgtccgg tcgatcaggg aggatatcgg ggaagaacag tatgtcgagc tattttttga 8640cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt 8700gttttagtac ctagatgtgg cgcaacgatg ccggcgacaa gcaggagcgc accgacttct 8760tccgcatcaa gtgttttggc tctcaggccg aggcccacgg caagtatttg ggcaaggggt 8820cgctggtatt cgtgcagggc aagattcgga ataccaagta cgagaaggac ggccagacgg 8880tctacgggac cgacttcatt gccgataagg tggattatct ggacaccaag gcaccaggcg 8940ggtcaaatca ggaataaggg cacattgccc cggcgtgagt cggggcaatc ccgcaaggag 9000ggtgaatgaa tcggacgttt gaccggaagg catacaggca agaactgatc gacgcggggt 9060tttccgccga ggatgccgaa accatcgcaa gccgcaccgt catgcgtgcg ccccgcgaaa 9120ccttccagtc cgtcggctcg atggtccagc aagctacggc caagatcgag cgcgacagcg 9180tgcaactggc tccccctgcc ctgcccgcgc catcggccgc cgtggagcgt tcgcgtcgtc 9240tcgaacagga ggcggcaggt ttggcgaagt cgatgaccat cgacacgcga ggaactatga 9300cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa acaggtcagc gaggccaagc 9360aggccgcgtt gctgaaacac acgaagcagc agatcaagga aatgcagctt tccttgttcg 9420atattgcgcc gtggccggac acgatgcgag cgatgccaaa cgacacggcc cgctctgccc 9480tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct gcaaaacaag gtcattttcc 9540acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga gctgcgggcc gacgatgacg 9600aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac ccctatcggc gagccgatca 9660ccttcacgtt ctacgagctt tgccaggacc tgggctggtc gatcaatggc cggtattaca 9720cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc gatgggcttc acgtccgacc 9780gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt ccgcgtcctg gaccgtggca 9840agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat cgtcgtgctg tttgctggcg 9900accactacac gaaattcata tgggagaagt accgcaagct gtcgccgacg gcccgacgga 9960tgttcgacta tttcagctcg caccgggagc cgtacccgct caagctggaa accttccgcc 10020tcatgtgcgg atcggattcc acccgcgtga agaagtggcg cgagcaggtc ggcgaagcct 10080gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg ggtcaatgat gacctggtgc 10140attgcaaacg ctagggcctt gtggggtcag ttccggctgg gggttcagca gccagcgctt 10200tactggcatt tcaggaacaa gcgggcactg ctcgacgcac ttgcttcgct cagtatcgct 10260cgggacgcac ggcgcgctct acgaactgcc gataaacaga ggattaaaat tgacaattgt 10320gattaaggct cagattcgac ggcttggagc ggccgacgtg caggatttcc gcgagatccg 10380attgtcggcc ctgaagaaag ctccagagat gttcgggtcc gtttacgagc acgaggagaa 10440aaagcccatg gaggcgttcg ctgaacggtt gcgagatgcc gtggcattcg gcgcctacat 10500cgacggcgag atcattgggc tgtcggtctt caaacaggag gacggcccca aggacgctca 10560caaggcgcat ctgtccggcg ttttcgtgga gcccgaacag cgaggccgag gggtcgccgg 10620tatgctgctg cgggcgttgc cggcgggttt attgctcgtg atgatcgtcc gacagattcc 10680aacgggaatc tggtggatgc gcatcttcat cctcggcgca cttaatattt cgctattctg 10740gagcttgttg tttatttcgg tctaccgcct gccgggcggg gtcgcggcga cggtaggcgc 10800tgtgcagccg ctgatggtcg tgttcatctc tgccgctctg ctaggtagcc cgatacgatt 10860gatggcggtc ctgggggcta tttgcggaac tgcgggcgtg gcgctgttgg tgttgacacc 10920aaacgcagcg ctagatcctg tcggcgtcgc agcgggcctg gcgggggcgg tttccatggc 10980gttcggaacc gtgctgaccc gcaagtggca acctcccgtg cctctgctca cctttaccgc 11040ctggcaactg gcggccggag gacttctgct cgttccagta gctttagtgt ttgatccgcc 11100aatcccgatg cctacaggaa ccaatgttct cggcctggcg tggctcggcc tgatcggagc 11160gggtttaacc tacttccttt ggttccgggg gatctcgcga ctcgaaccta cagttgtttc 11220cttactgggc tttctcagcc ccagatctgg ggtcgatcag ccggggatgc atcaggccga 11280cagtcggaac ttcgggtccc cgacctgtac cattcggtga gcaatggata ggggagttga 11340tatcgtcaac gttcacttct aaagaaatag cgccactcag cttcctcagc ggctttatcc 11400agcgatttcc tattatgtcg gcatagttct caagatcgac agcctgtcac ggttaagcga 11460gaaatgaata agaaggctga taattcggat ctctgcgagg gagatgatat ttgatcacag 11520gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc tccgcgagat catccgtgtt 11580tcaaacccgg cagcttagtt gccgttcttc cgaatagcat cggtaacatg agcaaagtct 11640gccgccttac aacggctctc ccgctgacgc cgtcccggac tgatgggctg cctgtatcga 11700gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg gctggctggt ggcaggatat 11760attgtggtgt aaacaaattg acgcttagac aacttaataa cacattgcgg acgtttttaa 11820tgtactgggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc 11880gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa 11940tcctgtttga tggtggttcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg 12000agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 12060ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 12120ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 12180gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 12240aagcgaaagg agcgggcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 12300gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 12360gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attaattccc 12420atcttgaaag aaatatagtt taaatattta ttgataaaat aacaagtcag gtattatagt 12480ccaagcaaaa acataaattt attgatgcaa gtttaaattc agaaatattt caataactga 12540ttatatcagc tggtacattg ccgtagatga aagactgagt gcgatattat gtgtaataca 12600taaattgatg atatagctag cttagctcat cgggggatcc gtcgaagcta gcttgggtcc 12660cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc 12720gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc 12780acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 12840gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 12900cacgacgaga tcctcgccgt cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg 12960cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 13020agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 13080aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 13140gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 13200ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 13260ccgcgctgcc tcgtcctgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 13320aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 13380ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 13440tccatcttgt tcaatccaag ctcccatggg ccctcgacta gagtcgagat ctggattgag 13500agtgaatatg agactctaat tggataccga ggggaattta tggaacgtca gtggagcatt 13560tttgacaaga aatatttgct agctgatagt gaccttaggc gacttttgaa cgcgcaataa 13620tggtttctga cgtatgtgct tagctcatta aactccagaa acccgcggct gagtggctcc 13680ttcaacgttg cggttctgtc agttccaaac gtaaaacggc ttgtcccgcg tcatcggcgg 13740gggtcataac gtgactccct taattctccg ctcatgatct tgatcccctg cgccatcaga 13800tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg 13860gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc 13920gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc 13980agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct 14040acgtgttccg cttcctttag cagcccttgc gccctgagtg cttgcggcag cgtgaagctc 14100tggacatcat gttggatatg aaacaactat tatttatcta catgttttag atgttatctg 14160attattttta taccgtagtc ttctattgat gaggagtcta aggctataga attatatatc 14220taaatgatta atatatatat tattaataat taacaataat taatatatta taatttatat 14280atatatattt tatattatta taataatatt cttacaaata taattattat attcgacggt 14340atcgatgatg gacagataat gagatgaatt agaaaaaaaa aattcgtgtt gtaagataga 14400atacttgcta tctactgatg aatgcagttc agttttcctc acgatcttaa agatcgcgca 14460ctatcctcag cttcactctg gaaattttga ttctcttctt ctgctcagca gcctcgactc 14520tgtctagggt ttcgtacaat cggacgccat tctacatgaa tcgagcacag ggaatgaaga 14580caattaggag atcctcgatg tcctccgact tacttgcatg acttgacggg gaagatctcg 14640agcagggaag cgacgcctct ccggaggact cgcctcgccg agaggacctc ctccgcgaca 14700cggaccatgg cctccacggg gtagaagctg gccctgttct ttattctctt gaggatcatc 14760ggccgaagcc tccgcaaatc catccccgag gagtagaatc tcgcctgcag gaagcatctg 14820tcgagatcct cgccgaggcg gcggagatac ctcgccggcg ccgccatggc gccggggacg 14880gagcaccacc acggagaaga agaaccctaa cccaaggcat taacgaagtt gcgcagatta 14940tacaaaagcc ctcaaatatc tttcattttc tatttcactg atacattttc attattgtat 15000atgagtgttt atttaaatta ttccgtatta gaaaagcacc tccagaaccc gacaaaatag 15060ggtgacgtca tcatggtgtc atgaccgccc aacagccgca gatttaaaat cggtggatga 15120gtgcggccac gccacgaaag cgatgggcct tcgtcgatgc cgtgagaatc catctgacat 15180aaagtaaacg gcgccgtcag tattgacggc gtatgacacg tggaaagaag ctattggttc 15240acgcatcggt ggttccgcta gcctccgtcc accgctagta ctataaatac ggtcccgagg 15300cctcctcacc actcgcacat atcctctttg ttttcctctc cgtgaaagaa gcgaggaagc 15360gcgtcgtctc tcccaaggta aggagcagat ctctttgatc gtttttgttc ttcttttgtt 15420ttgttttttt tttctgcgga tcttcggttg catcatgcct tggctgtttt tattagttta 15480ggatatcctc gtttggatct gagccgatca tatatgttaa aggttgtgtt cgatctcttt 15540gttcattttc gcatgaaaag gatgtatcct tttgatgtga ggcgatcttc tatggttaag 15600actttgttcg gtctattgat catttctgtt cttcgttttt gagttttttt ctgcggatat 15660cgcatcatcc ctaggttttt gctttggtta ggatgcatcc tttggatttg agccgatctc 15720ccttggttaa ggctgtgtct gttgcagagg agaaagtctg tcgaggtcct tatgcaggct 15780ttgtccagat gcgcgtgctc tctcatgcta tgaatttatg ttttgagaac tcctcccggt 15840ttttctagat ccggatttga agtattcatt gcggttcccc ttcggtttta tgtatttctc 15900gagttgattt ggtccatgat cgtgttctgt ccagatctct cttgatatgg atgagatatt 15960cgttacctct ttcaaacatc ggtggatgtt ctttttagtc ttggctcacc tttatctaga 16020aattaatttt cggtttgaaa cccctgcttg ttaaggtgat gtattccttc tttatagatt 16080tcggtgtgtt atttcttaac ggtgatctgt ccgatccatg tgttgcacct cttgttttct 16140gtgtaatcct ctgtgaatta taattatgtt ttgaaaacgt acttaagtaa ggggcatgtt 16200ccccgtttaa aacttttgtt ctatcaattt gtggttaata gatcctgatt tgtggtcgcc 16260ttattctgtc tttaatcgtg gattttattt atcttgagcg cgtccttttc ttttaaaatc 16320atgtgtttaa cctttcagtc gtcatatgtt ccatcaggtc gacgtacaag ggcgaattca 16380ccatgggggc cgggcagtcg tccccggcca cggggtccca gaaccagtcc ggcaacaccg 16440gcagcatcat caacaactac tacatgcagc agtaccagaa ctccatggac acccaactgg 16500gggacaacgc cacgagcggc ggcagcaatg agggctccac cgacaccacg agcacccaca 16560cgaccaacac ccagaacaac gactggttca gcaagctcgc gtccagcgcc ttctccgggc 16620tcttcggggc gctcctggcc gacaagaaga ccgaggagac cacgctgctc gaggaccgca 16680tcctcaccac gcggaacggc cacacgacct ccaccacgca gtcgagcgtc ggggtcactt 16740acggttacgc cacggccgag gacttcgtga gcggccccaa cacctccggc ctcgagactc 16800gggtcgccca ggccgagcgc ttcttcaaga cgcacctgtt cgactgggtg acctccgacc 16860ccttcggccg ctgccacctc ttggaactgc ccaccgacca caagggcgtg tacgggtcgt 16920tgacggacag ctacgcgtac atgaggaacg ggtgggacgt ggaggtcacc gccgtcggca 16980accagttcaa cgggggctgc ctcctggtgg ccatggtccc cgaactctgc tcgatccaga 17040agcgggagct gtaccagctg accctcttcc cgcaccagtt catcaacccg cgcaccaaca 17100tgaccgcgca catcaccgtc cccttcgtgg gcgtgaaccg ctacgaccag tacaaagtcc 17160acaagccctg gaccttggtg gtcatggtcg tcgcccccct caccgtcaac tcggaggggg 17220cgccccagat caaagtctac gccaacatcg cccccacgaa cgtgcacgtt gccggcgagt 17280tcccgagcaa ggagggcatc ttcccggttg cgtgctccga cggctacggg ggcctcgtga 17340cgaccgaccc gaagaccgcc gaccctgcct acggcaaggt gttcaacccg cccagaaaca 17400tgctccctgg gagattcacg aacttcctcg acgtggccga ggcctgcccc acgttcctgc 17460acttcgaggg ggacgtccct tacgtcacca cgaagactga ctcggatcgc gtgctggccc 17520agttcgacct gtccctcgcc gctaagcaca tgtccaacac cttcctcgcc ggcctcgcgc 17580aatactacac ccagtacagc ggcaccatca acctccactt catgttcacc ggccccaccg 17640acgccaaggc ccgctacatg atcgcttacg ccccgcccgg catggagccc ccgaagaccc 17700ccgaggcggc cgcgcactgc atccacgccg agtgggacac ggggctcaac tccaagttca 17760cgttctccat cccctacctc agcgccgcgg actacgcgta caccgccagc gacaccgcgg 17820agaccacgaa cgtgcagggc tgggtgtgcc tgttccagat cacccacggg aaggccgacg 17880gcgacgccct ggtcgtgctg gcctcggcgg ggaaggactt cgaactcagg ctgccggtgg 17940acgcccgcac ccagacgact agcgccggcg agtccgcgga cccggtgacc gcgaccgtcg 18000agaactacgg gggcgagacc caagtgcagc gcagacagca cacggacgtg tccttcatcc 18060tggatcgctt cgtgaaagtg accccgaagg accagatcaa cgtgctggac ctgatgcaga 18120cgccggcgca caccctcgtg ggggccctcc tgcgcaccgc cacgtactac ttcgcggacc 18180tcgaggtggc ggtgaagcac gagggcaacc tgacgtgggt gcccaacggt gcgccggagg 18240ccgcgctcga caacaccacg aaccccaccg cgtaccacaa ggcccccctg acccggctcg 18300ccctccccta caccgccccc cacagggtcc tcgccacggt ctacaacggc aacagcaagt 18360acggggacgg gaccgtcgcc aacgtgcgcg gggacctcca agtgctcgcc cagaaggccg 18420cgcgcgccct ccccactagc ttcaactacg gtgccatcaa ggccacgagg gtgacggagc 18480tgctctaccg catgaagcgc gccgagacct actgcccccg ccccctgctc gcgatccacc 18540ccgaccaggc cagacacaag cagaagatcg tcgcccctgt caagcagctc ctgaacttcg 18600acctcctgaa gctggccggc gacgtggaga gcaatccggg ctaagagct 186493616496DNAArtificial SequenceMERE01 Plasmid 36ggacatggca accgggcctg aatttcgcgg gtattctgtt tctattccaa ctttttcttg 60atccgcagcc attaacgact tttgaataga tacgctgaca cgccaagcct cgctagtcaa 120aagtgtacca aacaacgctt tacagcaaga acggaatgcg cgtgacgctc gcggtgacgc 180catttcgcct tttcagaaat ggataaatag ccttgcttcc tattatatct tcccccaaat 240taattaagaa actcccgagg tgagcaagga tccggagtcg agcgcgaaga agagaaagag 300ggaaagcgcg ggtaccgggc ccccccctcg acggatcaag tgcaaaggtc cgccttgttt 360ctcctctgtc tcttgatctg actaatcttg gtttatgatt cgttgagtaa ttttggggaa 420agctagcttc gtccacagtt tttttttcga tgaacagtgc cgcagtggcg ctgatcttgt 480atgctatcct gcaatcgtgg tgaacttatt tcttttatat ccttcactcc catgaaaagg 540ctagtaatct ttctcgatgt aacatcgtcc agcactgcta ttaccgtgtg gtccatccga 600cagtctggct gaacacatca tacgatattg agcaaagatc gatctatctt ccctgttctt 660taatgaaaga cgtcattttc atcagtatga tctaagaatg ttgcaacttg caaggaggcg 720tttctttctt tgaatttaac taactcgttg agtggccctg tttctcggac gtaaggcctt 780tgctgctcca cacatgtcca ttcgaatttt accgtgttta gcaagggcga aaagtttgca 840tcttgatgat ttagcttgac tatgcgattg ctttcctgga cccgtgcagc tgcggacgga 900tcccccgctc gaggtcgacg gtatcgataa gcttgatcag atctgatcga attcaccatg 960ggcgccgggc agtcctcgcc tgcgacgggg agccagaacc agtcgggcaa caccggctcg 1020atcatcaaca actactacat gcagcaatac cagaacagca tggacaccca gctcggcgat 1080aacgccatct ccggcgggtc caacgagggc tcgaccgaca cgacctccac ccacaccacg 1140aacacccaga acaatgactg gttcagcaag ctggcctcca gcgccttcac gggcctgttc 1200ggggcgctgc tcgcggacaa gaagaccgag gagaccacgc tgctcgagga ccggatcctc 1260accacgcgca acgggcacac gaccagcacg acccagtcca gcgtcggggt gacccacggc 1320tactccacgg aggaggacca cgtcgccggg ccgaacacga gcggcctcga gacccgcgtg 1380gtccaggcgg agcgcttcta caagaagtac ctgttcgact ggaccacgga caaggcgttc 1440gggcacctcg agaagctgga gctgccgagc gaccaccacg gcgtcttcgg ccacctcgtg 1500gactcctacg cgtacatgag aaatgggtgg gacgtcgagg tcagcgccgt cggcaaccag 1560ttcaacggcg ggtgcctgct cgtggcgatg gtcccggagt ggaaggagtt cgacacccgc 1620gagaagtatc agctgaccct cttcccccac cagttcatca gcccgcgcac caacatgacc 1680gcccacatca cggtgccgta cctcggggtg aaccgctacg accagtacaa gaagcacaag 1740ccctggaccc tcgtggtcat ggtggtctcc ccgctgactg tgaacaacac gtccgccgcg 1800cagatcaaag tctacgccaa catcgccccc acctacgtgc acgtcgccgg cgagctgccc 1860tccaaggagg gcatcttccc cgtcgcctgc gccgacgggt acgggggcct ggtgaccacc 1920gaccccaaga cggccgaccc ggcctacggc aaagtgtaca acccgcccag gacgaactac 1980cccggtcgct tcaccaacct cctggacgtg gcggaggcct gcccgacctt cctgtgcttc 2040gacgacggga agccctacgt caccacgcgc accgacgaca cgcgcctgct cgccaagttc 2100gacctcagcc tggccgctaa gcacatgagc aacacctacc tcagcggcat cgcccaatac 2160tatacccaat actcgggcac catcaacctg cacttcatgt tcacgggcag caccgacagc 2220aaggcccggt acatggtggc ctacatcccg ccgggcgtgg agacccctcc cgacacgccc 2280gagcgggctg cgcactgcat ccacgccgag tgggacaccg gcctcaacag caagttcacg 2340ttcagcatcc cctacgtgtc cgccgcggat tacgcttaca ccgcctcgga cacggccgag 2400acgatcaacg tccagggctg ggtctgcatc taccagatca ctcacggcaa ggccgagaac 2460gacaccctcg tcgtgagcgt ctccgccggg aaggacttcg agctgaggct gcccatcgac 2520cccaggcagc agaccacggc gaccggggag tccgccgacc ccgtgaccac gaccgtggag 2580aactacggcg gggagactca gatccagcgg cgccaccaca ccgacatcgg cttcatcatg 2640gaccgcttcg tgaagatcca gtccctgtcg cccacccacg ttatcgacct catgcaggcc 2700caccagcacg ggctcgtggg tgccctcctg cgcgcggcca cctactactt cagcgacctc 2760gagatcgttg tccgccacga ggggaacctc acctgggtcc cgaacggtgc ccccgagagc

