Self-containing lactococcus strain

Abstract

a recombinant Lactococcus strain, with environmentally limited growth and viability. More particularly, it relates to a recombinant Lactococcus that can only survive in a medium, where well-defined medium compounds are present. A preferred embodiment is a Lactococcus that may only survive in a host organism, where such medium compounds are present, but cannot survive outside the host organism in the absence of such medium compounds.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application is a continuation of U.S. patent application Ser. No. 10/687,996, filed Oct. 17, 2003, pending, which is a continuation of PCT International Patent Application No. PCT/EP02/04942, filed on May 3, 2002, designating the United States of America, and published, in English, as PCT International Publication No. WO 02/090551 A2 on Nov. 14, 2002, the contents of the entirety of which are incorporated herein by this reference.

TECHNICAL FIELD

[0002]The invention relates to a recombinant Lactococcus strain, with environmentally limited growth and viability. More particularly, it relates to a recombinant Lactococcus that can only survive in a medium, where well-defined medium compounds are present. A preferred embodiment is a Lactococcus that may only survive in a host organism, where the medium compounds are present, but cannot survive outside the host organism in an absence of the medium compounds. Moreover, the Lactococcus can be transformed with prophylactic and/or therapeutic molecules and can, as such, be used to treat diseases such as inflammatory bowel diseases.

BACKGROUND

[0003]Lactic acid bacteria have long been used in a wide variety of industrial fermentation processes. They have generally-regarded-as-safe ("GRAS") status, making them potentially useful organisms for the production of commercially important proteins. Indeed, several heterologous proteins, such as Interleukin-2, have been successfully produced in Lactococcus spp (Steidler et al., 1995). It is, however, undesirable that such genetically modified microorganisms survive and spread into the environment.

[0004]To avoid unintentional release of genetically modified microorganisms, special guidelines for safe handling and technical requirements for physical containment are used. Although this may be useful in industrial fermentations, the physical containment is generally considered as insufficient, and additional biological containment measures are taken to reduce the possibility of survival of the genetically modified microorganism in the environment.

[0005]Biological containment is extremely important in cases where physical containment is difficult or even inapplicable. This is, amongst others, the case in applications where genetically modified microorganisms are used as live vaccines or as a vehicle for delivery of therapeutic compounds. Such applications have been described, for example, in PCT International Publication Number WO 97/14806, which discloses the delivery of biologically active peptides, such as cytokines, to a subject by recombinant noninvasive or nonpathogenic bacteria. Further, PCT International Publication Number WO 96/11277 describes the delivery of therapeutic compounds to an animal or human by administering a recombinant bacterium encoding a therapeutic protein. Steidler et al. (2000) describe the treatment of colitis by administration of a recombinant Lactococcus lactis secreting Interleukin-10. Such a delivery may indeed be extremely useful to treat a disease in an affected human or animal, but the recombinant bacterium may act as a harmful and pathogenic microorganism when it enters a nonaffected subject, and an efficient biological containment that avoids such unintentional spreading of the microorganism is needed.

[0006]Biological containment systems for host organisms may be passive and based on a strict requirement of the host for a specific growth factor or a nutrient that is not present or is present in low concentrations in the outside environment. Alternatively, it may be active and, based on so-called suicidal genetic elements in the host, wherein the host is killed in the outside environment by a cell-killing function, encoded by a gene that is under the control of a promoter only being expressed under specific environmental conditions.

[0007]Passive biological containment systems are well known in microorganisms such as Escherichia coli or Saccharomyces cerevisiae. Such E. coli strains are disclosed, for example, in U.S. Pat. No. 4,190,495. Also, PCT International Publication Number WO 95/10621 discloses lactic acid bacterial suppressor mutants and their use as means of containment in lactic acid bacteria, but in that case, the containment is on the plasmid level, rather than on the level of the host strain and it stabilizes the plasmid in the host strain, but does not provide containment for the genetically modified host strain itself.

[0008]Active suicidal systems have been described by several authors. Such systems consist of two elements: a lethal gene and a control sequence that switches on the expression of the lethal gene under nonpermissive conditions. For example, PCT International Publication Number WO 95/10614 discloses the use of a cytoplasmatically active truncated and/or mutated Staphylococcus aureus nuclease as a lethal gene. PCT International Publication Number WO 96/40947 discloses a recombinant bacterial system with environmentally limited viability, based on the expression of either an essential gene, expressed when the cell is in the permissive environment and not expressed or temporarily expressed when the cell is in the nonpermissive environment, and/or a lethal gene, wherein expression of the gene is lethal to the cell and the lethal gene is expressed when the cell is in the nonpermissive environment but not when the cell is in the permissive environment. PCT International Publication Number WO 99/58652 describes a biological containment system based on the relE cytotoxin. However, most systems have been elaborated for Escherichia coli (Tedkin et al., 1995; Knudsen et al., 1995; Schweder et al., 1995) or for Pseudomonas (Kaplan et al., 1999; Molino et al., 1998). Although several of the containment systems theoretically can by applied to lactic acid bacteria, no specific biological containment system for Lactococcus has been described that allows the usage of a self-containing and transformed Lactococcus to deliver prophylactic and/or therapeutic molecules in order to prevent and/or treat diseases.

DISCLOSURE OF THE INVENTION

[0009]The invention includes a suitable biological containment system for Lactococcus. A first aspect of the invention is an isolated strain of Lactococcus sp. comprising a defective thymidylate synthase gene.

[0010]Another aspect of the invention is the use of a strain according to the invention as a host strain for transformation, wherein the transforming plasmid does not comprise an intact thymidylate synthase gene.

[0011]Still another aspect of the invention is a transformed strain of Lactococcus sp. according to the invention, comprising a plasmid that does not comprise an intact thymidylate synthase gene. Another aspect of the invention relates to a transformed strain of Lactococcus sp. comprising a gene or expression unit encoding a prophylactic and/or therapeutic molecule such as Interleukin-10. Consequently, the present invention also relates to the usage of a transformed strain of Lactococcus sp. to deliver prophylactic and/or therapeutic molecules and, as such, to treat diseases. Methods to deliver the molecules and methods to treat diseases such as inflammatory bowel diseases are explained in detail in PCT International Publication Numbers WO 97/14806 and WO 00/23471 to Steidler et al. and in Steidler et al. (Science 2000, 289: 1352), the contents of all of which are incorporated herein by this reference.

[0012]Another aspect of the invention is a medical preparation comprising a transformed strain of Lactococcus sp., according to the invention.

[0013]The invention further demonstrates that the transformed strains surprisingly pass the gut at the same speed as the control strains, showing that their loss of viability indeed is not different from that of the control strains. However, once the strain is secreted in the environment, for example, in the feces, it is not able to survive any longer.

[0014]The transforming plasmid can be any plasmid, as long as it does not complement the thyA mutation. It may be a self-replicating plasmid that preferably carries one or more genes of interest and one or more resistance markers, or it may be an integrative plasmid. In the latter case, the integrative plasmid itself may be used to create the mutation by causing integration at the thyA site, whereby the thyA gene is inactivated.

BRIEF DESCRIPTION OF THE FIGURES

[0015]FIG. 1: Map of the MG1363 thyA locus.

[0016]FIG. 2: Schematic representation of the different expression modules as present on pOThy plasmids ands genomic integrants of hIL-10. Black parts represent original L. lactis MG1363 genetic information; white parts represent recombinant genetic information.

[0017]FIGS. 3A and 3B: PCR identification of Thy11 (Thy11 1.1 and Thy11 7.1 represent individually obtained, identical clones). Standard PCR reactions were performed by using aliquots of saturated cultures of the indicated strains as a source of a DNA template. FIG. 3A shows an agarose gel of the products of the indicated PCR reactions. FIG. 3B shows the positions at which primers attach in the thyA (1), upstream (2) or downstream (3) PCR's. Oligonucleotide primers used:

TABLE-US-00001 (1): ATgACTTACgCAgATCAAgTTTTT (SEQ ID NO: 8 of the accompanying SEQUENCE LISTING, which is incorporated herein by this reference and (SEQ ID NO: 9) TTAAATTgCTAAATCAAATTTCAATTg (2): (SEQ ID NO: 10) TCTgATTgAgTACCTTgACC and (SEQ ID NO: 11) gCAATCATAATTggTTTTATTg (3): (SEQ ID NO: 12) CTTACATgACTATgAAAATCCg and (SEQ ID NO: 13) cTTTTTTATTATTAgggAAAgCA.

[0018]FIGS. 4A and 4B: PCR identification of Thy11, Thy12, Thy15 and Thy16. Standard PCR reactions were performed by using three-day old colonies of the indicated strains as a source of DNA template. FIG. 4A shows the positions at which primers attach in the upstream (1), downstream (2) or thyA (3), PCRs. Oligonucleotide primers used:

TABLE-US-00002 (1): ATgACTTACgCAgATCAAgTTTTT (SEQ ID NO: 8) and TTAAATTgCTAAATCAAATTTCAATTg (SEQ ID NO: 9) (2): TCTgATTgAgTACCTTgACC (SEQ ID NO: 10) and gCAATCATAATTggTTTTATTg (SEQ ID NO: 11) (3): CTTACATgACTATgAAAATCCg (SEQ ID NO: 12) and cTTTTTTATTATTAgggAAAgCA. (SEQ ID NO: 13)

FIG. 4B shows an agarose gel of the products of the indicated PCR reactions.