2820gccctgctca acacctccaa ccccacggcg tacaacaagg cccccttcac gcgcctcgcc 2880ctgccttaca ccgcgcccca ccgcgtgctg gccacggtgt acaacgggac ctccaagtac 2940gccgtgggcg ggagcggccg cagaggcgac atggggagcc tcgccgctag ggtcgtgaag 3000cagctcccgg cctccttcaa ctacggcgcc atcaaggccg acgccatcca cgaactcctg 3060gtccgcatga agcgcgccga actctactgc ccccggcccc tcctggccat cgaggtctcc 3120tcgcaggacc gccacaagca gaagatcatc gccccggcca agcagctgct caacttcgac 3180ctgctcaagc tcgcggggga cgtcgagtcg aaccccggtc ccttcttctt cgccgacgtg 3240cggtccaact tctcgaagct cgtggacacc atcaaccaga tgcaggagga catgagcacc 3300aagcacgggc cggacttcaa ccgcctcgtc tcggccttcg aggaactcgc caccggggtc 3360aaggccatca ggacgggcct ggacgaggcg aagccgtggt acaagctcat caagctcctg 3420tcgcgcctct cctgcatggc ggctgtcgcg gcccgctcca aggaccccgt cctcgtcgcc 3480atcatgctcg cggacaccgg gctcgagcgc cagcggcccc tgaaagtgcg cgccaagctg 3540ccgcagcagg agggccccta cgccggcccg ctcgagagac agaagccgct gaaagtcaag 3600gccaaggcgc ccgtggtcaa ggaggggccc tacgagggcc ccgtcaagaa gccggtggcg 3660ctgaaagtga aggccaagaa cctcatcgtg accgagtccg gtgcgcctcc caccgacctg 3720cagaagatgg tgatgggcaa caccaagccc gtcgagttga tcctcgacgg gaagaccgtg 3780gcgatctgct gcgccaccgg cgtgttcggc accgcctacc tcgtcccgag acacctgttc 3840gccgagaagt atgacaagat catgctggac gggcgggcca tgaccgactc ggactaccgg 3900gtcttcgagt tcgagatcaa agtgaagggc caggatatgc tctccgacgc cgcgctgatg 3960gtgctccaca gaggcaaccg cgtgcgggac atcaccaagc acttcaggga caccgcgcgc 4020atgaagaagg ggacccctgt ggtcggggtc gtgaacaacg ccgacgtcgg gcgcctcatc 4080ttctccggcg aggcgctgac ctacaaggac atcgtcgtgt gcatggacgg ggacacgatg 4140ccggggctct tcgcctacaa ggccgcgacc aaggccgggt actgcggggg tgcggtgctc 4200gccaaggacg gcgccgacac gttcatcgtg ggcacccact ccgccggcgg taacggcgtc 4260ggctactgct cctgcgtgtc ccggtccatg ctgctgcgga tgaaggccca cgttgacccc 4320gagccgcagc acgagtaaga gctcgaattt ccccgatcgt tcaaacattt ggcaataaag 4380tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa 4440ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 4500tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 4560aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatcggg aattaattca 4620gatcggctga gtggctcctt caacgttgcg gttctgtcag ttccaaacgt aaaacggctt 4680gtcccgcgtc atcggcgggg gtcataacgt gactccctta attctccgct catgatcaga 4740ttgtcgtttc ccgccttcag tttaaactat cagtgtttga caggatatat tggcgggtaa 4800acctaagaga aaagagcgtt tattagaata atcggatatt taaaagggcg tgaaaaggtt 4860tatccgttcg tccatttgta tgtgcatgcc aaccacaggg ttccccagat ctggcgccgg 4920ccagcgagac gagcaagatt ggccgccgcc cgaaacgatc cgacagcgcg cccagcacag 4980gtgcgcaggc aaattgcacc aacgcataca gcgccagcag aatgccatag tgggcggtga 5040cgtcgttcga gtgaaccaga tcgcgcagga ggcccggcag caccggcata atcaggccga 5100tgccgacagc gtcgagcgcg acagtgctca gaattacgat caggggtatg ttgggtttca 5160cgtctggcct ccggaccagc ctccgctggt ccgattgaac gcgcggattc tttatcactg 5220ataagttggt ggacatatta tgtttatcag tgataaagtg tcaagcatga caaagttgca 5280gccgaataca gtgatccgtg ccgccctgga cctgttgaac gaggtcggcg tagacggtct 5340gacgacacgc aaactggcgg aacggttggg ggttcagcag ccggcgcttt actggcactt 5400caggaacaag cgggcgctgc tcgacgcact ggccgaagcc atgctggcgg agaatcatac 5460gcattcggtg ccgagagccg acgacgactg gcgctcattt ctgatcggga atgcccgcag 5520cttcaggcag gcgctgctcg cctaccgcga tggcgcgcgc atccatgccg gcacgcgacc 5580gggcgcaccg cagatggaaa cggccgacgc gcagcttcgc ttcctctgcg aggcgggttt 5640ttcggccggg gacgccgtca atgcgctgat gacaatcagc tacttcactg ttggggccgt 5700gcttgaggag caggccggcg acagcgatgc cggcgagcgc ggcggcaccg ttgaacaggc 5760tccgctctcg ccgctgttgc gggccgcgat agacgccttc gacgaagccg gtccggacgc 5820agcgttcgag cagggactcg cggtgattgt cgatggattg gcgaaaagga ggctcgttgt 5880caggaacgtt gaaggaccga gaaagggtga cgattgatca ggaccgctgc cggagcgcaa 5940cccactcact acagcagagc catgtagaca acatcccctc cccctttcca ccgcgtcaga 6000cgcccgtagc agcccgctac gggctttttc atgccctgcc ctagcgtcca agcctcacgg 6060ccgcgctcgg cctctctggc ggccttctgg cgctcttccg cttcctcgct cactgactcg 6120ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 6180ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 6240gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 6300gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 6360taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 6420accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgcttttccg ctgcataacc 6480ctgcttcggg gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac 6540aggattttgc caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg 6600caggataggt gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc 6660gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt 6720aacagatgag ggcaagcgga tggctgatga aaccaagcca accaggaagg gcagcccacc 6780tatcaaggtg tactgccttc cagacgaacg aagagcgatt gaggaaaagg cggcggcggc 6840cggcatgagc ctgtcggcct acctgctggc cgtcggccag ggctacaaaa tcacgggcgt 6900cgtggactat gagcacgtcc gcgagctggc ccgcatcaat ggcgacctgg gccgcctggg 6960cggcctgctg aaactctggc tcaccgacga cccgcgcacg gcgcggttcg gtgatgccac 7020gatcctcgcc ctgctggcga agatcgaaga gaagcaggac gagcttggca aggtcatgat 7080gggcgtggtc cgcccgaggg cagagccatg acttttttag ccgctaaaac ggccgggggg 7140tgcgcgtgat tgccaagcac gtccccatgc gctccatcaa gaagagcgac ttcgcggagc 7200tggtgaagta catcaccgac gagcaaggca agaccgagcg cctttgcgac gctcaccggg 7260ctggttgccc tcgccgctgg gctggcggcc gtctatggcc ctgcaaacgc gccagaaacg 7320ccgtcgaagc cgtgtgcgag acaccgcggc cgccggcgtt gtggatacct cgcggaaaac 7380ttggccctca ctgacagatg aggggcggac gttgacactt gaggggccga ctcacccggc 7440gcggcgttga cagatgaggg gcaggctcga tttcggccgg cgacgtggag ctggccagcc 7500tcgcaaatcg gcgaaaacgc ctgattttac gcgagtttcc cacagatgat gtggacaagc 7560ctggggataa gtgccctgcg gtattgacac ttgaggggcg cgactactga cagatgaggg 7620gcgcgatcct tgacacttga ggggcagagt gctgacagat gaggggcgca cctattgaca 7680tttgaggggc tgtccacagg cagaaaatcc agcatttgca agggtttccg cccgtttttc 7740ggccaccgct aacctgtctt ttaacctgct tttaaaccaa tatttataaa ccttgttttt 7800aaccagggct gcgccctgtg cgcgtgaccg cgcacgccga aggggggtgc ccccccttct 7860cgaaccctcc cggcccgcta acgcgggcct cccatccccc caggggctgc gcccctcggc 7920cgcgaacggc ctcaccccaa aaatggcagc gctggcagtc cttgccattg ccgggatcgg 7980ggcagtaacg ggatgggcga tcagcccgag cgcgacgccc ggaagcattg acgtgccgca 8040ggtgctggca tcgacattca gcgaccaggt gccgggcagt gagggcggcg gcctgggtgg 8100cggcctgccc ttcacttcgg ccgtcggggc attcacggac ttcatggcgg ggccggcaat 8160ttttaccttg ggcattcttg gcatagtggt cgcgggtgcc gtgctcgtgt tcgggggtgc 8220gataaaccca gcgaaccatt tgaggtgata ggtaagatta taccgaggta tgaaaacgag 8280aattggacct ttacagaatt actctatgaa gcgccatatt taaaaagcta ccaagacgaa 8340gaggatgaag aggatgagga ggcagattgc cttgaatata ttgacaatac tgataagata 8400atatatcttt tatatagaag atatcgccgt atgtaaggat ttcagggggc aaggcatagg 8460cagcgcgctt atcaatatat ctatagaatg ggcaaagcat aaaaacttgc atggactaat 8520gcttgaaacc caggacaata accttatagc ttgtaaattc tatcataatt gggtaatgac 8580tccaacttat tgatagtgtt ttatgttcag ataatgcccg atgactttgt catgcagctc 8640caccgatttt gagaacgaca gcgacttccg tcccagccgt gccaggtgct gcctcagatt 8700caggttatgc cgctcaattc gctgcgtata tcgcttgctg attacgtgca gctttccctt 8760caggcgggat tcatacagcg gccagccatc cgtcatccat atcaccacgt caaagggtga 8820cagcaggctc ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata cgtgcgcaac 8880aaccgtcttc cggagactgt catacgcgta aaacagccag cgctggcgcg atttagcccc 8940gacatagccc cactgttcgt ccatttccgc gcagacgatg acgtcactgc ccggctgtat 9000gcgcgaggtt accgactgcg gcctgagttt tttaagtgac gtaaaatcgt gttgaggcca 9060acgcccataa tgcgggctgt tgcccggcat ccaacgccat tcatggccat atcaatgatt 9120ttctggtgcg taccgggttg agaagcggtg taagtgaact gcagttgcca tgttttacgg 9180cagtgagagc agagatagcg ctgatgtccg gcggtgcttt tgccgttacg caccaccccg 9240tcagtagctg aacaggaggg acagctgata gacacagaag ccactggagc acctcaaaaa 9300caccatcata cactaaatca gtaagttggc agcatcaccc ataattgtgg tttcaaaatc 9360ggctccgtcg atactatgtt atacgccaac tttgaaaaca actttgaaaa agctgttttc 9420tggtatttaa ggttttagaa tgcaaggaac agtgaattgg agttcgtctt gttataatta 9480gcttcttggg gtatctttaa atactgtaga aaagaggaag gaaataataa atggctaaaa 9540tgagaatatc accggaattg aaaaaactga tcgaaaaata ccgctgcgta aaagatacgg 9600aaggaatgtc tcctgctaag gtatataagc tggtgggaga aaatgaaaac ctatatttaa 9660aaatgacgga cagccggtat aaagggacca cctatgatgt ggaacgggaa aaggacatga 9720tgctatggct ggaaggaaag ctgcctgttc caaaggtcct gcactttgaa cggcatgatg 9780gctggagcaa tctgctcatg agtgaggccg atggcgtcct ttgctcggaa gagtatgaag 9840atgaacaaag ccctgaaaag attatcgagc tgtatgcgga gtgcatcagg ctctttcact 9900ccatcgacat atcggattgt ccctatacga atagcttaga cagccgctta gccgaattgg 9960attacttact gaataacgat ctggccgatg tggattgcga aaactgggaa gaagacactc 10020catttaaaga tccgcgcgag ctgtatgatt ttttaaagac ggaaaagccc gaagaggaac 10080ttgtcttttc ccacggcgac ctgggagaca gcaacatctt tgtgaaagat ggcaaagtaa 10140gtggctttat tgatcttggg agaagcggca gggcggacaa gtggtatgac attgccttct 10200gcgtccggtc gatcagggag gatatcgggg aagaacagta tgtcgagcta ttttttgact 10260tactggggat caagcctgat tgggagaaaa taaaatatta tattttactg gatgaattgt 10320tttagtacct agatgtggcg caacgatgcc ggcgacaagc aggagcgcac cgacttcttc 10380cgcatcaagt gttttggctc tcaggccgag gcccacggca agtatttggg caaggggtcg 10440ctggtattcg tgcagggcaa gattcggaat accaagtacg agaaggacgg ccagacggtc 10500tacgggaccg acttcattgc cgataaggtg gattatctgg acaccaaggc accaggcggg 10560tcaaatcagg aataagggca cattgccccg gcgtgagtcg gggcaatccc gcaaggaggg 10620tgaatgaatc ggacgtttga ccggaaggca tacaggcaag aactgatcga cgcggggttt 10680tccgccgagg atgccgaaac catcgcaagc cgcaccgtca tgcgtgcgcc ccgcgaaacc 10740ttccagtccg tcggctcgat ggtccagcaa gctacggcca agatcgagcg cgacagcgtg 10800caactggctc cccctgccct gcccgcgcca tcggccgccg tggagcgttc gcgtcgtctc 10860gaacaggagg cggcaggttt ggcgaagtcg atgaccatcg acacgcgagg aactatgacg 10920accaagaagc gaaaaaccgc cggcgaggac ctggcaaaac aggtcagcga ggccaagcag 10980gccgcgttgc tgaaacacac gaagcagcag atcaaggaaa tgcagctttc cttgttcgat 11040attgcgccgt ggccggacac gatgcgagcg atgccaaacg acacggcccg ctctgccctg 11100ttcaccacgc gcaacaagaa aatcccgcgc gaggcgctgc aaaacaaggt cattttccac 11160gtcaacaagg acgtgaagat cacctacacc ggcgtcgagc tgcgggccga cgatgacgaa 11220ctggtgtggc agcaggtgtt ggagtacgcg aagcgcaccc ctatcggcga gccgatcacc 11280ttcacgttct acgagctttg ccaggacctg ggctggtcga tcaatggccg gtattacacg 11340aaggccgagg aatgcctgtc gcgcctacag gcgacggcga tgggcttcac gtccgaccgc 11400gttgggcacc tggaatcggt gtcgctgctg caccgcttcc gcgtcctgga ccgtggcaag 11460aaaacgtccc gttgccaggt cctgatcgac gaggaaatcg tcgtgctgtt tgctggcgac 11520cactacacga aattcatatg ggagaagtac cgcaagctgt cgccgacggc ccgacggatg 11580ttcgactatt tcagctcgca ccgggagccg tacccgctca agctggaaac cttccgcctc 11640atgtgcggat cggattccac ccgcgtgaag aagtggcgcg agcaggtcgg cgaagcctgc 11700gaagagttgc gaggcagcgg cctggtggaa cacgcctggg tcaatgatga cctggtgcat 11760tgcaaacgct agggccttgt ggggtcagtt ccggctgggg gttcagcagc cagcgcttta 11820ctggcatttc aggaacaagc gggcactgct cgacgcactt gcttcgctca gtatcgctcg 11880ggacgcacgg cgcgctctac gaactgccga taaacagagg attaaaattg acaattgtga 11940ttaaggctca gattcgacgg cttggagcgg ccgacgtgca ggatttccgc gagatccgat 12000tgtcggccct gaagaaagct ccagagatgt tcgggtccgt ttacgagcac gaggagaaaa 12060agcccatgga ggcgttcgct gaacggttgc gagatgccgt ggcattcggc gcctacatcg 12120acggcgagat cattgggctg tcggtcttca aacaggagga cggccccaag gacgctcaca 12180aggcgcatct gtccggcgtt ttcgtggagc ccgaacagcg aggccgaggg gtcgccggta 12240tgctgctgcg ggcgttgccg gcgggtttat tgctcgtgat gatcgtccga cagattccaa 12300cgggaatctg gtggatgcgc atcttcatcc tcggcgcact taatatttcg ctattctgga 12360gcttgttgtt tatttcggtc taccgcctgc cgggcggggt cgcggcgacg gtaggcgctg 12420tgcagccgct gatggtcgtg ttcatctctg ccgctctgct aggtagcccg atacgattga 12480tggcggtcct gggggctatt tgcggaactg cgggcgtggc gctgttggtg ttgacaccaa 12540acgcagcgct agatcctgtc ggcgtcgcag cgggcctggc gggggcggtt tccatggcgt 12600tcggaaccgt gctgacccgc aagtggcaac ctcccgtgcc tctgctcacc tttaccgcct 12660ggcaactggc ggccggagga cttctgctcg ttccagtagc tttagtgttt gatccgccaa 12720tcccgatgcc tacaggaacc aatgttctcg gcctggcgtg gctcggcctg atcggagcgg 12780gtttaaccta cttcctttgg ttccggggga tctcgcgact cgaacctaca gttgtttcct 12840tactgggctt tctcagcccc agatctgggg tcgatcagcc ggggatgcat caggccgaca 12900gtcggaactt cgggtccccg acctgtacca ttcggtgagc aatggatagg ggagttgata 12960tcgtcaacgt tcacttctaa agaaatagcg ccactcagct tcctcagcgg ctttatccag 13020cgatttccta ttatgtcggc atagttctca agatcgacag cctgtcacgg ttaagcgaga 13080aatgaataag aaggctgata attcggatct ctgcgaggga gatgatattt gatcacaggc 13140agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca tccgtgtttc 13200aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag caaagtctgc 13260cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc tgtatcgagt 13320ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg caggatatat 13380tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac gtttttaatg 13440tactggggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc 13500ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc 13560ctgtttgatg gtggttccga aatcggcaaa atcccttata aatcaaaaga atagcccgag 13620atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 13680aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 13740aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 13800ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 13860gcgaaaggag cgggcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc 13920ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt 13980aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgccatcttg 14040aaagaaatat agtttaaata tttattgata aaataagtca ggtattatag tccaagcaaa 14100aacataattt attgatgcaa agtttaaatt cagaaatatt tcaataactg attatatcag 14160ctggtacatt gccgtagatg aaagactgag tgcgatatta tgtgtaatac ataaattgat 14220gatatagcta gcttagctca tcgggggatc cttaatcgac tctagctaga acgaattgtt 14280aggtggcggt acttgggtcg atatcaaagt gcatcacttc ttcccgtatg cccaactttg 14340tatagagagc cactgcggga tcgtcaccgt aatctgcttg cacgtagatc acataagcac 14400caagcgcgtt ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg ccctgcctcc 14460ggtgctcgcc ggagactgcg agatcataga tatagatctc actacgcggc tgctcaaacc 14520tgggcagaac gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag gcagcaagcg 14580cgatgaatgt cttactacgg agcaagttcc cgaggtaatc ggagtccggc tgatgttggg 14640agtaggtggc tacgtctccg aactcacgac cgaaaagatc aagagcagcc cgcatggatt 14700tgacttggtc agggccgagc ctacatgtgc gaatgatgcc catacttgag ccacctaact 14760ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctgcgtacc atggagatct 14820ggattgagag tgaatatgag actctaattg gataccgagg ggaatttatg gaagtcagtg 14880gagcattttt gacaagaaat atttgctagc tgatagtgac cttaggcgac ttttgaacgc 14940gcaataatgg tttctgacgt atgtgcttag ctcattaaac tccagaaacc cgcggctgag 15000tggctccttc aacgttgcgg ttctgtcagt tccaaacgta aaacggcttg tcccgcgtca 15060tcggcggggg tcataacgtg actcccttaa ttctccgctc atgatcttga tcccctgcgc 15120catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta 15180ccagagggcg ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct 15240agctatcgcc atgtaagccc actgcaagct acctgctttc tctttgcgct tgcgttttcc 15300cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt ctgcggactg 15360gctttctacg tgttccgctt cctttagcag cccttgcgcc ctgagtgctt gcggcagcgt 15420gaagctctgg acatcatgtt ggatatgaaa caactattat ttatctacat gttttagatg 15480ttatctgatt atttttatac cgtagtcttc tattgatgag gagtctaagg ctatagaatt 15540atatatctaa atgattaata tatatattat taataattaa caataattaa tatattataa 15600tttatatata tatattttat attattataa taatattctt acaaatataa ttattatatt 15660cgacggtatc gataagctcg ggatccctga aagcgacgtt ggatgttaac atctacaaat 15720tgccttttct tatcgaccat gtacgtaagc gcttacgttt ttggtggacc cttgaggaaa 15780ctggtagctg ttgtgggcct gtggtctcaa gatggatcat taatttccac cttcacctac 15840gatggggggc atcgcaccgg tgagtaatat tgtacggcta agagcgaatt tggcctgtag 15900gatccctgaa agcgacgttg gatgttaaca tctacaaatt gccttttctt atcgaccatg 15960tacgtaagcg cttacgtttt tggtggaccc ttgaggaaac tggtagctgt tgtgggcctg 16020tggtctcaag atggatcatt aatttccacc ttcacctacg atggggggca tcgcaccggt 16080gagtaatatt gtacggctaa gagcgaattt ggcctgtagg atccctgaaa gcgacgttgg 16140atgttaacat ctacaaattg ccttttctta tcgaccatgt acgtaagcgc ttacgttttt 16200ggtggaccct tgaggaaact ggtagctgtt gtgggcctgt ggtctcaaga tggatcatta 16260atttccacct tcacctacga tggggggcat cgcaccggtg agtaatattg tacggctaag 16320agcgaatttg gcctgtagga tccgcgagct ggtcaatccc attgcttttg aagcagctca 16380acattgatct ctttctcgat cgagggagat ttttcaaatc agtgcgcaag acgtgacgta 16440agtatccgag tcagttttta tttttctact aatttggtcg tttatttcgg cgtgta 164963715344DNAArtificial SequenceMERE02 Plasmid 37ggtcgtttat ttcggcgtgt aggacatggc aaccgggcct gaatttcgcg ggtattctgt 60ttctattcca actttttctt gatccgcagc cattaacgac ttttgaatag atacgctgac 120acgccaagcc tcgctagtca aaagtgtacc aaacaacgct ttacagcaag aacggaatgc 180gcgtgacgct cgcggtgacg ccatttcgcc ttttcagaaa tggataaata gccttgcttc 240ctattatatc ttcccccaaa ttaattaaga aactcccgag gtgagcaagg atccggagtc 300gagcgcgaag aagagaaaga gggaaagcgc gggtaccggg cccccccctc gacggatcaa 360gtgcaaaggt ccgccttgtt tctcctctgt ctcttgatct gactaatctt ggtttatgat 420tcgttgagta attttgggga aagctagctt cgtccacagt ttttttttcg atgaacagtg 480ccgcagtggc gctgatcttg tatgctatcc tgcaatcgtg gtgaacttat ttcttttata 540tccttcactc ccatgaaaag gctagtaatc tttctcgatg taacatcgtc cagcactgct 600attaccgtgt ggtccatccg acagtctggc tgaacacatc atacgatatt gagcaaagat 660cgatctatct tccctgttct ttaatgaaag acgtcatttt catcagtatg atctaagaat 720gttgcaactt gcaaggaggc gtttctttct ttgaatttaa ctaactcgtt gagtggccct 780gtttctcgga cgtaaggcct ttgctgctcc acacatgtcc attcgaattt taccgtgttt 840agcaagggcg aaaagtttgc atcttgatga tttagcttga ctatgcgatt gctttcctgg 900acccgtgcag ctgcggacgg atcccccgct cgaggtcgac ggtatcgata agcttgatca 960gatctgatcg aattcaccat gcaggtcctg aacacgatgg gcgccgggca gtcctcgcct 1020gcgacgggga gccagaacca gtcgggcaac accggctcga tcatcaacaa ctactacatg 1080cagcaatacc agaacagcat ggacacccag ctcggcgata acgccatctc cggcgggtcc 1140aacgagggct cgaccgacac gacctccacc cacaccacga acacccagaa caatgactgg 1200ttcagcaagc tggcctccag cgccttcacg ggcctgttcg gggcgctgct cgcggacaag 1260aagaccgagg agaccacgct gctcgaggac cggatcctca ccacgcgcaa cgggcacacg 1320accagcacga cccagtccag

cgtcggggtg acccacggct actccacgga ggaggaccac 1380gtcgccgggc cgaacacgag cggcctcgag acccgcgtgg tccaggcgga gcgcttctac 1440aagaagtacc tgttcgactg gaccacggac aaggcgttcg ggcacctcga gaagctggag 1500ctgccgagcg accaccacgg cgtcttcggc cacctcgtgg actcctacgc gtacatgaga 1560aatgggtggg acgtcgaggt cagcgccgtc ggcaaccagt tcaacggcgg gtgcctgctc 1620gtggcgatgg tcccggagtg gaaggagttc gacacccgcg agaagtatca gctgaccctc 1680ttcccccacc agttcatcag cccgcgcacc aacatgaccg cccacatcac ggtgccgtac 1740ctcggggtga accgctacga ccagtacaag aagcacaagc cctggaccct cgtggtcatg 1800gtggtctccc cgctgactgt gaacaacacg tccgccgcgc agatcaaagt ctacgccaac 1860atcgccccca cctacgtgca cgtcgccggc gagctgccct ccaaggaggg catcttcccc 1920gtcgcctgcg ccgacgggta cgggggcctg gtgaccaccg accccaagac ggccgacccg 1980gcctacggca aagtgtacaa cccgcccagg acgaactacc ccggtcgctt caccaacctc 2040ctggacgtgg cggaggcctg cccgaccttc ctgtgcttcg acgacgggaa gccctacgtc 2100accacgcgca ccgacgacac gcgcctgctc gccaagttcg acctcagcct ggccgctaag 2160cacatgagca acacctacct cagcggcatc gcccaatact atacccaata ctcgggcacc 2220atcaacctgc acttcatgtt cacgggcagc accgacagca aggcccggta catggtggcc 2280tacatcccgc cgggcgtgga gacccctccc gacacgcccg agcgggctgc gcactgcatc 2340cacgccgagt gggacaccgg cctcaacagc aagttcacgt tcagcatccc ctacgtgtcc 2400gccgcggatt acgcttacac cgcctcggac acggccgaga cgatcaacgt ccagggctgg 2460gtctgcatct accagatcac tcacggcaag gccgagaacg acaccctcgt cgtgagcgtc 2520tccgccggga aggacttcga gctgaggctg cccatcgacc ccaggcagca gaccacggcg 2580accggggagt ccgccgaccc cgtgaccacg accgtggaga actacggcgg ggagactcag 2640atccagcggc gccaccacac cgacatcggc ttcatcatgg accgcttcgt gaagatccag 2700tccctgtcgc ccacccacgt tatcgacctc atgcaggccc accagcacgg gctcgtgggt 2760gccctcctgc gcgcggccac ctactacttc agcgacctcg agatcgttgt ccgccacgag 2820gggaacctca cctgggtccc gaacggtgcc cccgagagcg ccctgctcaa cacctccaac 2880cccacggcgt acaacaaggc ccccttcacg cgcctcgccc tgccttacac cgcgccccac 2940cgcgtgctgg ccacggtgta caacgggacc tccaagtacg ccgtgggcgg gagcggccgc 3000agaggcgaca tggggagcct cgccgctagg gtcgtgaagc agctcccggc ctccttcaac 3060tacggcgcca tcaaggccga cgccatccac gaactcctgg tccgcatgaa gcgcgccgaa 3120ctctactgcc cccggcccct cctggccatc gaggtctcct cgcaggaccg ccacaagcag 3180aagatcatcg ccccggccaa gcagtaagag ctcgaatttc cccgatcgtt caaacatttg 3240gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt 3300tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag 3360atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat 3420atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcggga 3480attaattcag atcggctgag tggctccttc aacgttgcgg ttctgtcagt tccaaacgta 3540aaacggcttg tcccgcgtca tcggcggggg tcataacgtg actcccttaa ttctccgctc 3600atgatcagat tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt 3660ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt 3720gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tccccagatc 3780tggcgccggc cagcgagacg agcaagattg gccgccgccc gaaacgatcc gacagcgcgc 3840ccagcacagg tgcgcaggca aattgcacca acgcatacag cgccagcaga atgccatagt 3900gggcggtgac gtcgttcgag tgaaccagat cgcgcaggag gcccggcagc accggcataa 3960tcaggccgat gccgacagcg tcgagcgcga cagtgctcag aattacgatc aggggtatgt 4020tgggtttcac gtctggcctc cggaccagcc tccgctggtc cgattgaacg cgcggattct 4080ttatcactga taagttggtg gacatattat gtttatcagt gataaagtgt caagcatgac 4140aaagttgcag ccgaatacag tgatccgtgc cgccctggac ctgttgaacg aggtcggcgt 4200agacggtctg acgacacgca aactggcgga acggttgggg gttcagcagc cggcgcttta 4260ctggcacttc aggaacaagc gggcgctgct cgacgcactg gccgaagcca tgctggcgga 4320gaatcatacg cattcggtgc cgagagccga cgacgactgg cgctcatttc tgatcgggaa 4380tgcccgcagc ttcaggcagg cgctgctcgc ctaccgcgat ggcgcgcgca tccatgccgg 4440cacgcgaccg ggcgcaccgc agatggaaac ggccgacgcg cagcttcgct tcctctgcga 4500ggcgggtttt tcggccgggg acgccgtcaa tgcgctgatg acaatcagct acttcactgt 4560tggggccgtg cttgaggagc aggccggcga cagcgatgcc ggcgagcgcg gcggcaccgt 4620tgaacaggct ccgctctcgc cgctgttgcg ggccgcgata gacgccttcg acgaagccgg 4680tccggacgca gcgttcgagc agggactcgc ggtgattgtc gatggattgg cgaaaaggag 4740gctcgttgtc aggaacgttg aaggaccgag aaagggtgac gattgatcag gaccgctgcc 4800ggagcgcaac ccactcacta cagcagagcc atgtagacaa catcccctcc ccctttccac 4860cgcgtcagac gcccgtagca gcccgctacg ggctttttca tgccctgccc tagcgtccaa 4920gcctcacggc cgcgctcggc ctctctggcg gccttctggc gctcttccgc ttcctcgctc 4980actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 5040gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 5100cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 5160ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 5220ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 5280ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gcttttccgc 5340tgcataaccc tgcttcgggg tcattatagc gattttttcg gtatatccat cctttttcgc 5400acgatataca ggattttgcc aaagggttcg tgtagacttt ccttggtgta tccaacggcg 5460tcagccgggc aggataggtg aagtaggccc acccgcgagc gggtgttcct tcttcactgt 5520cccttattcg cacctggcgg tgctcaacgg gaatcctgct ctgcgaggct ggccggctac 5580cgccggcgta acagatgagg gcaagcggat ggctgatgaa accaagccaa ccaggaaggg 5640cagcccacct atcaaggtgt actgccttcc agacgaacga agagcgattg aggaaaaggc 5700ggcggcggcc ggcatgagcc tgtcggccta cctgctggcc gtcggccagg gctacaaaat 5760cacgggcgtc gtggactatg agcacgtccg cgagctggcc cgcatcaatg gcgacctggg 5820ccgcctgggc ggcctgctga aactctggct caccgacgac ccgcgcacgg cgcggttcgg 5880tgatgccacg atcctcgccc tgctggcgaa gatcgaagag aagcaggacg agcttggcaa 5940ggtcatgatg ggcgtggtcc gcccgagggc agagccatga cttttttagc cgctaaaacg 6000gccggggggt gcgcgtgatt gccaagcacg tccccatgcg ctccatcaag aagagcgact 6060tcgcggagct ggtgaagtac atcaccgacg agcaaggcaa gaccgagcgc ctttgcgacg 6120ctcaccgggc tggttgccct cgccgctggg ctggcggccg tctatggccc tgcaaacgcg 6180ccagaaacgc cgtcgaagcc gtgtgcgaga caccgcggcc gccggcgttg tggatacctc 6240gcggaaaact tggccctcac tgacagatga ggggcggacg ttgacacttg aggggccgac 6300tcacccggcg cggcgttgac agatgagggg caggctcgat ttcggccggc gacgtggagc 6360tggccagcct cgcaaatcgg cgaaaacgcc tgattttacg cgagtttccc acagatgatg 6420tggacaagcc tggggataag tgccctgcgg tattgacact tgaggggcgc gactactgac 6480agatgagggg cgcgatcctt gacacttgag gggcagagtg ctgacagatg aggggcgcac 6540ctattgacat ttgaggggct gtccacaggc agaaaatcca gcatttgcaa gggtttccgc 6600ccgtttttcg gccaccgcta acctgtcttt taacctgctt ttaaaccaat atttataaac 6660cttgttttta accagggctg cgccctgtgc gcgtgaccgc gcacgccgaa ggggggtgcc 6720cccccttctc gaaccctccc ggcccgctaa cgcgggcctc ccatcccccc aggggctgcg 6780cccctcggcc gcgaacggcc tcaccccaaa aatggcagcg ctggcagtcc ttgccattgc 6840cgggatcggg gcagtaacgg gatgggcgat cagcccgagc gcgacgcccg gaagcattga 6900cgtgccgcag gtgctggcat cgacattcag cgaccaggtg ccgggcagtg agggcggcgg 6960cctgggtggc ggcctgccct tcacttcggc cgtcggggca ttcacggact tcatggcggg 7020gccggcaatt tttaccttgg gcattcttgg catagtggtc gcgggtgccg tgctcgtgtt 7080cgggggtgcg ataaacccag cgaaccattt gaggtgatag gtaagattat accgaggtat 7140gaaaacgaga attggacctt tacagaatta ctctatgaag cgccatattt aaaaagctac 7200caagacgaag aggatgaaga ggatgaggag gcagattgcc ttgaatatat tgacaatact 7260gataagataa tatatctttt atatagaaga tatcgccgta tgtaaggatt tcagggggca 7320aggcataggc agcgcgctta tcaatatatc tatagaatgg gcaaagcata aaaacttgca 7380tggactaatg cttgaaaccc aggacaataa ccttatagct tgtaaattct atcataattg 7440ggtaatgact ccaacttatt gatagtgttt tatgttcaga taatgcccga tgactttgtc 7500atgcagctcc accgattttg agaacgacag cgacttccgt cccagccgtg ccaggtgctg 7560cctcagattc aggttatgcc gctcaattcg ctgcgtatat cgcttgctga ttacgtgcag 7620ctttcccttc aggcgggatt catacagcgg ccagccatcc gtcatccata tcaccacgtc 7680aaagggtgac agcaggctca taagacgccc cagcgtcgcc atagtgcgtt caccgaatac 7740gtgcgcaaca accgtcttcc ggagactgtc atacgcgtaa aacagccagc gctggcgcga 7800tttagccccg acatagcccc actgttcgtc catttccgcg cagacgatga cgtcactgcc 7860cggctgtatg cgcgaggtta ccgactgcgg cctgagtttt ttaagtgacg taaaatcgtg 7920ttgaggccaa cgcccataat gcgggctgtt gcccggcatc caacgccatt catggccata 7980tcaatgattt tctggtgcgt accgggttga gaagcggtgt aagtgaactg cagttgccat 8040gttttacggc agtgagagca gagatagcgc tgatgtccgg cggtgctttt gccgttacgc 8100accaccccgt cagtagctga acaggaggga cagctgatag acacagaagc cactggagca 8160cctcaaaaac accatcatac actaaatcag taagttggca gcatcaccca taattgtggt 8220ttcaaaatcg gctccgtcga tactatgtta tacgccaact ttgaaaacaa ctttgaaaaa 8280gctgttttct ggtatttaag gttttagaat gcaaggaaca gtgaattgga gttcgtcttg 8340ttataattag cttcttgggg tatctttaaa tactgtagaa aagaggaagg aaataataaa 8400tggctaaaat gagaatatca ccggaattga aaaaactgat cgaaaaatac cgctgcgtaa 8460aagatacgga aggaatgtct cctgctaagg tatataagct ggtgggagaa aatgaaaacc 8520tatatttaaa aatgacggac agccggtata aagggaccac ctatgatgtg gaacgggaaa 8580aggacatgat gctatggctg gaaggaaagc tgcctgttcc aaaggtcctg cactttgaac 8640ggcatgatgg ctggagcaat ctgctcatga gtgaggccga tggcgtcctt tgctcggaag 8700agtatgaaga tgaacaaagc cctgaaaaga ttatcgagct gtatgcggag tgcatcaggc 8760tctttcactc catcgacata tcggattgtc cctatacgaa tagcttagac agccgcttag 8820ccgaattgga ttacttactg aataacgatc tggccgatgt ggattgcgaa aactgggaag 8880aagacactcc atttaaagat ccgcgcgagc tgtatgattt tttaaagacg gaaaagcccg 8940aagaggaact tgtcttttcc cacggcgacc tgggagacag caacatcttt gtgaaagatg 9000gcaaagtaag tggctttatt gatcttggga gaagcggcag ggcggacaag tggtatgaca 9060ttgccttctg cgtccggtcg atcagggagg atatcgggga agaacagtat gtcgagctat 9120tttttgactt actggggatc aagcctgatt gggagaaaat aaaatattat attttactgg 9180atgaattgtt ttagtaccta gatgtggcgc aacgatgccg gcgacaagca ggagcgcacc 9240gacttcttcc gcatcaagtg ttttggctct caggccgagg cccacggcaa gtatttgggc 9300aaggggtcgc tggtattcgt gcagggcaag attcggaata ccaagtacga gaaggacggc 9360cagacggtct acgggaccga cttcattgcc gataaggtgg attatctgga caccaaggca 9420ccaggcgggt caaatcagga ataagggcac attgccccgg cgtgagtcgg ggcaatcccg 9480caaggagggt gaatgaatcg gacgtttgac cggaaggcat acaggcaaga actgatcgac 9540gcggggtttt ccgccgagga tgccgaaacc atcgcaagcc gcaccgtcat gcgtgcgccc 9600cgcgaaacct tccagtccgt cggctcgatg gtccagcaag ctacggccaa gatcgagcgc 9660gacagcgtgc aactggctcc ccctgccctg cccgcgccat cggccgccgt ggagcgttcg 9720cgtcgtctcg aacaggaggc ggcaggtttg gcgaagtcga tgaccatcga cacgcgagga 9780actatgacga ccaagaagcg aaaaaccgcc ggcgaggacc tggcaaaaca ggtcagcgag 9840gccaagcagg ccgcgttgct gaaacacacg aagcagcaga tcaaggaaat gcagctttcc 9900ttgttcgata ttgcgccgtg gccggacacg atgcgagcga tgccaaacga cacggcccgc 9960tctgccctgt tcaccacgcg caacaagaaa atcccgcgcg aggcgctgca aaacaaggtc 10020attttccacg tcaacaagga cgtgaagatc acctacaccg gcgtcgagct gcgggccgac 10080gatgacgaac tggtgtggca gcaggtgttg gagtacgcga agcgcacccc tatcggcgag 10140ccgatcacct tcacgttcta cgagctttgc caggacctgg gctggtcgat caatggccgg 10200tattacacga aggccgagga atgcctgtcg cgcctacagg cgacggcgat gggcttcacg 10260tccgaccgcg ttgggcacct ggaatcggtg tcgctgctgc accgcttccg cgtcctggac 10320cgtggcaaga aaacgtcccg ttgccaggtc ctgatcgacg aggaaatcgt cgtgctgttt 10380gctggcgacc actacacgaa attcatatgg gagaagtacc gcaagctgtc gccgacggcc 10440cgacggatgt tcgactattt cagctcgcac cgggagccgt acccgctcaa gctggaaacc 10500ttccgcctca tgtgcggatc ggattccacc cgcgtgaaga agtggcgcga gcaggtcggc 10560gaagcctgcg aagagttgcg aggcagcggc ctggtggaac acgcctgggt caatgatgac 10620ctggtgcatt gcaaacgcta gggccttgtg gggtcagttc cggctggggg ttcagcagcc 10680agcgctttac tggcatttca ggaacaagcg ggcactgctc gacgcacttg cttcgctcag 10740tatcgctcgg gacgcacggc gcgctctacg aactgccgat aaacagagga ttaaaattga 10800caattgtgat taaggctcag attcgacggc ttggagcggc cgacgtgcag gatttccgcg 10860agatccgatt gtcggccctg aagaaagctc cagagatgtt cgggtccgtt tacgagcacg 10920aggagaaaaa gcccatggag gcgttcgctg aacggttgcg agatgccgtg gcattcggcg 10980cctacatcga cggcgagatc attgggctgt cggtcttcaa acaggaggac ggccccaagg 11040acgctcacaa ggcgcatctg tccggcgttt tcgtggagcc cgaacagcga ggccgagggg 11100tcgccggtat gctgctgcgg gcgttgccgg cgggtttatt gctcgtgatg atcgtccgac 11160agattccaac gggaatctgg tggatgcgca tcttcatcct cggcgcactt aatatttcgc 11220tattctggag cttgttgttt atttcggtct accgcctgcc gggcggggtc gcggcgacgg 11280taggcgctgt gcagccgctg atggtcgtgt tcatctctgc cgctctgcta ggtagcccga 11340tacgattgat ggcggtcctg ggggctattt gcggaactgc gggcgtggcg ctgttggtgt 11400tgacaccaaa cgcagcgcta gatcctgtcg gcgtcgcagc gggcctggcg ggggcggttt 11460ccatggcgtt cggaaccgtg ctgacccgca agtggcaacc tcccgtgcct ctgctcacct 11520ttaccgcctg gcaactggcg gccggaggac ttctgctcgt tccagtagct ttagtgtttg 11580atccgccaat cccgatgcct acaggaacca atgttctcgg cctggcgtgg ctcggcctga 11640tcggagcggg tttaacctac ttcctttggt tccgggggat ctcgcgactc gaacctacag 11700ttgtttcctt actgggcttt ctcagcccca gatctggggt cgatcagccg gggatgcatc 11760aggccgacag tcggaacttc gggtccccga cctgtaccat tcggtgagca atggataggg 11820gagttgatat cgtcaacgtt cacttctaaa gaaatagcgc cactcagctt cctcagcggc 11880tttatccagc gatttcctat tatgtcggca tagttctcaa gatcgacagc ctgtcacggt 11940taagcgagaa atgaataaga aggctgataa ttcggatctc tgcgagggag atgatatttg 12000atcacaggca gcaacgctct gtcatcgtta caatcaacat gctaccctcc gcgagatcat 12060ccgtgtttca aacccggcag cttagttgcc gttcttccga atagcatcgg taacatgagc 12120aaagtctgcc gccttacaac ggctctcccg ctgacgccgt cccggactga tgggctgcct 12180gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc 12240aggatatatt gtggtgtaaa caaattgacg cttagacaac ttaataacac attgcggacg 12300tttttaatgt actggggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc 12360cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag 12420gcgaaaatcc tgtttgatgg tggttccgaa atcggcaaaa tcccttataa atcaaaagaa 12480tagcccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 12540gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 12600ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 12660aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 12720gggaagaaag cgaaaggagc gggcgccatt caggctgcgc aactgttggg aagggcgatc 12780ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 12840aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 12900gccatcttga aagaaatata gtttaaatat ttattgataa aataagtcag gtattatagt 12960ccaagcaaaa acataattta ttgatgcaaa gtttaaattc agaaatattt caataactga 13020ttatatcagc tggtacattg ccgtagatga aagactgagt gcgatattat gtgtaataca 13080taaattgatg atatagctag cttagctcat cgggggatcc ttaatcgact ctagctagaa 13140cgaattgtta ggtggcggta cttgggtcga tatcaaagtg catcacttct tcccgtatgc 13200ccaactttgt atagagagcc actgcgggat cgtcaccgta atctgcttgc acgtagatca 13260cataagcacc aagcgcgttg gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc 13320cctgcctccg gtgctcgccg gagactgcga gatcatagat atagatctca ctacgcggct 13380gctcaaacct gggcagaacg taagccgcga gagcgccaac aaccgcttct tggtcgaagg 13440cagcaagcgc gatgaatgtc ttactacgga gcaagttccc gaggtaatcg gagtccggct 13500gatgttggga gtaggtggct acgtctccga actcacgacc gaaaagatca agagcagccc 13560gcatggattt gacttggtca gggccgagcc tacatgtgcg aatgatgccc atacttgagc 13620cacctaactt tgttttaggg cgactgccct gctgcgtaac atcgttgctg ctgcgtacca 13680tggagatctg gattgagagt gaatatgaga ctctaattgg ataccgaggg gaatttatgg 13740aagtcagtgg agcatttttg acaagaaata tttgctagct gatagtgacc ttaggcgact 13800tttgaacgcg caataatggt ttctgacgta tgtgcttagc tcattaaact ccagaaaccc 13860gcggctgagt ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt 13920cccgcgtcat cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcttgat 13980cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc 14040ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc 14100gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt 14160gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc 14220tgcggactgg ctttctacgt gttccgcttc ctttagcagc ccttgcgccc tgagtgcttg 14280cggcagcgtg aagctctgga catcatgttg gatatgaaac aactattatt tatctacatg 14340ttttagatgt tatctgatta tttttatacc gtagtcttct attgatgagg agtctaaggc 14400tatagaatta tatatctaaa tgattaatat atatattatt aataattaac aataattaat 14460atattataat ttatatatat atattttata ttattataat aatattctta caaatataat 14520tattatattc gacggtatcg ataagctcgg gatccctgaa agcgacgttg gatgttaaca 14580tctacaaatt gccttttctt atcgaccatg tacgtaagcg cttacgtttt tggtggaccc 14640ttgaggaaac tggtagctgt tgtgggcctg tggtctcaag atggatcatt aatttccacc 14700ttcacctacg atggggggca tcgcaccggt gagtaatatt gtacggctaa gagcgaattt 14760ggcctgtagg atccctgaaa gcgacgttgg atgttaacat ctacaaattg ccttttctta 14820tcgaccatgt acgtaagcgc ttacgttttt ggtggaccct tgaggaaact ggtagctgtt 14880gtgggcctgt ggtctcaaga tggatcatta atttccacct tcacctacga tggggggcat 14940cgcaccggtg agtaatattg tacggctaag agcgaatttg gcctgtagga tccctgaaag 15000cgacgttgga tgttaacatc tacaaattgc cttttcttat cgaccatgta cgtaagcgct 15060tacgtttttg gtggaccctt gaggaaactg gtagctgttg tgggcctgtg gtctcaagat 15120ggatcattaa tttccacctt cacctacgat ggggggcatc gcaccggtga gtaatattgt 15180acggctaaga gcgaatttgg cctgtaggat ccgcgagctg gtcaatccca ttgcttttga 15240agcagctcaa cattgatctc tttctcgatc gagggagatt tttcaaatca gtgcgcaaga 15300cgtgacgtaa gtatccgagt cagtttttat ttttctacta attt 153443817728DNAArtificial SequenceMERE03 Plasmid 38ctaaggctat agaattatat atctaaatga ttaatatata tattattaat aattaacaat 60aattaatata ttataattta tatatatata ttttatatta ttataataat attcttacaa 120atataattat tatattcgac ggtatcgata agctcgggat ccctgaaagc gacgttggat 180gttaacatct acaaattgcc ttttcttatc gaccatgtac gtaagcgctt acgtttttgg 240tggacccttg aggaaactgg tagctgttgt gggcctgtgg tctcaagatg gatcattaat 300ttccaccttc acctacgatg gggggcatcg caccggtgag taatattgta cggctaagag 360cgaatttggc ctgtaggatc cctgaaagcg acgttggatg ttaacatcta caaattgcct 420tttcttatcg accatgtacg taagcgctta cgtttttggt ggacccttga ggaaactggt 480agctgttgtg ggcctgtggt ctcaagatgg atcattaatt tccaccttca cctacgatgg 540ggggcatcgc accggtgagt aatattgtac ggctaagagc gaatttggcc tgtaggatcc 600ctgaaagcga cgttggatgt taacatctac aaattgcctt ttcttatcga ccatgtacgt 660aagcgcttac gtttttggtg gacccttgag gaaactggta gctgttgtgg gcctgtggtc 720tcaagatgga tcattaattt ccaccttcac ctacgatggg gggcatcgca ccggtgagta 780atattgtacg gctaagagcg aatttggcct gtaggatccg cgagctggtc aatcccattg 840cttttgaagc agctcaacat tgatctcttt ctcgatcgag ggagattttt caaatcagtg 900cgcaagacgt gacgtaagta tccgagtcag tttttatttt tctactaatt tggtcgttta 960tttcggcgtg taggacatgg caaccgggcc tgaatttcgc