[0019]FIGS. 5A and 5B: Southern blot analysis of the indicated strains. Chromosomal DNA was extracted and digested with the indicated restriction enzymes. Following agarose gel electrophoresis, the DNA was transferred to a membrane and the chromosome structure around the thyA locus was revealed by use of DIG-labeled thyA or hIL-10 DNA fragments (FIG. 5A). FIG. 5B shows a schematic overview of the predicted structure of the thyA locus in both MG1363 and Thy11.

[0020]FIG. 6A shows a schematic overview of part of the predicted structure of the L. lactis chromosome at the thyA locus in MG1363, Thy11, Thy12, Thy15 and Thy16. Numbers indicate base pairs. FIG. 6B illustrates a Southern blot analysis of the indicated strains. Chromosomal DNA was extracted and digested with NdeI and SpeI restriction enzymes. Following agarose gel electrophoresis, the DNA was transferred to a membrane and the chromosome structure around the thyA locus was revealed by use of DIG-labeled thyA or hIL-10 DNA fragments.

[0021]FIGS. 7A and 7B: Production of hIL-10. FIG. 7A shows a western blot revealed with anti-hIL-10 antiserum of culture supernatant and cell-associated proteins of the indicated strains. FIG. 7B shows quantification (by ELISA) of hIL-10 present in the culture supernatant.

[0022]FIGS. 8A and 8B: Production of hIL-10. FIG. 8A shows quantification (by ELISA) of hIL-10 present in the culture supernatant of the indicated strains. FIG. 8B shows a western blot revealed with anti-hIL-10 antiserum of culture supernatant proteins of the indicated strains.

[0023]FIG. 9: Production of hIL-10 by the L. lactis strains LL108 carrying pOThy11, pOThy12, or pOThy16. Quantification (by ELISA) of hIL-10 present in the culture supernatant of the indicated strains is shown. The N-terminal protein sequence of the recombinant hIL-10 was determined by Edman degradation and was shown to be identical to the structure as predicted for the mature, recombinant hIL-10. The protein showed full biological activity.

[0024]FIGS. 10A and 10B: Growth rate of the indicated strains in GM17 containing 100 μg/ml (T100), 50 μg/ml (T50), 25 μg/ml (T25), or no (T0) extra thymidine and possibly supplemented with 5 μg/ml of erythromycin (E). Saturated overnight cultures (prepared in T50) were diluted 1:100 in the indicated culture media. FIG. 10A shows the kinetics of absorbance accumulation. FIG. 10B shows the kinetics of the number of colony-forming units (cfu) per ml of culture.

[0025]FIG. 11: Growth rate of MG1363 and Thy12 in thymidine-free medium (TFM). TFM was prepared by growing L. lactis Thy12 bacteria in GM17, removing the bacteria by subsequent centrifugation and filtration on a 0.22 μm pore size filter, adjusting the pH to 7.0 and autoclaving. MG1363 and Thy12 bacteria were collected from an overnight culture in GM17 or GM17+50 μg/ml of thymidine, respectively, and washed in M9 buffer (6 Na₂HPO₄, 3 g/l KH₂PO₄, 1 g/l NH₄Cl, 0.5 g/l NaCl in water). The suspensions of both were either diluted in TFM or TFM supplemented with 50 μg/ml of thymidine (T50). CFU counts were determined at different time points: t=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 20 hours. This shows that Thy12 viability is severely impaired in the absence of thymidine.

[0026]FIG. 12: Intestinal passage and viability: L. lactis MG1363 was transformed with the plasmid pLET2N, which carries a chloramphenicol (Cm) resistance marker. L. lactis Thy12 was transformed with the plasmid pT1NX, which carries an erythromycin (Em) resistance marker. Of both strains, 10⁹ bacteria were resuspended in BM9 (6 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 1 g/l NH₄Cl, 0.5 g/l NaCl in 25 mM NaHCO₃+25 mM Na₂CO₃), mixed and inoculated in three mice at t=0h. Feces were collected at the time intervals -1 to 0, 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10 and 10 to overnight. All samples were resuspended in isotonic buffer and appropriate dilutions were plated on GM17 (M17 medium, Difco, St. Louis, Mo., supplemented with 0.5% glucose) plates containing either Cm, Em or Em+50 μg/ml thymidine. Colony-forming units for the different plates are represented in the graph.

DETAILED DESCRIPTION OF THE INVENTION

[0027]As previously identified, the invention includes a suitable biological containment system for Lactococcus. In one aspect, the invention is an isolated strain of Lactococcus sp. comprising a defective thymidylate synthase gene. Preferably, the defective thymidylate synthase gene is inactivated by gene disruption. Even more preferably, the Lactococcus sp. is Lactococcus lactis. A special embodiment is a Lactococcus sp. strain, preferably Lactococcus lactis, more preferably a Lactococcus lactis MG1363 derivative, wherein the thymidylate synthase gene has been disrupted and replaced by an Interleukin-10 expression unit. The Interleukin-10 expression unit is preferably, but not limited to, a human Interleukin-10 expression unit or gene encoding for human Interleukin-10.

[0028]The Lactococcus lactis subsp. lactis thymidylate synthase gene (thyA) has been cloned by Ross et al. (1990a). Its sequence is comprised in SEQ ID NO:3 and SEQ ID NO:5. European Patent Application Publication Number 0406003 discloses a vector devoid of antibiotic resistance and bearing a thymidylate synthase gene as a selection marker; the same vector has been described by Ross et al. (1990b). However, this vector could not be used in a Lactococcus lactis strain due to the lack of a suitable thyA mutant that has never been described. The present invention discloses how to construct such a mutant by gene disruption using homologous recombination in Lactococcus. In a preferred embodiment, the thyA gene is disrupted by a functional human Interleukin-10 expression cassette. However, it is clear that any construct can be used for gene disruption, as long as it results in an inactivation of the thyA gene or in an inactive thymidylate synthase. As a nonlimiting example, the homologous recombination may result in a deletion of the gene, in one or more amino acid substitutions that lead to an inactive form of the thymidylate synthase, or in a frame shift mutation resulting in a truncated form of the protein.

[0029]Such a Lactococcus sp. thyA mutant is very useful as a host strain for transformation in situations where more severe containment than purely physical containment is needed. Indeed, thyA mutants cannot survive in an environment without or with only a limited concentration of thymidine and/or thymine. When such a strain is transformed with a plasmid that does not comprise an intact thyA gene and cannot complement the mutation, the transformed strain will become suicidal in a thymidine/thymine-poor environment. Such a strain can be used in a fermentor as an additional protection for the physical containment. Moreover, the present invention discloses that such a strain is especially useful in cases where the strain is used as a delivery vehicle in an animal body. Indeed, when such a transformed strain is given, for example, orally to an animal--including humans--it survives in the gut, provided that a sufficiently high concentration of thymidine/thymine is present, and produces homologous and/or heterologous proteins, such as human Interleukin-10, that may be beneficial for the animal.

[0030]The invention further demonstrates that the transformed strains surprisingly pass the gut at the same speed as the control strains, showing that their loss of viability indeed is not different from that of the control strains. However, once the strain is secreted in the environment, for example, in the feces, it is not able to survive any longer.

[0031]The transforming plasmid can be any plasmid, as long as it does not complement the thyA mutation. It may be a self-replicating plasmid that preferably carries one or more genes of interest and one or more resistance markers, or it may be an integrative plasmid. In the latter case, the integrative plasmid itself may be used to create the mutation by causing integration at the thyA site, whereby the thyA gene is inactivated. Preferably, the active thyA gene is replaced by double homologous recombination by a cassette comprising the gene or genes of interest, flanked by targeting sequences that target the insertion to the thyA target site. It is of extreme importance that these sequences are sufficiently long and sufficiently homologous to integrate the sequence into the target site. Preferably, the targeting sequences include at least 100 contiguous nucleotides of SEQ ID NO:1 at one side of the gene of interest and at least 100 contiguous nucleotides of SEQ ID NO:2 at the other side. More preferably, the targeting sequences consist of at least 500 contiguous nucleotides of SEQ ID NO:1 at one side of the gene of interest and at least 500 contiguous nucleotides of SEQ ID NO:2 at the other side. Most preferably, the targeting sequences consist of SEQ ID NO:1 at one side of the gene of interest and SEQ ID NO:2 at the other side, or the targeting sequences consist of at least 100 nucleotides that are at least 80% identical, preferably 90% identical to a region of SEQ ID NO:1 at one side of the gene of interest and at least 100 nucleotides that are at least 80% identical, preferably 90% identical to a region of SEQ ID NO:2 at the other side of the gene of interest. Preferably, the targeting sequences consist of at least 500 nucleotides that are at least 80% identical, preferably 90% identical to a region of SEQ ID NO:1 at one side of the gene of interest and at least 500 nucleotides that are at least 80% identical, preferably 90% identical to a region of SEQ ID NO:2 at the other side of the gene of interest. Most preferably, the targeting sequences consist of at least 1000 nucleotides that are at least 80% identical, preferably 90% identical to a region of SEQ ID NO:1 at one side of the gene of interest and at least 1000 nucleotides that are at least 80% identical, preferably 90% identical to a region of SEQ ID NO:2 at the other side of the gene of interest. The percentage identity is measured with BLAST, according to Altschul et al. (1997). A preferred example of a sequence homologous to SEQ ID NO:1 is given in SEQ ID NO:7. For the purpose of the invention, SEQ ID NO:1 and SEQ ID NO:7 are interchangeable.

[0032]Transformation methods of Lactococcus are known to the person skilled in the art and include, but are not limited to, protoplast transformation and electroporation.

[0033]A transformed Lactococcus sp. strain according to the invention is useful for the delivery of prophylactic and/or therapeutic molecules and can be used in a pharmaceutical composition. The delivery of such molecules has been disclosed, as a nonlimiting example, in PCT International Publication Numbers WO 97/14806 and WO 98/31786. Prophylactic and/or therapeutic molecules include, but are not limited to, polypeptides such as insulin, growth hormone, prolactin, calcitonin, group 1 cytokines, group 2 cytokines and group 3 cytokines and polysaccharides such as polysaccharide antigens from pathogenic bacteria. A preferred embodiment is the use of a Lactococcus sp. strain according to the invention to deliver human Interleukin-10. This strain can be used in the manufacture of a medicament to treat Crohn's disease as indicated herein.

[0034]The invention is further explained with the use of the following illustrative examples.

EXAMPLES

[0035]From L. lactis MG1363 (Gasson, 1983) regions flanking the sequence according to Ross et al. (1990a) have been cloned.

[0036]The knowledge of these sequences is of critical importance for the genetic engineering of any Lactococcus strain in a way as described below, as the strategy will employ double homologous recombination in the areas of 1000 by at the 5' end (SEQ ID NO:1) and 1000 by at the 3' end (SEQ ID NO:2) of thyA, the "thyA target." These sequences are not available from any public source to date. These flanking DNA fragments have been cloned and their sequence has been identified. The sequence of the whole locus is shown in SEQ ID NO:3; a mutant version of this sequence is shown in SEQ ID NO:5. Both the 5' and 3' sequences are different from the sequence at GenBank AE006385 describing the L. lactis IL1403 sequence (Bolotin, in press) or at AF336368 describing the L. lactis subsp. lactis CHCC373 sequence. From the literature, it is apparent that homologous recombination by use of the published sequences adjacent to thyA (Ross et al., 1990a) (86 by at the 5' end and 31 by at the 3' end) is virtually impossible due to the shortness of the sequences. Indeed, Biswas et al. (1993) describe a logarithmically decreasing correlation between the length of the homologous sequences and the frequency of integration. The sequences of L. lactis Thy 11, Thy 12, Thy 15 and Thy 16 at the thyA locus as determined in the present invention are given by SEQ ID NOS:19, 20, 21, 22 respectively.

[0037]The thyA replacement is performed by making suitable replacements in a plasmid-borne version of the thyA target, as described below. The carrier plasmid is a derivative of pORI19 (Law et al., 1995), a replication-defective plasmid, which only transfers the erythromycin resistance to a given strain when a first homologous recombination, at either the 5' 1000 by or at the 3' 1000 by of the thyA target. A second homologous recombination at the 3' 1000 by or at the 5' 1000 by of the thyA target yields the desired strain.

[0038]The thyA gene is replaced by a synthetic gene encoding a protein which has the L. lactis Usp45 secretion leader (van Asseldonk et al., 1990) fused to a protein of an identical amino acid sequence when: (a) the mature part of human-Interleukin 10 (hIL-10) or (b) the mature part of hIL-10 in which proline at position 2 has been replaced with alanine or (c) the mature part of hIL-10 in which the first two amino acids have been deleted; (a), (b) and (c) are called hIL-10 analogs, the fusion products are called Usp45-hIL-10.

[0039]The thyA gene is replaced by an expression unit comprising the lactococcal P1 promoter (Waterfield et al., 1995), the E. coli bacteriophage T7 expression signals, putative RNA stabilizing sequence and modified gene10 ribosomal binding site (Wells and Schofield, 1996).

[0040]At the 5' end, the insertion is performed in such way that the ATG of thyA is fused to the P1-T7Usp45-hIL-10 expression unit.

TABLE-US-00003 (SEQ ID NO: 27) 5' agataggaaaatttcatgacttacgcagatcaagttttt . . . thyA wild-type (SEQ ID NO: 14) gattaagtcatcttacctctt . . . P1-T7-usp45-hIL10 (SEQ ID NO: 15) 5' agataggaaaatttcatggattaagtcatcttacctctt . . . thyA, P1-T7-usp45-hIL10

[0041]Alternatively, at the 5' end, the insertion is performed in such a way that the thyA ATG is not included:

TABLE-US-00004 (SEQ ID NO: 28) 5' agataggaaaatttcacttacgcagatcaagttttt . . . thyA wild-type (SEQ ID NO: 14) gattaagtcatcttacctctt . . . P1-T7-usp45-hIL10 (SEQ ID NO: 16) 5' agataggaaaatttcgattaagtcatcttacctctt . . . thyA, P1-T7-usp-hIL10

[0042]Alternatively, at the 5' end, the insertion is performed in such a way that the thyA promoter (Ross, 1990 a) is not included:

TABLE-US-00005 (SEQ ID NO: 29) 5' tctgagaggttattttgggaaatactattgaaccatatcgaggtg tgtggtataatgaagggaattaaaaaagataggaaaatttcatg . . . thyA wild-type (SEQ ID NO: 29) gattaagtcatcttacctctt . . . P1-T7-usp45-hIL10 (SEQ ID NO: 14) 5' tctgagaggttattttgggaaatactagattaagtcatcttacct ctt . . . thyA, P1-T7-usp45-hIL10

[0043]At the 3' end, an ACTAGT SpeI restriction site was engineered immediately adjacent to the TAA stop codon of the usp45-hIL-10 sequence. This was ligated in a TCTAGA XbaI restriction site, which was engineered immediately following the thyA stop codon

TABLE-US-00006 (SEQ ID NO: 30) aaaatccgtaactaactagt 3' . . . usp45-hIL10 (SEQ ID NO: 31) gatttagcaatttaaattaaattaatctataagtt 3' . . . thyA-wild-type (SEQ ID NO: 32) tctagaattaatctataagttactga 3' . . . engineered thyA target (SEQ ID NO: 18) aaaatccgtaactaactagaattaatctataagttactga 3' . . . thyA, usp45-hIL10

[0044]These constructs are depicted in FIG. 2. The sequences of pOThy11, pOThy12 pOThy15 and pOThy16 are given by SEQ ID NOs: 23, 24, 25, and 26 respectively. The resulting strains are thyA deficient, a mutant not yet described for L. lactis. It is strictly dependent upon the addition of thymine or thymidine for growth.

[0045]The map of the deletion, as well as the PCR analysis of all the isolates/mutants of the present invention, is shown in FIGS. 3A-4B. The presence of the thymidylate synthase and the Interleukin 10 (IL-10) gene in the wild-type strain and in the independent isolates/mutant was analyzed by Southern analysis as shown in FIGS. 5A-6B. The region around the inserted hIL-10 gene was isolated by PCR and the DNA sequence was verified. The structure is identical to the predicted sequence.

[0046]Human Interleukin-10 (hIL-10) production in the mutants was checked by western blot analysis and compared with the parental strain, transformed with pTREX1 as negative control, and the parental strain, transformed with the IL10-producing plasmid pT1HIL10apxa as a positive control (FIG. 7A). The concentration in the culture supernatant was quantified using ELISA. As shown in FIG. 7B, both isolates of the mutant produce a comparable, significant amount of hIL-10, be it far less than the strain, transformed with the nonintegrative plasmid pT1HIL10apxa. FIGS. 8A and 8B further demonstrate that all mutants produce a significant amount of hIL-10.

[0047]FIG. 9 shows the production of hIL-10 by the L. lactis strains LL108 carrying pOThy11, pOThy12, or pOThy16. Quantification (by ELISA) of hIL-10 present in the culture supernatant of the indicated strains is shown. The N-terminal protein sequence of the recombinant hIL-10 was determined by Edman degradation and was shown to be identical to the structure as predicted for the mature, recombinant hIL-10. The protein showed full biological activity. LL108 is an L. lactis strain carrying a genomic integration of the repA gene, required for replication of pORI19 derived plasmids such as pOThy 11, pOThy12, pOThy15 or pOThy16. This strain was kindly donated by Dr. Jan Kok, University of Groningen, The Netherlands. The plasmids pOThy11, pOThy12, pOThy15 and pOThy16 carry the synthetic human IL-10 gene in different promoter configurations (see FIG. 2), flanked by approximately 1 kB of genomic DNA derived from the thyA locus, upstream and downstream from thyA. These plasmids were used for the construction of the genomic integration as described.

[0048]The effect of the thymidylate synthase deletion on the growth in thymidine less and thymidine-supplemented media was tested; the results are summarized in FIGS. 10 and 11. An absence of thymidine in the medium strongly limits the growth of the mutant and even results in a decrease of colony-forming units after four hours of cultivation. The addition of thymidine to the medium results in an identical growth curve and amount of colony-forming units, compared to the wild-type strain, indicating that the mutant does not affect the growth or viability in thymidine-supplemented medium. FIG. 11 clearly demonstrates that Thy12 viability is severely impaired in the absence of thymidine.

[0049]FIG. 12 finally shows that L. lactis Thy12 passes through the intestine of the mice at the same speed as MG1363. Loss of viability does not appear to differ between Thy12 and MG1363. Thy12 appears fully dependent on thymidine for growth, indicating that no Thy12 bacteria had taken up a foreign thyA gene.