gggtattctg tttctattcc 1020aactttttct tgatccgcag ccattaacga cttttgaata gatacgctga cacgccaagc 1080ctcgctagtc aaaagtgtac caaacaacgc tttacagcaa gaacggaatg cgcgtgacgc 1140tcgcggtgac gccatttcgc cttttcagaa atggataaat agccttgctt cctattatat 1200cttcccccaa attaattaag aaactcccga ggtgagcaag gatccggagt cgagcgcgaa 1260gaagagaaag agggaaagcg cgggtaccgg gcccccccct cgacggatca agtgcaaagg 1320tccgccttgt ttctcctctg tctcttgatc tgactaatct tggtttatga ttcgttgagt 1380aattttgggg aaagctagct tcgtccacag tttttttttc gatgaacagt gccgcagtgg 1440cgctgatctt gtatgctatc ctgcaatcgt ggtgaactta tttcttttat atccttcact 1500cccatgaaaa ggctagtaat ctttctcgat gtaacatcgt ccagcactgc tattaccgtg 1560tggtccatcc gacagtctgg ctgaacacat catacgatat tgagcaaaga tcgatctatc 1620ttccctgttc tttaatgaaa gacgtcattt tcatcagtat gatctaagaa tgttgcaact 1680tgcaaggagg cgtttctttc tttgaattta actaactcgt tgagtggccc tgtttctcgg 1740acgtaaggcc tttgctgctc cacacatgtc cattcgaatt ttaccgtgtt tagcaagggc 1800gaaaagtttg catcttgatg atttagcttg actatgcgat tgctttcctg gacccgtgca 1860gctgcggacg gatcccccgc tcgaagctag cttgatcaga tctgatcgaa ttcaccatgg 1920gcgccgggca gtcctcgcct gcgacgggga gccagaacca gtcgggcaac accggctcga 1980tcatcaacaa ctactacatg cagcaatacc agaacagcat ggacacccag ctcggcgata 2040acgccatctc cggcgggtcc aacgagggct cgaccgacac gacctccacc cacaccacga 2100acacccagaa caatgactgg ttcagcaagc tggcctccag cgccttcacg ggcctgttcg 2160gggcgctgct cgcggacaag aagaccgagg agaccacgct gctcgaggac cggatcctca 2220ccacgcgcaa cgggcacacg accagcacga cccagtccag cgtcggggtg acccacggct 2280actccacgga ggaggaccac gtcgccgggc cgaacacgag cggcctcgag acccgcgtgg 2340tccaggcgga gcgcttctac aagaagtacc tgttcgactg gaccacggac aaggcgttcg 2400ggcacctcga gaagctggag ctgccgagcg accaccacgg cgtcttcggc cacctcgtgg 2460actcctacgc gtacatgaga aatgggtggg acgtcgaggt cagcgccgtc ggcaaccagt 2520tcaacggcgg gtgcctgctc gtggcgatgg tcccggagtg gaaggagttc gacacccgcg 2580agaagtatca gctgaccctc ttcccccacc agttcatcag cccgcgcacc aacatgaccg 2640cccacatcac ggtgccgtac ctcggggtga accgctacga ccagtacaag aagcacaagc 2700cctggaccct cgtggtcatg gtggtctccc cgctgactgt gaacaacacg tccgccgcgc 2760agatcaaagt ctacgccaac atcgccccca cctacgtgca cgtcgccggc gagctgccct 2820ccaaggaggg catcttcccc gtcgcctgcg ccgacgggta cgggggcctg gtgaccaccg 2880accccaagac ggccgacccg gcctacggca aagtgtacaa cccgcccagg acgaactacc 2940ccggtcgctt caccaacctc ctggacgtgg cggaggcctg cccgaccttc ctgtgcttcg 3000acgacgggaa gccctacgtc accacgcgca ccgacgacac gcgcctgctc gccaagttcg 3060acctcagcct ggccgctaag cacatgagca acacctacct cagcggcatc gcccaatact 3120atacccaata ctcgggcacc atcaacctgc acttcatgtt cacgggcagc accgacagca 3180aggcccggta catggtggcc tacatcccgc cgggcgtgga gacccctccc gacacgcccg 3240agcgggctgc gcactgcatc cacgccgagt gggacaccgg cctcaacagc aagttcacgt 3300tcagcatccc ctacgtgtcc gccgcggatt acgcttacac cgcctcggac acggccgaga 3360cgatcaacgt ccagggctgg gtctgcatct accagatcac tcacggcaag gccgagaacg 3420acaccctcgt cgtgagcgtc tccgccggga aggacttcga gctgaggctg cccatcgacc 3480ccaggcagca gaccacggcg accggggagt ccgccgaccc cgtgaccacg accgtggaga 3540actacggcgg ggagactcag atccagcggc gccaccacac cgacatcggc ttcatcatgg 3600accgcttcgt gaagatccag tccctgtcgc ccacccacgt tatcgacctc atgcaggccc 3660accagcacgg gctcgtgggt gccctcctgc gcgcggccac ctactacttc agcgacctcg 3720agatcgttgt ccgccacgag gggaacctca cctgggtccc gaacggtgcc cccgagagcg 3780ccctgctcaa cacctccaac cccacggcgt acaacaaggc ccccttcacg cgcctcgccc 3840tgccttacac cgcgccccac cgcgtgctgg ccacggtgta caacgggacc tccaagtacg 3900ccgtgggcgg gagcggccgc agaggcgaca tggggagcct cgccgctagg gtcgtgaagc 3960agctcccggc ctccttcaac tacggcgcca tcaaggccga cgccatccac gaactcctgg 4020tccgcatgaa gcgcgccgaa ctctactgcc cccggcccct cctggccatc gaggtctcct 4080cgcaggaccg ccacaagcag aagatcatcg ccccggccaa gcagctgctc aacttcgacc 4140tgctcaagct cgcgggggac gtcgagtcga accccggtta agagctcaga gctcgaattt 4200ccccgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 4260tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 4320atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 4380atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 4440atctgtacat ctagatgctt gcggcagcgt gaagctctgg acatcatgtt ggatatgaaa 4500caactattat ttatctacat gttttagatg ttatctgatt atttttatac cgtagtcttc 4560tattgatgag gagtctaagg ctatagaatt atatatctaa atgattaata tatatattat 4620taataattaa caataattaa tatattataa tttatatata tatattttat attattataa 4680taatattctt acaaatataa ttattatatt cgacgcccgg gctagtacta taaatacggt 4740cccgaggcct cctcaccact cgcacatatc ctctttgttt tcctctccgt gaaagaagcg 4800aggaagcgcg tcgtctctcc caaggtaagg agcagatctc tttgatcgtt tttgttcttc 4860ttttgttttg tttttttttt ctgcggatct tcggttgcat catgccttgg ctgtttttat 4920tagtttagga tatcctcgtt tggatctgag ccgatcatat atgttaaagg ttgtgttcga 4980tctctttgtt cattttcgca tgaaaaggat gtatcctttt gatgtgaggc gatcttctat 5040ggttaagact ttgttcggtc tattgatcat ttctgttctt cgtttttgag tttttttctg 5100cggatatcgc atcatcccta ggtttttgct ttggttagga tgcatccttt ggatttgagc 5160cgatctccct tggttaaggc tgtgtctgtt gcagaggaga aagtctgtcg aggtccttat 5220gcaggctttg tccagatgcg cgtgctctct catgctatga atttatgttt tgagaactcc 5280tcccggtttt tctagatccg gatttgaagt attcattgcg gttccccttc ggttttatgt 5340atttctcgag ttgatttggt ccatgatcgt gttctgtcca gatctctctt gatatggatg 5400agatattcgt tacctctttc aaacatcggt ggatgttctt tttagtcttg gctcaccttt 5460atctagaaat taattttcgg tttgaaaccc ctgcttgtta aggtgatgta ttccttcttt 5520atagatttcg gtgtgttatt tcttaacggt gatctgtccg atccatgtgt tgcacctctt 5580gttttctgtg taatcctctg tgaattataa ttatgttttg aaaacgtact taagtaaggg 5640gcatgttccc cgtttaaaac ttttgttcta tcaatttgtg gttaatagat cctgatttgt 5700ggtcgcctta ttctgtcttt aatcgtggat tttatttatc ttgagcgcgt ccttttcttt 5760taaaatcatg tgtttaacct ttcagtcgtc atatgttcca tcagggtacc gtcgacacca 5820tgtccggggc gccccccacc gacctgcaga agatggtgat gggcaacacc aagcccgtcg 5880agttgatcct cgacgggaag accgtggcga tctgctgcgc caccggcgtg ttcggcaccg 5940cctacctcgt cccgagacac ctgttcgccg agaagtatga caagatcatg ctggacgggc 6000gggccatgac cgactcggac taccgggtct tcgagttcga gatcaaagtg aagggccagg 6060atatgctctc cgacgccgcg ctgatggtgc tccacagagg caaccgcgtg cgggacatca 6120ccaagcactt cagggacacc gcgcgcatga agaaggggac ccctgtggtc ggggtcgtga 6180acaacgccga cgtcgggcgc ctcatcttct ccggcgaggc gctgacctac aaggacatcg 6240tcgtgtgcat ggacggggac acgatgccgg ggctcttcgc ctacaaggcc gcgaccaagg 6300ccgggtactg cgggggtgcg gtgctcgcca aggacggcgc cgacacgttc atcgtgggca 6360cccactccgc cggcggtaac ggcgtcggct actgctcctg cgtgtcccgg tccatgctgc 6420tgcggatgaa ggcccacgtt gaccccgagc cgcagcacga gtaaaagctt atcgatgtgc 6480acgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 6540cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 6600gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 6660acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 6720ctatgttact agatcgggtg gacaactcgt cacgtactag tggcgcgccc acgtgattta 6780aatcgtacgg gccggccgta tactctagtg gatcccccaa ttcagatcgg ctgagtggct 6840ccttcaacgt tgcggttctg tcagttccaa acgtaaaacg gcttgtcccg cgtcatcggc 6900gggggtcata acgtgactcc cttaattctc cgctcatgat cagattgtcg tttcccgcct 6960tcagtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag 7020cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt 7080tgtatgtgca tgccaaccac agggttcccc agatctggcg ccggccagcg agacgagcaa 7140gattggccgc cgcccgaaac gatccgacag cgcgcccagc acaggtgcgc aggcaaattg 7200caccaacgca tacagcgcca gcagaatgcc atagtgggcg gtgacgtcgt tcgagtgaac 7260cagatcgcgc aggaggcccg gcagcaccgg cataatcagg ccgatgccga cagcgtcgag 7320cgcgacagtg ctcagaatta cgatcagggg tatgttgggt ttcacgtctg gcctccggac 7380cagcctccgc tggtccgatt gaacgcgcgg attctttatc actgataagt tggtggacat 7440attatgttta tcagtgataa agtgtcaagc atgacaaagt tgcagccgaa tacagtgatc 7500cgtgccgccc tggacctgtt gaacgaggtc ggcgtagacg gtctgacgac acgcaaactg 7560gcggaacggt tgggggttca gcagccggcg ctttactggc acttcaggaa caagcgggcg 7620ctgctcgacg cactggccga agccatgctg gcggagaatc atacgcattc ggtgccgaga 7680gccgacgacg actggcgctc atttctgatc gggaatgccc gcagcttcag gcaggcgctg 7740ctcgcctacc gcgatggcgc gcgcatccat gccggcacgc gaccgggcgc accgcagatg 7800gaaacggccg acgcgcagct tcgcttcctc tgcgaggcgg gtttttcggc cggggacgcc 7860gtcaatgcgc tgatgacaat cagctacttc actgttgggg ccgtgcttga ggagcaggcc 7920ggcgacagcg atgccggcga gcgcggcggc accgttgaac aggctccgct ctcgccgctg 7980ttgcgggccg cgatagacgc cttcgacgaa gccggtccgg acgcagcgtt cgagcaggga 8040ctcgcggtga ttgtcgatgg attggcgaaa aggaggctcg ttgtcaggaa cgttgaagga 8100ccgagaaagg gtgacgattg atcaggaccg ctgccggagc gcaacccact cactacagca 8160gagccatgta gacaacatcc cctccccctt tccaccgcgt cagacgcccg tagcagcccg 8220ctacgggctt tttcatgccc tgccctagcg tccaagcctc acggccgcgc tcggcctctc 8280tggcggcctt ctggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 8340ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 8400gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 8460aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 8520gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 8580ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 