REFERENCES

[0050]Altschul S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389-3402. [0051]Biswas I., A. Gruss, S. D. Ehrlich, et al. (1993). High-efficiency gene inactivation and replacement system for gram-positive bacteria. J. Bacteriol. 175, 3628-3635. [0052]Gasson M. J. (1983). Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154, 1-9. [0053]Kaplan D. L., C. Mello, T. Sano, C. Cantor, and C. Smith (1999). Streptavidin-based containment system for genetically engineered microorganisms. Biomol. Eng. 31, 135-140. [0054]Knudsen S., P. Saadbye, L. H. Hansen, A. Collier, B. L. Jacobsen, J. Schlundt, and O. H. Karlstrom (1995). Development and testing of improved suicide functions for biological containment of bacteria. Appl. Environ. Microbiol. 61, 985-991. [0055]Law J., G. Buist, A. Haandrikman, et al. (1995). A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J. Bacteriol. 177, 7011-7018. [0056]Molina L., C. Ramos, M. C. Ronchel, S. Molin, and J. L. Ramos (1998). Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays. Appl. Environ. Microbiol. 64, 2072-2078. [0057]Ross P., F. O'Gara, and S. Condon (1990a). Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. Lactis. Appl. Environ. Microbiol. 56, 2156-2163. [0058]Ross P., F. O'Gara, and S. Condon (1990b). Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance. Appl. Environ. Microbiol. 56, 2164-2169. [0059]Schweder T., K. Hofmann, and M. Hecker (1995). Escherichia coli K₁₂ relA strains as safe hosts for expression of recombinant DNA. Appl. Environ. Microbiol. 42, 718-723. [0060]Steidler L., W. Hans, L. Schotte, S, Neirynck, F. Obermeier, W. Falk, W. Fiers, and E. Remaut (2000). Treatment of murine colitis by Lactococcus lactis secreting Interleukin-10. Science 289, 1352-1355. [0061]Steidler L., J. M. Wells, A. Raeymaekers, J. Vandekerckhove, W. Fiers, and E. Remaut (1995). Secretion of biologically active murine Interleukin-2 by Lactococcus lactis subsp. Lactis. Appl. Environ. Microbiol. 61, 1627-1629. [0062]Tedin K., A. Witte, G. Reisinger, W. Lubitz, and U. Basi (1995). Evaluation of the E. coli ribosomal rrnB P1 promoter and phage derived lysis genes for the use in biological containment system: a concept study. J. Biotechnol. 39, 137-148. [0063]van Asseldonk M., G. Rutten, M. Oteman, et al. (1990). Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95, 155-160. [0064]Waterfield N. R., R. W. Le Page, P. W. Wilson, et al. (1995). The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis. Gene 165, 9-15. [0065]Wells J. M. and K. M. Schofield (1996). Cloning and expression vectors for Lactococci. Nato ASI series H 98, 37-62.