8640cctttctccc ttcgggaagc gtggcgcttt tccgctgcat aaccctgctt cggggtcatt 8700atagcgattt tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg 8760gttcgtgtag actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta 8820ggcccacccg cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc 8880aacgggaatc ctgctctgcg aggctggccg gctaccgccg gcgtaacaga tgagggcaag 8940cggatggctg atgaaaccaa gccaaccagg aagggcagcc cacctatcaa ggtgtactgc 9000cttccagacg aacgaagagc gattgaggaa aaggcggcgg cggccggcat gagcctgtcg 9060gcctacctgc tggccgtcgg ccagggctac aaaatcacgg gcgtcgtgga ctatgagcac 9120gtccgcgagc tggcccgcat caatggcgac ctgggccgcc tgggcggcct gctgaaactc 9180tggctcaccg acgacccgcg cacggcgcgg ttcggtgatg ccacgatcct cgccctgctg 9240gcgaagatcg aagagaagca ggacgagctt ggcaaggtca tgatgggcgt ggtccgcccg 9300agggcagagc catgactttt ttagccgcta aaacggccgg ggggtgcgcg tgattgccaa 9360gcacgtcccc atgcgctcca tcaagaagag cgacttcgcg gagctggtga agtacatcac 9420cgacgagcaa ggcaagaccg agcgcctttg cgacgctcac cgggctggtt gccctcgccg 9480ctgggctggc ggccgtctat ggccctgcaa acgcgccaga aacgccgtcg aagccgtgtg 9540cgagacaccg cggccgccgg cgttgtggat acctcgcgga aaacttggcc ctcactgaca 9600gatgaggggc ggacgttgac acttgagggg ccgactcacc cggcgcggcg ttgacagatg 9660aggggcaggc tcgatttcgg ccggcgacgt ggagctggcc agcctcgcaa atcggcgaaa 9720acgcctgatt ttacgcgagt ttcccacaga tgatgtggac aagcctgggg ataagtgccc 9780tgcggtattg acacttgagg ggcgcgacta ctgacagatg aggggcgcga tccttgacac 9840ttgaggggca gagtgctgac agatgagggg cgcacctatt gacatttgag gggctgtcca 9900caggcagaaa atccagcatt tgcaagggtt tccgcccgtt tttcggccac cgctaacctg 9960tcttttaacc tgcttttaaa ccaatattta taaaccttgt ttttaaccag ggctgcgccc 10020tgtgcgcgtg accgcgcacg ccgaaggggg gtgccccccc ttctcgaacc ctcccggccc 10080gctaacgcgg gcctcccatc cccccagggg ctgcgcccct cggccgcgaa cggcctcacc 10140ccaaaaatgg cagcgctggc agtccttgcc attgccggga tcggggcagt aacgggatgg 10200gcgatcagcc cgagcgcgac gcccggaagc attgacgtgc cgcaggtgct ggcatcgaca 10260ttcagcgacc aggtgccggg cagtgagggc ggcggcctgg gtggcggcct gcccttcact 10320tcggccgtcg gggcattcac ggacttcatg gcggggccgg caatttttac cttgggcatt 10380cttggcatag tggtcgcggg tgccgtgctc gtgttcgggg gtgcgataaa cccagcgaac 10440catttgaggt gataggtaag attataccga ggtatgaaaa cgagaattgg acctttacag 10500aattactcta tgaagcgcca tatttaaaaa gctaccaaga cgaagaggat gaagaggatg 10560aggaggcaga ttgccttgaa tatattgaca atactgataa gataatatat cttttatata 10620gaagatatcg ccgtatgtaa ggatttcagg gggcaaggca taggcagcgc gcttatcaat 10680atatctatag aatgggcaaa gcataaaaac ttgcatggac taatgcttga aacccaggac 10740aataacctta tagcttgtaa attctatcat aattgggtaa tgactccaac ttattgatag 10800tgttttatgt tcagataatg cccgatgact ttgtcatgca gctccaccga ttttgagaac 10860gacagcgact tccgtcccag ccgtgccagg tgctgcctca gattcaggtt atgccgctca 10920attcgctgcg tatatcgctt gctgattacg tgcagctttc ccttcaggcg ggattcatac 10980agcggccagc catccgtcat ccatatcacc acgtcaaagg gtgacagcag gctcataaga 11040cgccccagcg tcgccatagt gcgttcaccg aatacgtgcg caacaaccgt cttccggaga 11100ctgtcatacg cgtaaaacag ccagcgctgg cgcgatttag ccccgacata gccccactgt 11160tcgtccattt ccgcgcagac gatgacgtca ctgcccggct gtatgcgcga ggttaccgac 11220tgcggcctga gttttttaag tgacgtaaaa tcgtgttgag gccaacgccc ataatgcggg 11280ctgttgcccg gcatccaacg ccattcatgg ccatatcaat gattttctgg tgcgtaccgg 11340gttgagaagc ggtgtaagtg aactgcagtt gccatgtttt acggcagtga gagcagagat 11400agcgctgatg tccggcggtg cttttgccgt tacgcaccac cccgtcagta gctgaacagg 11460agggacagct gatagacaca gaagccactg gagcacctca aaaacaccat catacactaa 11520atcagtaagt tggcagcatc acccataatt gtggtttcaa aatcggctcc gtcgatacta 11580tgttatacgc caactttgaa aacaactttg aaaaagctgt tttctggtat ttaaggtttt 11640agaatgcaag gaacagtgaa ttggagttcg tcttgttata attagcttct tggggtatct 11700ttaaatactg tagaaaagag gaaggaaata ataaatggct aaaatgagaa tatcaccgga 11760attgaaaaaa ctgatcgaaa aataccgctg cgtaaaagat acggaaggaa tgtctcctgc 11820taaggtatat aagctggtgg gagaaaatga aaacctatat ttaaaaatga cggacagccg 11880gtataaaggg accacctatg atgtggaacg ggaaaaggac atgatgctat ggctggaagg 11940aaagctgcct gttccaaagg tcctgcactt tgaacggcat gatggctgga gcaatctgct 12000catgagtgag gccgatggcg tcctttgctc ggaagagtat gaagatgaac aaagccctga 12060aaagattatc gagctgtatg cggagtgcat caggctcttt cactccatcg acatatcgga 12120ttgtccctat acgaatagct tagacagccg cttagccgaa ttggattact tactgaataa 12180cgatctggcc gatgtggatt gcgaaaactg ggaagaagac actccattta aagatccgcg 12240cgagctgtat gattttttaa agacggaaaa gcccgaagag gaacttgtct tttcccacgg 12300cgacctggga gacagcaaca tctttgtgaa agatggcaaa gtaagtggct ttattgatct 12360tgggagaagc ggcagggcgg acaagtggta tgacattgcc ttctgcgtcc ggtcgatcag 12420ggaggatatc ggggaagaac agtatgtcga gctatttttt gacttactgg ggatcaagcc 12480tgattgggag aaaataaaat attatatttt actggatgaa ttgttttagt acctagatgt 12540ggcgcaacga tgccggcgac aagcaggagc gcaccgactt cttccgcatc aagtgttttg 12600gctctcaggc cgaggcccac ggcaagtatt tgggcaaggg gtcgctggta ttcgtgcagg 12660gcaagattcg gaataccaag tacgagaagg acggccagac ggtctacggg accgacttca 12720ttgccgataa ggtggattat ctggacacca aggcaccagg cgggtcaaat caggaataag 12780ggcacattgc cccggcgtga gtcggggcaa tcccgcaagg agggtgaatg aatcggacgt 12840ttgaccggaa ggcatacagg caagaactga tcgacgcggg gttttccgcc gaggatgccg 12900aaaccatcgc aagccgcacc gtcatgcgtg cgccccgcga aaccttccag tccgtcggct 12960cgatggtcca gcaagctacg gccaagatcg agcgcgacag cgtgcaactg gctccccctg 13020ccctgcccgc gccatcggcc gccgtggagc gttcgcgtcg tctcgaacag gaggcggcag 13080gtttggcgaa gtcgatgacc atcgacacgc gaggaactat gacgaccaag aagcgaaaaa 13140ccgccggcga ggacctggca aaacaggtca gcgaggccaa gcaggccgcg ttgctgaaac 13200acacgaagca gcagatcaag gaaatgcagc tttccttgtt cgatattgcg ccgtggccgg 13260acacgatgcg agcgatgcca aacgacacgg cccgctctgc cctgttcacc acgcgcaaca 13320agaaaatccc gcgcgaggcg ctgcaaaaca aggtcatttt ccacgtcaac aaggacgtga 13380agatcaccta caccggcgtc gagctgcggg ccgacgatga cgaactggtg tggcagcagg 13440tgttggagta cgcgaagcgc acccctatcg gcgagccgat caccttcacg ttctacgagc 13500tttgccagga cctgggctgg tcgatcaatg gccggtatta cacgaaggcc gaggaatgcc 13560tgtcgcgcct acaggcgacg gcgatgggct tcacgtccga ccgcgttggg cacctggaat 13620cggtgtcgct gctgcaccgc ttccgcgtcc tggaccgtgg caagaaaacg tcccgttgcc 13680aggtcctgat cgacgaggaa atcgtcgtgc tgtttgctgg cgaccactac acgaaattca 13740tatgggagaa gtaccgcaag ctgtcgccga cggcccgacg gatgttcgac tatttcagct 13800cgcaccggga gccgtacccg ctcaagctgg aaaccttccg cctcatgtgc ggatcggatt 13860ccacccgcgt gaagaagtgg cgcgagcagg tcggcgaagc ctgcgaagag ttgcgaggca 13920gcggcctggt ggaacacgcc tgggtcaatg atgacctggt gcattgcaaa cgctagggcc 13980ttgtggggtc agttccggct gggggttcag cagccagcgc tttactggca tttcaggaac 14040aagcgggcac tgctcgacgc acttgcttcg ctcagtatcg ctcgggacgc acggcgcgct 14100ctacgaactg ccgataaaca gaggattaaa attgacaatt gtgattaagg ctcagattcg 14160acggcttgga gcggccgacg tgcaggattt ccgcgagatc cgattgtcgg ccctgaagaa 14220agctccagag atgttcgggt ccgtttacga gcacgaggag aaaaagccca tggaggcgtt 14280cgctgaacgg ttgcgagatg ccgtggcatt cggcgcctac atcgacggcg agatcattgg 14340gctgtcggtc ttcaaacagg aggacggccc caaggacgct cacaaggcgc atctgtccgg 14400cgttttcgtg gagcccgaac agcgaggccg aggggtcgcc ggtatgctgc tgcgggcgtt 14460gccggcgggt ttattgctcg tgatgatcgt ccgacagatt ccaacgggaa tctggtggat 14520gcgcatcttc atcctcggcg cacttaatat ttcgctattc tggagcttgt tgtttatttc 14580ggtctaccgc ctgccgggcg gggtcgcggc gacggtaggc gctgtgcagc cgctgatggt 14640cgtgttcatc tctgccgctc tgctaggtag cccgatacga ttgatggcgg tcctgggggc 14700tatttgcgga actgcgggcg tggcgctgtt ggtgttgaca ccaaacgcag cgctagatcc 14760tgtcggcgtc gcagcgggcc tggcgggggc ggtttccatg gcgttcggaa ccgtgctgac 14820ccgcaagtgg caacctcccg tgcctctgct cacctttacc gcctggcaac tggcggccgg 14880aggacttctg ctcgttccag tagctttagt gtttgatccg ccaatcccga tgcctacagg 14940aaccaatgtt ctcggcctgg cgtggctcgg cctgatcgga gcgggtttaa cctacttcct 15000ttggttccgg gggatctcgc gactcgaacc tacagttgtt tccttactgg gctttctcag 15060ccccagatct ggggtcgatc agccggggat gcatcaggcc gacagtcgga acttcgggtc 15120cccgacctgt accattcggt gagcaatgga taggggagtt gatatcgtca acgttcactt 15180ctaaagaaat agcgccactc agcttcctca gcggctttat ccagcgattt cctattatgt 15240cggcatagtt ctcaagatcg acagcctgtc acggttaagc gagaaatgaa taagaaggct 15300gataattcgg atctctgcga gggagatgat atttgatcac aggcagcaac gctctgtcat 15360cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc ggcagcttag 15420ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt acaacggctc 15480tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat tttgtgccga 15540gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt gtaaacaaat 15600tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactgg ggtggttttt 15660cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt 15720tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt 15780ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagataggg ttgagtgttg 15840ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa 15900aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca agttttttgg 15960ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt 16020gacggggaaa gccggcgaac gtggcgagaa aggaagggaa