Sequence CWU 1

3211000DNALactococcus lactis 1tatatacaat tgagcaaaag aaatttagtt attaaattac cagctggagt tcctccaatg 60gttgtagatt cactaagtcc agcaattatt tcaatggtga ttttctgttt gatgttcggg 120attcgtgtgg gattctctta tacgccattc catgatattt tcaatttctc aacacaacta 180attcaagcac cgttgactgg tgctgtggca aatccatggg ttcttatggg catctttacc 240tttggtaatt tcttatggtt ctttggtatc caccctaatt taattggggg aattttaaat 300ccattgttat taacaatgtc atatgctaat attgatgcct atgctgccgg aaaacctgta 360ccatacttac aaatgatgat tgtgtttgct gtgggtgcga acgcatgggg cggaagtgga 420aatacttatg ggttagttat ttcaatgttt acggcaaaat ctgaacgcta taaacaatta 480ttaaaattag gtgcaattcc tagtattttc aatatcagtg aaccattact ttttggtctt 540ccaatgatgt taaatcctct tttctttatt cctttggttt tccaaccagc aattttagga 600actgtagcat tgggcttggc aaagatatta tatattacaa atctgaatcc aatgacggca 660cttcttcctt ggacgacacc agcacctgtg agaatggcca tttcaggtgg acttccattt 720ttgattattt ttgcaatctg tttagtcttg aatgttctta tttactaccc attctttaag 780gtggcgtata ataaagcttt agaagaagaa aaagcagctg ttgaattaga gggttcagaa 840actgcctgat ggatattttt tataaatctg gtttgaacaa attatattga catctctttt 900tctatcctga taattctgag aggttatttt gggaaatact attgaaccat atcgaggtgt 960gtggtataat gaagggaatt aaaaaagata ggaaaatttc 100021000DNALactococcus lactis 2taaattaatc tataagttac tgacaaaact gtcagtaact ttttttgtgg gaaaaatgta 60tttttatgac cgtaaagaat ctgtcagtag aagtctgaaa ttcgtttaaa aatcgactag 120aataggcttt aacgacaaga tgttttaaag agtacgctct aaatgtattt ttgtattttt 180gtttgattac gaagtttaaa tttaattgac aaatgtttta aaatgagtat aataggactt 240gtaaccgatt ttatttttat aaaggagaaa gaaagatgaa caaactttta cttggaacag 300cctttatagg ggctagctta ctgattggtg ggggtgctca tgcagatcaa atgtttatcg 360tttgtataat cataatactg gtgagcactc tatacaacta gtgggacacc aaaagaatgc 420taatgtaagt gcgggttgga cttatgaagg tgtcggttgg atcgcaccaa caacaagttc 480aagcccagtt taccgtgtgt acaatccaaa tgcattatta cacaaaaagc aagtatgaag 540cccaaagttt agtaaataag ggttggaaat gggataataa cggaaaggcg gtcttctatt 600ctggaggttc tcaagccgta tatgtcgctt ataatcccaa tgcacaatct ggcgctcaca 660attacacgga aagtagcttt gagcaaaata gcttattgaa tactggttgg aaatatgggg 720cagtagcttg gtacgggatt ggagtaaaaa acgaaatgtt aaacattgct caaattgtta 780gtggtaattt ttctagtatt gttggaactt ggaaagatac ttctggaaat atgcttgaaa 840ttaatgcaat gggaaatctt actttaatat ggaaaggggc aaagaatcaa acctttgaac 900ttggcgcagg tcaacaattt aatggaactg cagatattgc cttaaaaaat ggagagattt 960cccctggtag tccacttaac atttttgttg taccaacaga 100037157DNALactococcus lactisCDS(4473)..(5312) 3gnagnggttt tcccagtccg acgttgtaaa acgacggcca gtgaattcat taacagcctt 60ttgagcagct agctcattat tttgaaataa atcataaatt tctttcccac tatctgattt 120atgattgcta gcatatttgt tgtataatcg aacgagtcca ttttgaacag atccatatag 180attgagtgaa ctataaaata catctatatc atagttgagt ttgttcacaa tcatgagacc 240aaattctcca gcatttcgtg tagaaccacg ataaagctgt ttatttagca aaatggcacc 300tccgacacct gtacctaaag tcatgcaaat aaaattttgg ctttcttgtc cattccctag 360ccaaagttca gctagacctg cacaattggc atcattttca acataaaccg gaagatttaa 420atgtttttgt agttctgtcc ccaatggata gccataaaga tcagttagag ctcctgccag 480taataatgtt ccctttttgt cagaagttcc gggaacactt acaccaattg cagatactga 540atgatgagct tttaactgat gaatatttgt gagcaagcta tccataattt tttctttttt 600taatggggtt ggaacttgta aatgttgtat gatcgttcca tcactagtta caagaccaaa 660ttttataaat gtaccaccga tatcaattcc tattgaataa tgcatctttt attacctctt 720tctctaattt gttttagtat agcaaaatca aaaaattaat tatggtatgc attatagata 780tgttgtataa ttttcacaaa aacggagaaa actatgaaaa caatagaaca gctcatgata 840gattcagcag atttaatgtc agattttatt caattgacaa tttttatatt ccgcaaggag 900gattttcaac ttttttatag gagtgatgaa gaagagcaag ctttttcaag gtaatgactc 960caacttattg atagtgtttt atgttcagat aatgcccgat gactttgtca tgcagctcca 1020ccgattttga gaacgacagc gacttccgtc ccagccgtgc caggtgctgc ctcagattca 1080ggttatgccg ctcaattcgc tgcgtatatc gcttgctgat tacgtgcagc tttcccttca 1140ggcgggattc atacagcggc cagccatccg tcatccatat caccacgtca aagggtgaca 1200gcaggctcat aagacgcccc agcgtcgcca tagtgcgttc accgaatacg tgcgcaacaa 1260ccgtcttccg gagactgtca tacgcgtaaa acagccagcg ctggcgcgat ttagccccga 1320catagcccca ctgttcgtcc atttccgcgc agacgatgac gtcactgccc ggctgtatgc 1380gcgaggttac cgactgcggc ctgagttttt taagtgacgt aaaatcgtgt tgaggccaac 1440gcccataatg cgggctgttg cccggcatcc aacgccattc atggccatat caatgatttt 1500ctggtgcgta ccgggttgag aagcggtgta agtgaactgc agttgccatg ttttacggca 1560gtgagagcag agatagcgct gatgtccggc ggtgcttttg ccgttacgca ccaccccgtc 1620agtagctgaa caggagggac agctgataga aacagaagcc actggagcac ctcaaaaaca 1680ccatcataca ctaaatcagt aagttggcag catcaccctt tttcaaaaga aatcatcgct 1740catttatctc agttgccctt gaaggaagag gtgaatttat tttatatgcc taagataaaa 1800ggatatatta cttatttttc tgtatttggt aaagaggagt atcttctact tatttttaaa 1860ggacaagaaa aacttgcaaa taatcctttc cccgttgaag taaaacaatt attaaaaagt 1920ggtattttac tctatcaaat gatttttcaa gaaaaattag attatgaaga attatttgag 1980aaaaatcagc atattatttc tccattgctt gctgctaaac caattgaatg gaatgattcc 2040aatacgtgag gaaagtaaat tcccataaaa catatctttt tgaaaaatat ttgggggaat 2100gtgttattcg tggagatgtt gcagagttaa aaaaagcttt ttcaaattat atgaataaag 2160gaactgctgg aaaattatct aataattcaa tgcgacataa gaaaaacatt ttgatttcag 2220tcatcactat gactactcgt tcggctatac agggaggatt acctgaagaa gaagcttttt 2280tgatgagtga tttatatatt caagagcttg aagaattaac ggaattagaa gaaattagaa 2340cgcttgccta taatgtgatg atcgattttg cagataaagt gaaacagcat cgatattgtc 2400aggtttctta taaaatatta tcttgtcaaa agtatattgt taatcattta tacgaaaaac 2460taagtgtgag tgaaattgca gaagagctac acatgaatat ttcttattta tcttcacaat 2520tcaaaaaaga gacagggcaa acaattacaa actttattca ggagaagcga atagaagaag 2580ctagagaatt aatccttttc tcagactatc ctttttcaag aatttatacc ttgttggttt 2640tactgccaaa gtcattttat aaaaatattt aaaaaatata ctggaataac tcccaaaaag 2700tttcaagatc agtatattta tcatgcctct acatcaatat atgattgaaa ttaaaaaaag 2760acctagaatt tcaaaattga taaaatacat acctaaaata ttaattctgt actattacgg 2820gtggagtatc tactgtataa tgagggtata aattatggaa gaagggagta aaactaaatt 2880tattgatggt tttacgaatt aattaggata ttttttttaa aaaccaaaga aaacgcttac 2940aaacgttaaa ggagtgaatc taaagatgga caaatttgaa aaatggctaa ataagacctt 3000gatgccactt gcctcaaaaa tgaataaaaa tcatttcatt tcggcattaa gtgaagcatt 3060tatgagatgt atgcccttaa cattagggat tgcattattg acaattatag gatactttcc 3120agttcctgcc tgggtagatt tcttaaactc tattggactg gctcagcatt tttcagcagt 3180tattggtgca gttaccagtg cgctagcaat ttatgtaact tataattttg cttattctta 3240tgtaaatcgt catgaatata atggccatac ggccggttta ttatcaatcg caagtttgtt 3300aatgctaatg ccacaaatta ttactgtccc tgtagtaaaa aacattccaa ccgaatttcc 3360gaaatccgcg gtagttgaca gtgtgtcaaa tgttgaagca tttcaaacgg tatacacggg 3420tagcacagga ttaattgtag caatcataat tggttttatt gtttcattag tctatataca 3480attgagcaaa agaaatttag ttattaaatt accagctgga gttcctccaa tggttgtaga 3540ttcactaagt ccagcaatta tttcaatggt gattttctgt ttgatgttcg ggattcgtgt 3600gggattctct tatacgccat tccatgatat tttcaatttc tcaacacaac taattcaagc 3660accgttgact ggtgctgtgg caaatccatg ggttcttatg ggcatcttta cctttggtaa 3720tttcttatgg ttctttggta tccaccctaa tttaattggg ggaattttaa atccattgtt 3780attaacaatg tcatatgcta atattgatgc ctatgctgcc ggaaaacctg taccatactt 3840acaaatgatg attgtgtttg ctgtgggtgc gaacgcatgg ggcggaagtg gaaatactta 3900tgggttagtt atttcaatgt ttacggcaaa atctgaacgc tataaacaat tattaaaatt 3960aggtgcaatt cctagtattt tcaatatcag tgaaccatta ctttttggtc ttccaatgat 4020gttaaatcct cttttcttta ttcctttggt tttccaacca gcaattttag gaactgtagc 4080attgggcttg gcaaagatat tatatattac aaatctgaat ccaatgacgg cacttcttcc 4140ttggacgaca ccagcacctg tgagaatggc catttcaggt ggacttccat ttttgattat 4200ttttgcaatc tgtttagtct tgaatgttct tatttactac ccattcttta aggtggcgta 4260taataaagct ttagaagaag aaaaagcagc tgttgaatta gagggttcag aaactgcctg 4320atggatattt tttataaatc tggtttgaac aaattatatt gacatctctt tttctatcct 4380gataattctg agaggttatt ttgggaaata ctattgaacc atatcgaggt gtgtggtata 4440atgaagggaa ttaaaaaaga taggaaaatt tc atg act tac gca gat caa gtt 4493 Met Thr Tyr Ala Asp Gln Val 1 5ttt aaa caa aat atc caa aat atc cta gat aat ggt gtt ttt tca gaa 4541Phe Lys Gln Asn Ile Gln Asn Ile Leu Asp Asn Gly Val Phe Ser Glu 10 15 20aat gca aga cca aag tat aag gat ggt caa atg gcg aat agc aaa tat 4589Asn Ala Arg Pro Lys Tyr Lys Asp Gly Gln Met Ala Asn Ser Lys Tyr 25 30 35gtc act ggt tca ttc gtt act tat gat ttg caa aag ggg gag ttt cca 4637Val Thr Gly Ser Phe Val Thr Tyr Asp Leu Gln Lys Gly Glu Phe Pro40 45 50 55att acc act ttg cgt cca att cca atc aaa tct gct att aaa gaa ttg 4685Ile Thr Thr Leu Arg Pro Ile Pro Ile Lys Ser Ala Ile Lys Glu Leu 60 65 70atg tgg ata tac caa gac caa aca agt gaa ctt tct gtt ctc gaa gag 4733Met Trp Ile Tyr Gln Asp Gln Thr Ser Glu Leu Ser Val Leu Glu Glu 75 80 85aag tat gga gtc aaa tac tgg gga gaa tgg gga att ggt gat ggt acg 4781Lys Tyr Gly Val Lys Tyr Trp Gly Glu Trp Gly Ile Gly Asp Gly Thr 90 95 100att ggg caa cgt tat ggt gca aca gtc aaa aaa tat aat atc att ggt 4829Ile Gly Gln Arg Tyr Gly Ala Thr Val Lys Lys Tyr Asn Ile Ile Gly 105 110 115aaa tta tta gaa ggc ttg gcc aaa aat cca tgg aat cgt cgt aat atc 4877Lys Leu Leu Glu Gly Leu Ala Lys Asn Pro Trp Asn Arg Arg Asn Ile120 125 130 135atc aac ctt tgg cag tat gaa gat ttt gag gaa aca gaa ggt ctt tta 4925Ile Asn Leu Trp Gln Tyr Glu Asp Phe Glu Glu Thr Glu Gly Leu Leu 140 145 150cca tgt gct ttc caa acg atg ttt gat gtc cgt cga gaa aaa gat ggt 4973Pro Cys Ala Phe Gln Thr Met Phe Asp Val Arg Arg Glu Lys Asp Gly 155 160 165cag att tat ttg gat gcc aca ctg att caa cgt tca aac gat atg ctt 5021Gln Ile Tyr Leu Asp Ala Thr Leu Ile Gln Arg Ser Asn Asp Met Leu 170 175 180gta gcc cac cat atc aat gcg atg caa tat gtt gct ttg caa atg atg 5069Val Ala His His Ile Asn Ala Met Gln Tyr Val Ala Leu Gln Met Met 185 190 195att gca aaa cat ttt tct tgg aaa gtt ggg aaa ttc ttt tat ttt gta 5117Ile Ala Lys His Phe Ser Trp Lys Val Gly Lys Phe Phe Tyr Phe Val200 205 210 215aat aat tta cat att tat gat aat cag ttt gag cag gca aat gaa tta 5165Asn Asn Leu His Ile Tyr Asp Asn Gln Phe Glu Gln Ala Asn Glu Leu 220 225 230atg aag cga aca gct tct gaa aaa gaa cct cgt ttg gtc ctt aat gtt 5213Met Lys Arg Thr Ala Ser Glu Lys Glu Pro Arg Leu Val Leu Asn Val 235 240 245cct gat ggt aca aac ttt ttc gat att aaa cct gaa gat ttt gaa ctt 5261Pro Asp Gly Thr Asn Phe Phe Asp Ile Lys Pro Glu Asp Phe Glu Leu 250 255 260gtg gac tat gag cca gta aaa cct caa ttg aaa ttt gat tta gca att 5309Val Asp Tyr Glu Pro Val Lys Pro Gln Leu Lys Phe Asp Leu Ala Ile 265 270 275taa attaatctat aagttactga caaaactgtc agtaactttt tttgtgggaa 5362aaatgtattt ttatgaccgt aaagaatctg tcagtagaag tctgaaattc gtttaaaaat 5422cgactagaat aggctttaac gacaagatgt tttaaagagt acgctctaaa tgtatttttg 5482tatttttgtt tgattacgaa gtttaaattt aattgacaaa tgttttaaaa tgagtataat 5542aggacttgta accgatttta tttttataaa ggagaaagaa agatgaacaa acttttactt 5602ggaacagcct ttataggggc tagcttactg attggtgggg gtgctcatgc agatcaaatg 5662tttatcgttt gtataatcat aatactggtg agcactctat acaactagtg ggacaccaaa 5722agaatgctaa tgtaagtgcg ggttggactt atgaaggtgt cggttggatc gcaccaacaa 5782caagttcaag cccagtttac cgtgtgtaca atccaaatgc attattacac aaaaagcaag 5842tatgaagccc aaagtttagt aaataagggt tggaaatggg ataataacgg aaaggcggtc 5902ttctattctg gaggttctca agccgtatat gtcgcttata atcccaatgc acaatctggc 5962gctcacaatt acacggaaag tagctttgag caaaatagct tattgaatac tggttggaaa 6022tatggggcag tagcttggta cgggattgga gtaaaaaacg aaatgttaaa cattgctcaa 6082attgttagtg gtaatttttc tagtattgtt ggaacttgga aagatacttc tggaaatatg 6142cttgaaatta atgcaatggg aaatcttact ttaatatgga aaggggcaaa gaatcaaacc 6202tttgaacttg gcgcaggtca acaatttaat ggaactgcag atattgcctt aaaaaatgga 6262gagatttccc ctggtagtcc acttaacatt tttgttgtac caacagaagt tgctttccct 6322aataataaaa aagtagacga ttcaactggg caacaacgaa tttttgtgaa ttattctggt 6382acaagccctc aaatggcgaa tagtatggca gcggtggctt tttttagagt tattccatga 6442ttatattaaa gttagaattg aataaaatgt attattaaaa agataatatt atatcacgac 6502aaggcgacat ctatcaactt taccactggt atggaagtga ccattattac atcaggaaac 6562gctaaaacgg ttgtttttac acccgtaaaa taaataataa aataatgtgn aattactgac 6622agcattttgt cagtaatttt ttttatcaaa atcacacaaa aatgttcgtt gacgaacaaa 6682aaaaactatg ttataataat tcgtatgcga actaaaaaag aagcgattgg ccgactttta 6742aaagtagcca gcaaccaaat gtctcgagaa tttgataatt ttgcagctca acttgatttg 6802acaggtcagc aaatgtcaat tttagatttt cttggaaatc aaagcgaaga aggttcagga 6862aaagaaatta gtcagacgat gattgaatta gaatttaata tccgacgttc aacaacgacg 6922gaaattttac agcgcatgga aaagcggctt ttaattaatc gaagaacaag cctgaccgat 6982gcccgccaaa aatcagttga attaactgaa gaagggaaaa gatatttacc tgaaatcagg 7042gcttatatcc aagcacataa taaaaaagct tggcgtaatc atggtcatag ctgtttncct 7102ggttaggngg gccannnnnn nnnnnnnnnn nnncnnnnnc nnnnncnnnn cnnnc 71574279PRTLactococcus lactismisc_feature(2)..(2)'n' may be any base 4Met Thr Tyr Ala Asp Gln Val Phe Lys Gln Asn Ile Gln Asn Ile Leu1 5 10 15Asp Asn Gly Val Phe Ser Glu Asn Ala Arg Pro Lys Tyr Lys Asp Gly 20 25 30Gln Met Ala Asn Ser Lys Tyr Val Thr Gly Ser Phe Val Thr Tyr Asp 35 40 45Leu Gln Lys Gly Glu Phe Pro Ile Thr Thr Leu Arg Pro Ile Pro Ile 50 55 60Lys Ser Ala Ile Lys Glu Leu Met Trp Ile Tyr Gln Asp Gln Thr Ser65 70 75 80Glu Leu Ser Val Leu Glu Glu Lys Tyr Gly Val Lys Tyr Trp Gly Glu 85 90 95Trp Gly Ile Gly Asp Gly Thr Ile Gly Gln Arg Tyr Gly Ala Thr Val 100 105 110Lys Lys Tyr Asn Ile Ile Gly Lys Leu Leu Glu Gly Leu Ala Lys Asn 115 120 125Pro Trp Asn Arg Arg Asn Ile Ile Asn Leu Trp Gln Tyr Glu Asp Phe 130 135 140Glu Glu Thr Glu Gly Leu Leu Pro Cys Ala Phe Gln Thr Met Phe Asp145 150 155 160Val Arg Arg Glu Lys Asp Gly Gln Ile Tyr Leu Asp Ala Thr Leu Ile 165 170 175Gln Arg Ser Asn Asp Met Leu Val Ala His His Ile Asn Ala Met Gln 180 185 190Tyr Val Ala Leu Gln Met Met Ile Ala Lys His Phe Ser Trp Lys Val 195 200 205Gly Lys Phe Phe Tyr Phe Val Asn Asn Leu His Ile Tyr Asp Asn Gln 210 215 220Phe Glu Gln Ala Asn Glu Leu Met Lys Arg Thr Ala Ser Glu Lys Glu225 230 235 240Pro Arg Leu Val Leu Asn Val Pro Asp Gly Thr Asn Phe Phe Asp Ile 245 250 255Lys Pro Glu Asp Phe Glu Leu Val Asp Tyr Glu Pro Val Lys Pro Gln 260 265 270Leu Lys Phe Asp Leu Ala Ile 27557094DNALactococcus lactisCDS(4469)..(5305) 5ggttttccca gtccgacgtt gtaaaacgac ggccagtgaa ttcattaaca gccttttgag 60cagctagctc attattttga aataaatcat aaatttcttt cccactatct gatttatgat 120tgctagcata tttgttgtat aatcgaacga gtccattttg aacagatcca tatagattga 180gtgaactata aaatacatct atatcatagt tgagtttgtt cacaatcatg agaccaaatt 240ctccagcatt tcgtgtagaa ccacgataaa gctgtttatt tagcaaaatg gcacctccga 300cacctgtacc taaagtcatg caaataaaat tttggctttc ttgtccattc cctagccaaa 360gttcagctag acctgcacaa ttggcatcat tttcaacata aaccggaaga tttaaatgtt 420tttgtagttc tgtccccaat ggatagccat aaagatcagt tagagctcct gccagtaata 480atgttccctt tttgtcagaa gttccgggaa cacttacacc aattgcagat actgaatgat 540gagcttttaa ctgatgaata tttgtgagca agctatccat aattttttct ttttttaatg 600gggttggaac ttgtaaatgt tgtatgatcg ttccatcact agttacaaga ccaaatttta 660taaatgtacc accgatatca attcctattg aataatgcat cttttattac ctctttctct 720aatttgtttt agtatagcaa aatcaaaaaa ttaattatgg tatgcattat agatatgttg 780tataattttc acaaaaacgg agaaaactat gaaaacaata gaacagctca tgatagattc 840agcagattta atgtcagatt ttattcaatt gacaattttt atattccgca aggaggattt 900tcaacttttt tataggagtg atgaagaaga gcaagctttt tcaaggtaat gactccaact 960tattgatagt gttttatgtt cagataatgc ccgatgactt tgtcatgcag ctccaccgat 1020tttgagaacg acagcgactt ccgtcccagc cgtgccaggt gctgcctcag attcaggtta 1080tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt gcagctttcc cttcaggcgg 1140gattcataca gcggccagcc atccgtcatc catatcacca cgtcaaaggg tgacagcagg 1200ctcataagac gccccagcgt cgccatagtg cgttcaccga atacgtgcgc aacaaccgtc 1260ttccggagac tgtcatacgc gtaaaacagc cagcgctggc gcgatttagc cccgacatag 1320ccccactgtt cgtccatttc cgcgcagacg atgacgtcac tgcccggctg tatgcgcgag 1380gttaccgact gcggcctgag ttttttaagt gacgtaaaat cgtgttgagg ccaacgccca 1440taatgcgggc tgttgcccgg catccaacgc cattcatggc catatcaatg attttctggt 1500gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg ccatgtttta cggcagtgag 1560agcagagata gcgctgatgt ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag 1620ctgaacagga gggacagctg atagaaacag aagccactgg agcacctcaa aaacaccatc 1680atacactaaa tcagtaagtt ggcagcatca ccctttttca aaagaaatca tcgctcattt 1740atctcagttg cccttgaagg aagaggtgaa tttattttat atgcctaaga taaaaggata 1800tattacttat ttttctgtat ttggtaaaga ggagtatctt ctacttattt ttaaaggaca 1860agaaaaactt gcaaataatc ctttccccgt tgaagtaaaa caattattaa aaagtggtat 1920tttactctat caaatgattt ttcaagaaaa attagattat gaagaattat ttgagaaaaa 1980tcagcatatt atttctccat