gaaagcgaaa ggagcgggcg 16080ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc 16140cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc 16200cagtcacgac gttgtaaaac gacggccagt gaattgccat cttgaaagaa atatagttta 16260aatatttatt gataaaataa gtcaggtatt atagtccaag caaaaacata atttattgat 16320gcaaagttta aattcagaaa tatttcaata actgattata tcagctggta cattgccgta 16380gatgaaagac tgagtgcgat attatgtgta atacataaat tgatgatata gctagcttag 16440ctcatcgggg gatccttaat cgactctagc tagaacgaat tgttaggtgg cggtacttgg 16500gtcgatatca aagtgcatca cttcttcccg tatgcccaac tttgtataga gagccactgc 16560gggatcgtca ccgtaatctg cttgcacgta gatcacataa gcaccaagcg cgttggcctc 16620atgcttgagg agattgatga gcgcggtggc aatgccctgc ctccggtgct cgccggagac 16680tgcgagatca tagatataga tctcactacg cggctgctca aacctgggca gaacgtaagc 16740cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca agcgcgatga atgtcttact 16800acggagcaag ttcccgaggt aatcggagtc cggctgatgt tgggagtagg tggctacgtc 16860tccgaactca cgaccgaaaa gatcaagagc agcccgcatg gatttgactt ggtcagggcc 16920gagcctacat gtgcgaatga tgcccatact tgagccacct aactttgttt tagggcgact 16980gccctgctgc gtaacatcgt tgctgctgcg taccatggag atctggattg agagtgaata 17040tgagactcta attggatacc gaggggaatt tatggaagtc agtggagcat ttttgacaag 17100aaatatttgc tagctgatag tgaccttagg cgacttttga acgcgcaata atggtttctg 17160acgtatgtgc ttagctcatt aaactccaga aacccgcggc tgagtggctc cttcaacgtt 17220gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc gtcatcggcg ggggtcataa 17280cgtgactccc ttaattctcc gctcatgatc ttgatcccct gcgccatcag atccttggcg 17340gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag 17400ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa 17460gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc 17520agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc 17580gcttccttta gcagcccttg cgccctgagt gcttgcggca gcgtgaagct ctggacatca 17640tgttggatat gaaacaacta ttatttatct acatgtttta gatgttatct gattattttt 17700ataccgtagt cttctattga tgaggagt 177283917746DNAArtificial SequenceMERE04 Plasmid 39tattattaat aattaacaat aattaatata ttataattta tatatatata ttttatatta 60ttataataat attcttacaa atataattat tatattcgac ggtatcgata agctcgggat 120ccctgaaagc gacgttggat gttaacatct acaaattgcc ttttcttatc gaccatgtac 180gtaagcgctt acgtttttgg tggacccttg aggaaactgg tagctgttgt gggcctgtgg 240tctcaagatg gatcattaat ttccaccttc acctacgatg gggggcatcg caccggtgag 300taatattgta cggctaagag cgaatttggc ctgtaggatc cctgaaagcg acgttggatg 360ttaacatcta caaattgcct tttcttatcg accatgtacg taagcgctta cgtttttggt 420ggacccttga ggaaactggt agctgttgtg ggcctgtggt ctcaagatgg atcattaatt 480tccaccttca cctacgatgg ggggcatcgc accggtgagt aatattgtac ggctaagagc 540gaatttggcc tgtaggatcc ctgaaagcga cgttggatgt taacatctac aaattgcctt 600ttcttatcga ccatgtacgt aagcgcttac gtttttggtg gacccttgag gaaactggta 660gctgttgtgg gcctgtggtc tcaagatgga tcattaattt ccaccttcac ctacgatggg 720gggcatcgca ccggtgagta atattgtacg gctaagagcg aatttggcct gtaggatccg 780cgagctggtc aatcccattg cttttgaagc agctcaacat tgatctcttt ctcgatcgag 840ggagattttt caaatcagtg cgcaagacgt gacgtaagta tccgagtcag tttttatttt 900tctactaatt tggtcgttta tttcggcgtg taggacatgg caaccgggcc tgaatttcgc 960gggtattctg tttctattcc aactttttct tgatccgcag ccattaacga cttttgaata 1020gatacgctga cacgccaagc ctcgctagtc aaaagtgtac caaacaacgc tttacagcaa 1080gaacggaatg cgcgtgacgc tcgcggtgac gccatttcgc cttttcagaa atggataaat 1140agccttgctt cctattatat cttcccccaa attaattaag aaactcccga ggtgagcaag 1200gatccggagt cgagcgcgaa gaagagaaag agggaaagcg cgggtaccgg gcccccccct 1260cgacggatca agtgcaaagg tccgccttgt ttctcctctg tctcttgatc tgactaatct 1320tggtttatga ttcgttgagt aattttgggg aaagctagct tcgtccacag tttttttttc 1380gatgaacagt gccgcagtgg cgctgatctt gtatgctatc ctgcaatcgt ggtgaactta 1440tttcttttat atccttcact cccatgaaaa ggctagtaat ctttctcgat gtaacatcgt 1500ccagcactgc tattaccgtg tggtccatcc gacagtctgg ctgaacacat catacgatat 1560tgagcaaaga tcgatctatc ttccctgttc tttaatgaaa gacgtcattt tcatcagtat 1620gatctaagaa tgttgcaact tgcaaggagg cgtttctttc tttgaattta actaactcgt 1680tgagtggccc tgtttctcgg acgtaaggcc tttgctgctc cacacatgtc cattcgaatt 1740ttaccgtgtt tagcaagggc gaaaagtttg catcttgatg atttagcttg actatgcgat 1800tgctttcctg gacccgtgca gctgcggacg gatcccccgc tcgaagctag cttgatcaga 1860tctgatcgaa ttcaccatgc aggtcctgaa cacgatgggc gccgggcagt cctcgcctgc 1920gacggggagc cagaaccagt cgggcaacac cggctcgatc atcaacaact actacatgca 1980gcaataccag aacagcatgg acacccagct cggcgataac gccatctccg gcgggtccaa 2040cgagggctcg accgacacga cctccaccca caccacgaac acccagaaca atgactggtt 2100cagcaagctg gcctccagcg ccttcacggg cctgttcggg gcgctgctcg cggacaagaa 2160gaccgaggag accacgctgc tcgaggaccg gatcctcacc acgcgcaacg ggcacacgac 2220cagcacgacc cagtccagcg tcggggtgac ccacggctac tccacggagg aggaccacgt 2280cgccgggccg aacacgagcg gcctcgagac ccgcgtggtc caggcggagc gcttctacaa 2340gaagtacctg ttcgactgga ccacggacaa ggcgttcggg cacctcgaga agctggagct 2400gccgagcgac caccacggcg tcttcggcca cctcgtggac tcctacgcgt acatgagaaa 2460tgggtgggac gtcgaggtca gcgccgtcgg caaccagttc aacggcgggt gcctgctcgt 2520ggcgatggtc ccggagtgga aggagttcga cacccgcgag aagtatcagc tgaccctctt 2580cccccaccag ttcatcagcc cgcgcaccaa catgaccgcc cacatcacgg tgccgtacct 2640cggggtgaac cgctacgacc agtacaagaa gcacaagccc tggaccctcg tggtcatggt 2700ggtctccccg ctgactgtga acaacacgtc cgccgcgcag atcaaagtct acgccaacat 2760cgcccccacc tacgtgcacg tcgccggcga gctgccctcc aaggagggca tcttccccgt 2820cgcctgcgcc gacgggtacg ggggcctggt gaccaccgac cccaagacgg ccgacccggc 2880ctacggcaaa gtgtacaacc cgcccaggac gaactacccc ggtcgcttca ccaacctcct 2940ggacgtggcg gaggcctgcc cgaccttcct gtgcttcgac gacgggaagc cctacgtcac 3000cacgcgcacc gacgacacgc gcctgctcgc caagttcgac ctcagcctgg ccgctaagca 3060catgagcaac acctacctca gcggcatcgc ccaatactat acccaatact cgggcaccat 3120caacctgcac ttcatgttca cgggcagcac cgacagcaag gcccggtaca tggtggccta 3180catcccgccg ggcgtggaga cccctcccga cacgcccgag cgggctgcgc actgcatcca 3240cgccgagtgg gacaccggcc tcaacagcaa gttcacgttc agcatcccct acgtgtccgc 3300cgcggattac gcttacaccg cctcggacac ggccgagacg atcaacgtcc agggctgggt 3360ctgcatctac cagatcactc acggcaaggc cgagaacgac accctcgtcg tgagcgtctc 3420cgccgggaag gacttcgagc tgaggctgcc catcgacccc aggcagcaga ccacggcgac 3480cggggagtcc gccgaccccg tgaccacgac cgtggagaac tacggcgggg agactcagat 3540ccagcggcgc caccacaccg acatcggctt catcatggac cgcttcgtga agatccagtc 3600cctgtcgccc acccacgtta tcgacctcat gcaggcccac cagcacgggc tcgtgggtgc 3660cctcctgcgc gcggccacct actacttcag cgacctcgag atcgttgtcc gccacgaggg 3720gaacctcacc tgggtcccga acggtgcccc cgagagcgcc ctgctcaaca cctccaaccc 3780cacggcgtac aacaaggccc ccttcacgcg cctcgccctg ccttacaccg cgccccaccg 3840cgtgctggcc acggtgtaca acgggacctc caagtacgcc gtgggcggga gcggccgcag 3900aggcgacatg gggagcctcg ccgctagggt cgtgaagcag ctcccggcct ccttcaacta 3960cggcgccatc aaggccgacg ccatccacga actcctggtc cgcatgaagc gcgccgaact 4020ctactgcccc cggcccctcc tggccatcga ggtctcctcg caggaccgcc acaagcagaa 4080gatcatcgcc ccggccaagc agctgctcaa cttcgacctg ctcaagctcg cgggggacgt 4140cgagtcgaac cccggttaag agctcagagc tcgaatttcc ccgatcgttc aaacatttgg 4200caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 4260ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 4320tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 4380tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctgtacatct agatgcttgc 4440ggcagcgtga agctctggac atcatgttgg atatgaaaca actattattt atctacatgt 4500tttagatgtt atctgattat ttttataccg tagtcttcta ttgatgagga gtctaaggct 4560atagaattat atatctaaat gattaatata tatattatta ataattaaca ataattaata 4620tattataatt tatatatata tattttatat tattataata atattcttac aaatataatt 4680attatattcg acgcccgggc tagtactata aatacggtcc cgaggcctcc tcaccactcg 4740cacatatcct ctttgttttc ctctccgtga aagaagcgag gaagcgcgtc gtctctccca 4800aggtaaggag cagatctctt tgatcgtttt tgttcttctt ttgttttgtt ttttttttct 4860gcggatcttc ggttgcatca tgccttggct gtttttatta gtttaggata tcctcgtttg 4920gatctgagcc gatcatatat gttaaaggtt gtgttcgatc tctttgttca ttttcgcatg 4980aaaaggatgt atccttttga tgtgaggcga tcttctatgg ttaagacttt gttcggtcta 5040ttgatcattt ctgttcttcg tttttgagtt tttttctgcg gatatcgcat catccctagg 5100tttttgcttt ggttaggatg catcctttgg atttgagccg atctcccttg gttaaggctg 5160tgtctgttgc agaggagaaa gtctgtcgag gtccttatgc aggctttgtc cagatgcgcg 5220tgctctctca tgctatgaat ttatgttttg agaactcctc ccggtttttc tagatccgga 5280tttgaagtat tcattgcggt tccccttcgg ttttatgtat ttctcgagtt gatttggtcc 5340atgatcgtgt tctgtccaga tctctcttga tatggatgag atattcgtta cctctttcaa 5400acatcggtgg atgttctttt tagtcttggc tcacctttat ctagaaatta attttcggtt 5460tgaaacccct gcttgttaag gtgatgtatt ccttctttat agatttcggt gtgttatttc 5520ttaacggtga tctgtccgat ccatgtgttg cacctcttgt tttctgtgta atcctctgtg 5580aattataatt atgttttgaa aacgtactta agtaaggggc atgttccccg tttaaaactt 5640ttgttctatc aatttgtggt taatagatcc tgatttgtgg tcgccttatt ctgtctttaa 5700tcgtggattt tatttatctt gagcgcgtcc ttttctttta aaatcatgtg tttaaccttt 5760cagtcgtcat atgttccatc agggtaccgt cgacaccatg tccggggcgc cccccaccga 5820cctgcagaag atggtgatgg gcaacaccaa gcccgtcgag ttgatcctcg acgggaagac 5880cgtggcgatc tgctgcgcca ccggcgtgtt cggcaccgcc tacctcgtcc cgagacacct 5940gttcgccgag aagtatgaca agatcatgct ggacgggcgg gccatgaccg actcggacta 6000ccgggtcttc gagttcgaga tcaaagtgaa gggccaggat atgctctccg acgccgcgct 6060gatggtgctc cacagaggca accgcgtgcg ggacatcacc aagcacttca gggacaccgc 6120gcgcatgaag aaggggaccc ctgtggtcgg ggtcgtgaac aacgccgacg tcgggcgcct 6180catcttctcc ggcgaggcgc tgacctacaa ggacatcgtc gtgtgcatgg acggggacac 6240gatgccgggg ctcttcgcct acaaggccgc gaccaaggcc gggtactgcg ggggtgcggt 6300gctcgccaag gacggcgccg acacgttcat cgtgggcacc cactccgccg gcggtaacgg 6360cgtcggctac tgctcctgcg tgtcccggtc catgctgctg cggatgaagg cccacgttga 6420ccccgagccg cagcacgagt aaaagcttat cgatgtgcac gatcgttcaa acatttggca 6480ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct 6540gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg 6600ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata 6660gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggtgga 6720caactcgtca cgtactagtg gcgcgcccac gtgatttaaa tcgtacgggc cggccgtata 6780ctctagtgga tcccccaatt cagatcggct gagtggctcc ttcaacgttg cggttctgtc 6840agttccaaac gtaaaacggc ttgtcccgcg tcatcggcgg gggtcataac gtgactccct 6900taattctccg ctcatgatca gattgtcgtt tcccgccttc agtttaaact atcagtgttt 6960gacaggatat attggcgggt aaacctaaga gaaaagagcg tttattagaa taatcggata 7020tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg ccaaccacag 7080ggttccccag atctggcgcc ggccagcgag acgagcaaga ttggccgccg cccgaaacga 7140tccgacagcg cgcccagcac aggtgcgcag gcaaattgca ccaacgcata cagcgccagc 7200agaatgccat agtgggcggt gacgtcgttc gagtgaacca gatcgcgcag gaggcccggc 7260agcaccggca taatcaggcc gatgccgaca gcgtcgagcg cgacagtgct cagaattacg 7320atcaggggta tgttgggttt cacgtctggc ctccggacca gcctccgctg gtccgattga 7380acgcgcggat tctttatcac tgataagttg gtggacatat tatgtttatc agtgataaag 7440tgtcaagcat gacaaagttg cagccgaata cagtgatccg tgccgccctg gacctgttga 7500acgaggtcgg cgtagacggt ctgacgacac gcaaactggc ggaacggttg ggggttcagc 7560agccggcgct ttactggcac ttcaggaaca agcgggcgct gctcgacgca ctggccgaag 7620ccatgctggc ggagaatcat acgcattcgg tgccgagagc cgacgacgac tggcgctcat 7680ttctgatcgg gaatgcccgc agcttcaggc aggcgctgct cgcctaccgc gatggcgcgc 7740gcatccatgc cggcacgcga ccgggcgcac cgcagatgga aacggccgac gcgcagcttc 7800gcttcctctg cgaggcgggt ttttcggccg gggacgccgt caatgcgctg atgacaatca 7860gctacttcac tgttggggcc gtgcttgagg agcaggccgg cgacagcgat gccggcgagc 7920gcggcggcac cgttgaacag gctccgctct cgccgctgtt gcgggccgcg atagacgcct 7980tcgacgaagc cggtccggac gcagcgttcg agcagggact cgcggtgatt gtcgatggat 8040tggcgaaaag gaggctcgtt gtcaggaacg ttgaaggacc gagaaagggt gacgattgat 8100caggaccgct gccggagcgc aacccactca ctacagcaga gccatgtaga caacatcccc 8160tccccctttc caccgcgtca gacgcccgta gcagcccgct acgggctttt tcatgccctg 8220ccctagcgtc caagcctcac ggccgcgctc ggcctctctg gcggccttct ggcgctcttc 8280cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 8340tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 8400gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 8460ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 8520aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 8580tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 8640ggcgcttttc cgctgcataa ccctgcttcg gggtcattat agcgattttt tcggtatatc 8700catccttttt cgcacgatat acaggatttt gccaaagggt tcgtgtagac tttccttggt 8760gtatccaacg gcgtcagccg ggcaggatag gtgaagtagg cccacccgcg agcgggtgtt 8820ccttcttcac tgtcccttat tcgcacctgg cggtgctcaa cgggaatcct gctctgcgag 8880gctggccggc taccgccggc gtaacagatg agggcaagcg gatggctgat gaaaccaagc 8940caaccaggaa gggcagccca cctatcaagg tgtactgcct tccagacgaa cgaagagcga 9000ttgaggaaaa ggcggcggcg gccggcatga gcctgtcggc ctacctgctg gccgtcggcc 9060agggctacaa aatcacgggc gtcgtggact atgagcacgt ccgcgagctg gcccgcatca 9120atggcgacct gggccgcctg ggcggcctgc tgaaactctg gctcaccgac gacccgcgca 9180cggcgcggtt cggtgatgcc acgatcctcg ccctgctggc gaagatcgaa gagaagcagg 9240acgagcttgg caaggtcatg atgggcgtgg tccgcccgag ggcagagcca tgactttttt 9300agccgctaaa acggccgggg ggtgcgcgtg attgccaagc acgtccccat gcgctccatc 9360aagaagagcg acttcgcgga gctggtgaag tacatcaccg acgagcaagg caagaccgag 9420cgcctttgcg acgctcaccg ggctggttgc cctcgccgct gggctggcgg ccgtctatgg 9480ccctgcaaac gcgccagaaa cgccgtcgaa gccgtgtgcg agacaccgcg gccgccggcg 9540ttgtggatac ctcgcggaaa acttggccct cactgacaga tgaggggcgg acgttgacac 9600ttgaggggcc gactcacccg gcgcggcgtt gacagatgag gggcaggctc gatttcggcc 9660ggcgacgtgg agctggccag cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt 9720cccacagatg atgtggacaa gcctggggat aagtgccctg cggtattgac acttgagggg 9780cgcgactact gacagatgag gggcgcgatc cttgacactt gaggggcaga gtgctgacag 9840atgaggggcg cacctattga catttgaggg gctgtccaca ggcagaaaat ccagcatttg 9900caagggtttc cgcccgtttt tcggccaccg ctaacctgtc ttttaacctg cttttaaacc 9960aatatttata aaccttgttt ttaaccaggg ctgcgccctg tgcgcgtgac cgcgcacgcc 10020gaaggggggt gccccccctt ctcgaaccct cccggcccgc taacgcgggc ctcccatccc 10080cccaggggct gcgcccctcg gccgcgaacg gcctcacccc aaaaatggca gcgctggcag 10140tccttgccat tgccgggatc ggggcagtaa cgggatgggc gatcagcccg agcgcgacgc 10200ccggaagcat tgacgtgccg caggtgctgg catcgacatt cagcgaccag gtgccgggca 10260gtgagggcgg cggcctgggt ggcggcctgc ccttcacttc ggccgtcggg gcattcacgg 10320acttcatggc ggggccggca atttttacct tgggcattct tggcatagtg gtcgcgggtg 10380ccgtgctcgt gttcgggggt gcgataaacc cagcgaacca tttgaggtga taggtaagat 10440tataccgagg tatgaaaacg agaattggac ctttacagaa ttactctatg aagcgccata 10500tttaaaaagc taccaagacg aagaggatga agaggatgag gaggcagatt gccttgaata 10560tattgacaat actgataaga taatatatct tttatataga agatatcgcc gtatgtaagg 10620atttcagggg gcaaggcata ggcagcgcgc ttatcaatat atctatagaa tgggcaaagc 10680ataaaaactt gcatggacta atgcttgaaa cccaggacaa taaccttata gcttgtaaat 10740tctatcataa ttgggtaatg actccaactt attgatagtg ttttatgttc agataatgcc 10800cgatgacttt gtcatgcagc tccaccgatt ttgagaacga cagcgacttc cgtcccagcc 10860gtgccaggtg ctgcctcaga ttcaggttat gccgctcaat tcgctgcgta tatcgcttgc 10920tgattacgtg cagctttccc ttcaggcggg attcatacag cggccagcca tccgtcatcc 10980atatcaccac gtcaaagggt gacagcaggc tcataagacg ccccagcgtc gccatagtgc 11040gttcaccgaa tacgtgcgca acaaccgtct tccggagact gtcatacgcg taaaacagcc 11100agcgctggcg cgatttagcc ccgacatagc cccactgttc gtccatttcc gcgcagacga 11160tgacgtcact gcccggctgt atgcgcgagg ttaccgactg cggcctgagt tttttaagtg 11220acgtaaaatc gtgttgaggc caacgcccat aatgcgggct gttgcccggc atccaacgcc 11280attcatggcc atatcaatga ttttctggtg cgtaccgggt tgagaagcgg tgtaagtgaa 11340ctgcagttgc catgttttac ggcagtgaga gcagagatag cgctgatgtc cggcggtgct 11400tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagacacaga 11460agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac 11520ccataattgt ggtttcaaaa tcggctccgt cgatactatg ttatacgcca actttgaaaa 11580caactttgaa aaagctgttt tctggtattt aaggttttag aatgcaagga acagtgaatt 11640ggagttcgtc ttgttataat tagcttcttg gggtatcttt aaatactgta gaaaagagga 11700aggaaataat aaatggctaa aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa 11760taccgctgcg taaaagatac ggaaggaatg tctcctgcta aggtatataa gctggtggga 11820gaaaatgaaa acctatattt aaaaatgacg gacagccggt ataaagggac cacctatgat 11880gtggaacggg aaaaggacat gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc 11940ctgcactttg aacggcatga tggctggagc aatctgctca tgagtgaggc cgatggcgtc 12000ctttgctcgg aagagtatga agatgaacaa agccctgaaa agattatcga gctgtatgcg 12060gagtgcatca ggctctttca ctccatcgac atatcggatt gtccctatac gaatagctta 12120gacagccgct tagccgaatt ggattactta ctgaataacg atctggccga tgtggattgc 12180gaaaactggg aagaagacac tccatttaaa gatccgcgcg agctgtatga ttttttaaag 12240acggaaaagc ccgaagagga acttgtcttt tcccacggcg acctgggaga cagcaacatc 12300tttgtgaaag atggcaaagt aagtggcttt attgatcttg ggagaagcgg cagggcggac 12360aagtggtatg acattgcctt ctgcgtccgg tcgatcaggg aggatatcgg ggaagaacag 12420tatgtcgagc tattttttga cttactgggg atcaagcctg attgggagaa aataaaatat 12480tatattttac tggatgaatt gttttagtac ctagatgtgg cgcaacgatg ccggcgacaa 12540gcaggagcgc accgacttct tccgcatcaa gtgttttggc tctcaggccg aggcccacgg 12600caagtatttg ggcaaggggt cgctggtatt cgtgcagggc aagattcgga ataccaagta 12660cgagaaggac ggccagacgg tctacgggac cgacttcatt gccgataagg tggattatct 12720ggacaccaag gcaccaggcg ggtcaaatca ggaataaggg cacattgccc cggcgtgagt 12780cggggcaatc ccgcaaggag ggtgaatgaa tcggacgttt gaccggaagg catacaggca 12840agaactgatc gacgcggggt tttccgccga ggatgccgaa accatcgcaa gccgcaccgt 12900catgcgtgcg ccccgcgaaa ccttccagtc cgtcggctcg atggtccagc aagctacggc 12960caagatcgag cgcgacagcg tgcaactggc tccccctgcc ctgcccgcgc catcggccgc 13020cgtggagcgt tcgcgtcgtc tcgaacagga ggcggcaggt ttggcgaagt cgatgaccat 13080cgacacgcga ggaactatga cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa 13140acaggtcagc gaggccaagc aggccgcgtt gctgaaacac acgaagcagc agatcaagga 13200aatgcagctt tccttgttcg atattgcgcc gtggccggac acgatgcgag cgatgccaaa 13260cgacacggcc cgctctgccc tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct

13320gcaaaacaag gtcattttcc acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga 13380gctgcgggcc gacgatgacg aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac 13440ccctatcggc gagccgatca ccttcacgtt ctacgagctt tgccaggacc tgggctggtc 13500gatcaatggc cggtattaca cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc 13560gatgggcttc acgtccgacc gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt 13620ccgcgtcctg gaccgtggca agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat 13680cgtcgtgctg tttgctggcg accactacac gaaattcata tgggagaagt accgcaagct 13740gtcgccgacg gcccgacgga tgttcgacta tttcagctcg caccgggagc cgtacccgct 13800caagctggaa accttccgcc tcatgtgcgg atcggattcc acccgcgtga agaagtggcg 13860cgagcaggtc ggcgaagcct gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg 13920ggtcaatgat gacctggtgc attgcaaacg ctagggcctt gtggggtcag ttccggctgg 13980gggttcagca gccagcgctt tactggcatt tcaggaacaa gcgggcactg ctcgacgcac 14040ttgcttcgct cagtatcgct cgggacgcac ggcgcgctct acgaactgcc gataaacaga 14100ggattaaaat tgacaattgt gattaaggct cagattcgac ggcttggagc ggccgacgtg 14160caggatttcc gcgagatccg attgtcggcc ctgaagaaag ctccagagat gttcgggtcc 14220gtttacgagc acgaggagaa aaagcccatg gaggcgttcg ctgaacggtt gcgagatgcc 14280gtggcattcg gcgcctacat cgacggcgag atcattgggc tgtcggtctt caaacaggag 14340gacggcccca aggacgctca caaggcgcat ctgtccggcg ttttcgtgga gcccgaacag 14400cgaggccgag gggtcgccgg tatgctgctg cgggcgttgc cggcgggttt attgctcgtg 14460atgatcgtcc gacagattcc aacgggaatc tggtggatgc gcatcttcat cctcggcgca 14520cttaatattt cgctattctg gagcttgttg tttatttcgg tctaccgcct gccgggcggg 14580gtcgcggcga cggtaggcgc tgtgcagccg ctgatggtcg tgttcatctc tgccgctctg 14640ctaggtagcc cgatacgatt gatggcggtc ctgggggcta tttgcggaac tgcgggcgtg 14700gcgctgttgg tgttgacacc aaacgcagcg ctagatcctg tcggcgtcgc agcgggcctg 14760gcgggggcgg tttccatggc gttcggaacc gtgctgaccc gcaagtggca acctcccgtg 14820cctctgctca cctttaccgc ctggcaactg gcggccggag gacttctgct cgttccagta 14880gctttagtgt ttgatccgcc aatcccgatg cctacaggaa ccaatgttct cggcctggcg 14940tggctcggcc tgatcggagc gggtttaacc tacttccttt ggttccgggg gatctcgcga 15000ctcgaaccta cagttgtttc cttactgggc tttctcagcc ccagatctgg ggtcgatcag 15060ccggggatgc atcaggccga cagtcggaac ttcgggtccc cgacctgtac cattcggtga 15120gcaatggata ggggagttga tatcgtcaac gttcacttct aaagaaatag cgccactcag 15180cttcctcagc ggctttatcc agcgatttcc tattatgtcg gcatagttct caagatcgac 15240agcctgtcac ggttaagcga gaaatgaata agaaggctga taattcggat ctctgcgagg 15300gagatgatat ttgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc 15360tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat 15420cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac 15480tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg 15540gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa 15600cacattgcgg acgtttttaa tgtactgggg tggtttttct tttcaccagt gagacgggca 15660acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg 15720tttgccccag caggcgaaaa tcctgtttga tggtggttcc gaaatcggca aaatccctta 15780taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 15840actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 15900cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact 15960aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 16020ggcgagaaag gaagggaaga aagcgaaagg agcgggcgcc attcaggctg cgcaactgtt 16080gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg 16140ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga 16200cggccagtga attgccatct tgaaagaaat atagtttaaa tatttattga taaaataagt 16260caggtattat agtccaagca aaaacataat ttattgatgc aaagtttaaa ttcagaaata 16320tttcaataac tgattatatc agctggtaca ttgccgtaga tgaaagactg agtgcgatat 16380tatgtgtaat acataaattg atgatatagc tagcttagct catcggggga tccttaatcg 16440actctagcta gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact 16500tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct 16560tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc 16620gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc 16680tcactacgcg gctgctcaaa cctgggcaga acgtaagccg cgagagcgcc aacaaccgct 16740tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa 16800tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga 16860tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg 16920cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt aacatcgttg 16980ctgctgcgta ccatggagat ctggattgag agtgaatatg agactctaat tggataccga 17040ggggaattta tggaagtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg 17100accttaggcg acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa 17160actccagaaa cccgcggctg agtggctcct tcaacgttgc ggttctgtca gttccaaacg 17220taaaacggct tgtcccgcgt catcggcggg ggtcataacg tgactccctt aattctccgc 17280tcatgatctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 17340tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 17400tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 17460tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 17520tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc agcccttgcg 17580ccctgagtgc ttgcggcagc gtgaagctct ggacatcatg ttggatatga aacaactatt 17640atttatctac atgttttaga tgttatctga ttatttttat accgtagtct tctattgatg 17700aggagtctaa ggctatagaa ttatatatct aaatgattaa tatata 17746