tgcttgctgc taaaccaatt gaatggaatg attccaatac 2040gtgaggaaag taaattccca taaaacatat ctttttgaaa aatatttggg ggaatgtgtt 2100attcgtggag atgttgcaga gttaaaaaaa gctttttcaa attatatgaa taaaggaact 2160gctggaaaat tatctaataa ttcaatgcga cataagaaaa acattttgat ttcagtcatc 2220actatgacta ctcgttcggc tatacaggga ggattacctg aagaagaagc ttttttgatg 2280agtgatttat atattcaaga gcttgaagaa ttaacggaat tagaagaaat tagaacgctt 2340gcctataatg tgatgatcga ttttgcagat aaagtgaaac agcatcgata ttgtcaggtt 2400tcttataaaa tattatcttg tcaaaagtat attgttaatc atttatacga aaaactaagt 2460gtgagtgaaa ttgcagaaga gctacacatg aatatttctt atttatcttc acaattcaaa 2520aaagagacag ggcaaacaat tacaaacttt attcaggaga agcgaataga agaagctaga 2580gaattaatcc ttttctcaga ctatcctttt tcaagaattt ataccttgtt ggttttactg 2640ccaaagtcat tttataaaaa tatttaaaaa atatactgga ataactccca aaaagtttca 2700agatcagtat atttatcatg cctctacatc aatatatgat tgaaattaaa aaaagaccta 2760gaatttcaaa attgataaaa tacataccta aaatattaat tctgtactat tacgggtgga 2820gtatctactg tataatgagg gtataaatta tggaagaagg gagtaaaact aaatttattg 2880atggttttac gaattaatta ggatattttt tttaaaaacc aaagaaaacg cttacaaacg 2940ttaaaggagt gaatctaaag atggacaaat ttgaaaaatg gctaaataag accttgatgc 3000cacttgcctc aaaaatgaat aaaaatcatt tcatttcggc attaagtgaa gcatttatga 3060gatgtatgcc cttaacatta gggattgcat tattgacaat tataggatac tttccagttc 3120ctgcctgggt agatttctta aactctattg gactggctca gcatttttca gcagttattg 3180gtgcagttac cagtgcgcta gcaatttatg taacttataa ttttgcttat tcttatgtaa 3240atcgtcatga atataatggc catacggccg gtttattatc aatcgcaagt ttgttaatgc 3300taatgccaca aattattact gtccctgtag taaaaaacat tccaaccgaa tttccgaaat 3360ccgcggtagt tgacagtgtg tcaaatgttg aagcatttca aacggtatac acgggtagca 3420caggattaat tgtagcaatc ataattggtt ttattgtttc attagtctat atacaattga 3480gcaaaagaaa tttagttatt aaattaccag ctggagttcc tccaatggtt gtagattcac 3540taagtccagc aattatttca atggtgattt tctgtttgat gttcgggatt cgtgtgggat 3600tctcttatac gccattccat gatattttca atttctcaac acaactaatt caagcaccgt 3660tgactggtgc tgtggcaaat ccatgggttc ttatgggcat ctttaccttt ggtaatttct 3720tatggttctt tggtatccac cctaatttaa ttgggggaat tttaaatcca ttgttattaa 3780caatgtcata tgctaatatt gatgcctatg ctgccggaaa acctgtacca tacttacaaa 3840tgatgattgt gtttgctgtg ggtgcgaacg catggggcgg aagtggaaat acttatgggt 3900tagttatttc aatgtttacg gcaaaatctg aacgctataa acaattatta aaattaggtg 3960caattcctag tattttcaat atcagtgaac cattactttt tggtcttcca atgatgttaa 4020atcctctttt ctttattcct ttggttttcc aaccagcaat tttaggaact gtagcattgg 4080gcttggcaaa gatattatat attacaaatc tgaatccaat gacggcactt cttccttgga 4140cgacaccagc acctgtgaga atggccattt caggtggact tccatttttg attatttttg 4200caatctgttt agtcttgaat gttcttattt actacccatt ctttaaggtg gcgtataata 4260aagctttaga agaagaaaaa gcagctgttg aattagaggg ttcagaaact gcctgatgga 4320tattttttat aaatctggtt tgaacaaatt atattgacat ctctttttct atcctgataa 4380ttctgagagg ttattttggg aaatactatt gaaccatatc gaggtggtgt ggtataatga 4440agggaattaa aaaagatagg aaaatttc atg act tac gca gat caa gtt ttt 4492 Met Thr Tyr Ala Asp Gln Val Phe 1 5aaa caa aat atc caa aat atc cta gat aat ggt gtt ttt tca gaa aat 4540Lys Gln Asn Ile Gln Asn Ile Leu Asp Asn Gly Val Phe Ser Glu Asn 10 15 20gca aga cca aag tat aag gat ggt caa atg gcg aat agc aaa tat gtc 4588Ala Arg Pro Lys Tyr Lys Asp Gly Gln Met Ala Asn Ser Lys Tyr Val25 30 35 40act ggt tca ttc gtt act tat gat ttg caa aag ggg gag ttt cca att 4636Thr Gly Ser Phe Val Thr Tyr Asp Leu Gln Lys Gly Glu Phe Pro Ile 45 50 55acc act ttg cgt cca att cca atc aaa tct gct att aaa gaa ttg atg 4684Thr Thr Leu Arg Pro Ile Pro Ile Lys Ser Ala Ile Lys Glu Leu Met 60 65 70tgg ata tac caa gac caa aca agt gaa ctt tct gtt ctc gaa gag aag 4732Trp Ile Tyr Gln Asp Gln Thr Ser Glu Leu Ser Val Leu Glu Glu Lys 75 80 85tat gga gtc aaa tac tgg gga gaa tgg gga att ggt gat ggt acg att 4780Tyr Gly Val Lys Tyr Trp Gly Glu Trp Gly Ile Gly Asp Gly Thr Ile 90 95 100ggg caa cgt tat ggt gca aca gtc aaa aaa tat aat atc att ggt aaa 4828Gly Gln Arg Tyr Gly Ala Thr Val Lys Lys Tyr Asn Ile Ile Gly Lys105 110 115 120tta tta gaa ggc ttg gcc aaa aat cca tgg aat cgt cgt aat atc atc 4876Leu Leu Glu Gly Leu Ala Lys Asn Pro Trp Asn Arg Arg Asn Ile Ile 125 130 135aac ctt tgg cag tat gaa gat ttt gag gaa aca gaa ggt ctt tta cca 4924Asn Leu Trp Gln Tyr Glu Asp Phe Glu Glu Thr Glu Gly Leu Leu Pro 140 145 150tgt gct ttc caa acg atg ttt gat gtc cgt cga gaa aaa gat ggt cag 4972Cys Ala Phe Gln Thr Met Phe Asp Val Arg Arg Glu Lys Asp Gly Gln 155 160 165att tat ttg gat gcc aca ctg att caa cgt tca aac gat atg ctt gta 5020Ile Tyr Leu Asp Ala Thr Leu Ile Gln Arg Ser Asn Asp Met Leu Val 170 175 180gcc cac cat atc aat gcg atg caa tat gtt gct ttg caa atg atg att 5068Ala His His Ile Asn Ala Met Gln Tyr Val Ala Leu Gln Met Met Ile185 190 195 200gca aaa cat ttt tct tgg aaa gtt ggg aaa ttc ttt tat ttt gta aat 5116Ala Lys His Phe Ser Trp Lys Val Gly Lys Phe Phe Tyr Phe Val Asn 205 210 215aat tta cat att tat gat aat cag ttt gag cag gca aat gaa tta atg 5164Asn Leu His Ile Tyr Asp Asn Gln Phe Glu Gln Ala Asn Glu Leu Met 220 225 230aag cga aca gct tct gaa aaa gaa cct cgt ttg gtc ctt aat gtt cct 5212Lys Arg Thr Ala Ser Glu Lys Glu Pro Arg Leu Val Leu Asn Val Pro 235 240 245gat ggt aca aac ttt ttc gat att aaa cct gaa gat ttt gaa ctt gtg 5260Asp Gly Thr Asn Phe Phe Asp Ile Lys Pro Glu Asp Phe Glu Leu Val 250 255 260gac tat gag cca gta aaa cct caa ttg aaa ttt gat tta gca att 5305Asp Tyr Glu Pro Val Lys Pro Gln Leu Lys Phe Asp Leu Ala Ile265 270 275taaattaatc tataagttac tgacaaaact gtcagtaact ttttttgtgg gaaaaatgta 5365tttttatgac cgtaaagaat ctgtcagtag aagtctgaaa ttcgtttaaa aatcgactag 5425aataggcttt aacgacaaga tgttttaaag agtacgctct aaatgtattt ttgtattttt 5485gtttgattac gaagtttaaa tttaattgac aaatgtttta aaatgagtat aataggactt 5545gtaaccgatt ttatttttat aaaggagaaa gaaagatgaa caaactttta cttggaacag 5605cctttatagg ggctagctta ctgattggtg ggggtgctca tgcagatcaa atgtttatcg 5665tttgtataat cataatactg gtgagcactc tatacaacta gtgggacacc aaaagaatgc 5725taatgtaagt gcgggttgga cttatgaagg tgtcggttgg atcgcaccaa caacaagttc 5785aagcccagtt taccgtgtgt acaatccaaa tgcattatta cacaaaaagc aagtatgaag 5845cccaaagttt agtaaataag ggttggaaat gggataataa cggaaaggcg gtcttctatt 5905ctggaggttc tcaagccgta tatgtcgctt ataatcccaa tgcacaatct ggcgctcaca 5965attacacgga aagtagcttt gagcaaaata gcttattgaa tactggttgg aaatatgggg 6025cagtagcttg gtacgggatt ggagtaaaaa acgaaatgtt aaacattgct caaattgtta 6085gtggtaattt ttctagtatt gttggaactt ggaaagatac ttctggaaat atgcttgaaa 6145ttaatgcaat gggaaatctt actttaatat ggaaaggggc aaagaatcaa acctttgaac 6205ttggcgcagg tcaacaattt aatggaactg cagatattgc cttaaaaaat ggagagattt 6265cccctggtag tccacttaac atttttgttg taccaacaga agttgctttc cctaataata 6325aaaaagtaga cgattcaact gggcaacaac gaatttttgt gaattattct ggtacaagcc 6385ctcaaatggc gaatagtatg gcagcggtgg ctttttttag agttattcca tgattatatt 6445aaagttagaa ttgaataaaa tgtattatta aaaagataat attatatcac gacaaggcga 6505catctatcaa ctttaccact ggtatggaag tgaccattat tacatcagga aacgctaaaa 6565cggttgtttt