Claims

1. A composition comprising an FMDV antigen and a pharmaceutical or veterinarily acceptable carrier, excipient, or vehicle.

2. The composition of claim 1, wherein the FMDV antigen is selected from the group consisting of FMDV P1-3C, FMDV P1, FMDV VP0, FMDV VP1, FMDV VP3, FMDV VP2, and FMDV VP4.

3. The composition of claim 1 or 2, wherein the FMDV antigen is expressed in a plant or microalga.

4. The composition of any one of claims 1-3, wherein the FMDV antigen is partially purified.

5. The composition of any one of claims 1-3, wherein the FMDV antigen is substantially purified.

6. The composition of any one of claims 1-5, wherein the FMDV antigen has at least 80% sequence identity to a polypeptide having a sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29.

7. The composition of any one of claims 1-6, wherein the FMDV antigen is encoded by a polynucleotide having at least 70% sequence identity to the sequence as set forth in SEQ ID NOs:1, 2, 4, 8, 9, 11, 13, 14, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30-35.

8. The composition of any one of claims 1-7, wherein the pharmaceutical or veterinarily acceptable carrier, excipient, or vehicle is a water-in-oil emulsion or an oil-in-water emulsion.

9. A method of vaccinating a host susceptible to ovine, bovine, caprine, or porcine FMDV comprising at least one administration of the composition according to any one of claims 1 to 8.

10. A method of vaccinating a host susceptible to ovine, bovine, caprine, or porcine FMDV comprising a prime-boost administration protocol.

11. The method of claim 10, wherein said prime-boost administration comprises a prime-administration of the composition of any one of claims 1-8, and a boost-administration of a vaccine or composition comprising a recombinant viral vector that contains and expresses the FMDV antigen in vivo, or an inactivated viral vaccine comprising the FMDV antigen, or a DNA plasmid vaccine or composition that contains or expresses the FMDV antigen.

12. The method of claim 10, wherein the prime-boost administration comprises a prime-administration of a vaccine or composition comprising a recombinant viral vector that contains and expresses the FMDV antigen in vivo, or an inactivated viral vaccine comprising the FMDV, or a DNA plasmid vaccine or composition that contains or expresses the FMDV antigen, and a boost-administration of the composition of any one of claims 1-8.

13. The method of claim 10, wherein the prime-boost administration comprises a prime-administration of the composition of any one of claims 1-8, and a boost-administration of the composition of any one of claims 1-8.

14. The method of any one of claims 9-13, wherein the host is ovine, bovine, caprine, or porcine.

15. A substantially purified FMDV polypeptide expressed in a plant or microalga, wherein the polypeptide comprises: a) an amino acid sequence having at least 80% sequence identity to a polypeptide having the sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29; b) a conservative variant of the amino acid sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29; c) an immunogenic fragment comprising at least eight consecutive amino acids of the amino acid sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29, that specifically binds to an antibody that specifically binds to the amino acid sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29.

16. A plasmid comprising a DNA fragment having at least 70% sequence identity to the sequence as set forth in SEQ ID NO:1, 2, 4, 8, 9, 11, 13, 14, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30-35.

17. The plasmid of claim 16, wherein the plasmid is for plant transformation.

18. A host cell transformed with the plasmid of claim 17.

19. A stably transformed duckweed plant or microalga culture transformed with a gene expressing an FMDV antigen or fragment or variant thereof.

20. The duckweed plant or microalga culture of claim 19, wherein the antigen or fragment or variant thereof has at least 80% sequence identity to the sequence as set forth in SEQ ID NOs:3, 10, 12, 17, 20, 23, 26, or 29.

21. A method of producing an FMDV antigen comprising: (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein the duckweed plant culture or the duckweed nodule culture is stably transformed to express the antigen, and wherein the antigen is expressed from a nucleotide sequence comprising a coding sequence for the antigen; and (b) collecting the antigen from the culture biomass.