tacacccgta aaataaataa taaaataatg tgaaattact gacagcattt 6625tgtcagtaat tttttttatc aaaatcacac aaaaatgttc gttgacgaac aaaaaaaact 6685atgttataat aattcgtatg cgaactaaaa aagaagcgat tggccgactt ttaaaagtag 6745ccagcaacca aatgtctcga gaatttgata attttgcagc tcaacttgat ttgacaggtc 6805agcaaatgtc aattttagat tttcttggaa atcaaagcga agaaggttca ggaaaagaaa 6865ttagtcagac gatgattgaa ttagaattta atatccgacg ttcaacaacg acggaaattt 6925tacagcgcat ggaaaagcgg cttttaatta atcgaagaac aagcctgacc gatgcccgcc 6985aaaaatcagt tgaattaact gaagaaggga aaagatattt acctgaaatc agggcttata 7045tccaagcaca taataaaaaa gcttggcgta atcatggtca tagctgttt 70946279PRTLactococcus lactis 6Met Thr Tyr Ala Asp Gln Val Phe Lys Gln Asn Ile Gln Asn Ile Leu1 5 10 15Asp Asn Gly Val Phe Ser Glu Asn Ala Arg Pro Lys Tyr Lys Asp Gly 20 25 30Gln Met Ala Asn Ser Lys Tyr Val Thr Gly Ser Phe Val Thr Tyr Asp 35 40 45Leu Gln Lys Gly Glu Phe Pro Ile Thr Thr Leu Arg Pro Ile Pro Ile 50 55 60Lys Ser Ala Ile Lys Glu Leu Met Trp Ile Tyr Gln Asp Gln Thr Ser65 70 75 80Glu Leu Ser Val Leu Glu Glu Lys Tyr Gly Val Lys Tyr Trp Gly Glu 85 90 95Trp Gly Ile Gly Asp Gly Thr Ile Gly Gln Arg Tyr Gly Ala Thr Val 100 105 110Lys Lys Tyr Asn Ile Ile Gly Lys Leu Leu Glu Gly Leu Ala Lys Asn 115 120 125Pro Trp Asn Arg Arg Asn Ile Ile Asn Leu Trp Gln Tyr Glu Asp Phe 130 135 140Glu Glu Thr Glu Gly Leu Leu Pro Cys Ala Phe Gln Thr Met Phe Asp145 150 155 160Val Arg Arg Glu Lys Asp Gly Gln Ile Tyr Leu Asp Ala Thr Leu Ile 165 170 175Gln Arg Ser Asn Asp Met Leu Val Ala His His Ile Asn Ala Met Gln 180 185 190Tyr Val Ala Leu Gln Met Met Ile Ala Lys His Phe Ser Trp Lys Val 195 200 205Gly Lys Phe Phe Tyr Phe Val Asn Asn Leu His Ile Tyr Asp Asn Gln 210 215 220Phe Glu Gln Ala Asn Glu Leu Met Lys Arg Thr Ala Ser Glu Lys Glu225 230 235 240Pro Arg Leu Val Leu Asn Val Pro Asp Gly Thr Asn Phe Phe Asp Ile 245 250 255Lys Pro Glu Asp Phe Glu Leu Val Asp Tyr Glu Pro Val Lys Pro Gln 260 265 270Leu Lys Phe Asp Leu Ala Ile 27571000DNALactococcus lactis 7atatacaatt gagcaaaaga aatttagtta ttaaattacc agctggagtt cctccaatgg 60ttgtagattc actaagtcca gcaattattt caatggtgat tttctgtttg atgttcggga 120ttcgtgtggg attctcttat acgccattcc atgatatttt caatttctca acacaactaa 180ttcaagcacc gttgactggt gctgtggcaa atccatgggt tcttatgggc atctttacct 240ttggtaattt cttatggttc tttggtatcc accctaattt aattggggga attttaaatc 300cattgttatt aacaatgtca tatgctaata ttgatgccta tgctgccgga aaacctgtac 360catacttaca aatgatgatt gtgtttgctg tgggtgcgaa cgcatggggc ggaagtggaa 420atacttatgg gttagttatt tcaatgttta cggcaaaatc tgaacgctat aaacaattat 480taaaattagg tgcaattcct agtattttca atatcagtga accattactt tttggtcttc 540caatgatgtt aaatcctctt ttctttattc ctttggtttt ccaaccagca attttaggaa 600ctgtagcatt gggcttggca aagatattat atattacaaa tctgaatcca atgacggcac 660ttcttccttg gacgacacca gcacctgtga gaatggccat ttcaggtgga cttccatttt 720tgattatttt tgcaatctgt ttagtcttga atgttcttat ttactaccca ttctttaagg 780tggcgtataa taaagcttta gaagaagaaa aagcagctgt tgaattagag ggttcagaaa 840ctgcctgatg gatatttttt ataaatctgg tttgaacaaa ttatattgac atctcttttt 900ctatcctgat aattctgaga ggttattttg ggaaatacta ttgaaccata tcgaggtggt 960gtggtataat gaagggaatt aaaaaagata ggaaaatttc 1000824DNAArtificial Sequenceoligonucleotide primer 8atgacttacg cagatcaagt tttt 24927DNAArtificial Sequenceoligonucleotide primer 9ttaaattgct aaatcaaatt tcaattg 271020DNAArtificial Sequenceoligonucleotide primer 10tctgattgag taccttgacc 201122DNAArtificial Sequenceoligonucleotide primer 11gcaatcataa ttggttttat tg 221222DNAArtificial Sequenceoligonucleotide primer 12cttacatgac tatgaaaatc cg 221323DNAArtificial Sequenceoligonucleotide primer 13cttttttatt attagggaaa gca 231421DNAArtificial Sequenceexpression unit comprising the lactococcal P1 promoter, the E.col i bacteriophage T7 expression signals, putative RNA stabilisingsequence and modified gene10 ribosomal binding site 14gattaagtca tcttacctct t 211539DNAArtificial SequencethyA-, P1-T7-usp45-hIL10 15agataggaaa atttcatgga ttaagtcatc ttacctctt 391636DNAArtificial SequenceATG not included, thyA-,P1-T7-usp45-hIL10 16agataggaaa atttcgatta agtcatctta cctctt 361748DNAArtificial SequencethyA promoter not included, theA-, P1-T7-usp45-hIL10 17tctgagaggt tattttggga aatactagat taagtcatct tacctctt 481840DNAArtificial SequencethyA-, usp45-hIL10 18aaaatccgta actaactaga attaatctat aagttactga 40196967DNALactococcus lactismisc_featureThy11 19attaacagcc ttttgagcag ctagctcatt attttgaaat aaatcataaa tttctttccc 60actatctgat ttatgattgc tagcatattt gttgtataat cgaacgagtc cattttgaac 120agatccatat agattgagtg aactataaaa tacatctata tcatagttga gtttgttcac 180aatcatgaga ccaaattctc cagcatttcg tgtagaacca cgataaagct gtttatttag 240caaaatggca cctccgacac ctgtacctaa agtcatgcaa ataaaatttt ggctttcttg 300tccattccct agccaaagtt cagctagacc tgcacaattg gcatcatttt caacataaac 360cggaagattt aaatgttttt gtagttctgt ccccaatgga tagccataaa gatcagttag 420agctcctgcc agtaataatg ttcccttttt gtcagaagtt ccgggaacac ttacaccaat 480tgcagatact gaatgatgag cttttaactg atgaatattt gtgagcaagc tatccataat 540tttttctttt tttaatgggg ttggaacttg taaatgttgt atgatcgttc catcactagt 600tacaagacca aattttataa atgtaccacc gatatcaatt cctattgaat aatgcatctt 660ttattacctc tttctctaat ttgttttagt atagcaaaat caaaaaatta attatggtat 720gcattataga tatgttgtat aattttcaca aaaacggaga aaactatgaa aacaatagaa 780cagctcatga tagattcagc agatttaatg tcagatttta ttcaattgac aatttttata 840ttccgcaagg aggattttca acttttttat aggagtgatg aagaagagca agctttttca 900aggtaatgac tccaacttat tgatagtgtt ttatgttcag ataatgcccg atgactttgt 960catgcagctc caccgatttt gagaacgaca gcgacttccg tcccagccgt gccaggtgct 1020gcctcagatt caggttatgc cgctcaattc gctgcgtata tcgcttgctg attacgtgca 1080gctttccctt caggcgggat tcatacagcg gccagccatc cgtcatccat atcaccacgt 1140caaagggtga cagcaggctc ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata 1200cgtgcgcaac aaccgtcttc cggagactgt catacgcgta aaacagccag cgctggcgcg 1260atttagcccc gacatagccc cactgttcgt ccatttccgc gcagacgatg acgtcactgc 1320ccggctgtat gcgcgaggtt accgactgcg gcctgagttt tttaagtgac gtaaaatcgt 1380gttgaggcca acgcccataa tgcgggctgt tgcccggcat ccaacgccat tcatggccat 1440atcaatgatt ttctggtgcg taccgggttg agaagcggtg taagtgaact gcagttgcca 1500tgttttacgg cagtgagagc agagatagcg ctgatgtccg gcggtgcttt tgccgttacg 1560caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag ccactggagc 1620acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc tttttcaaaa 1680gaaatcatcg ctcatttatc tcagttgccc ttgaaggaag aggtgaattt attttatatg 1740cctaagataa aaggatatat tacttatttt tctgtatttg gtaaagagga gtatcttcta 1800cttattttta aaggacaaga aaaacttgca aataatcctt tccccgttga agtaaaacaa 1860ttattaaaaa gtggtatttt actctatcaa atgatttttc aagaaaaatt agattatgaa 1920gaattatttg agaaaaatca gcatattatt tctccattgc ttgctgctaa accaattgaa 1980tggaatgatt ccaatacgtg aggaaagtaa attcccataa aacatatctt tttgaaaaat 2040atttggggga atgtgttatt cgtggagatg ttgcagagtt aaaaaaagct ttttcaaatt 2100atatgaataa aggaactgct ggaaaattat ctaataattc aatgcgacat aagaaaaaca 2160ttttgatttc agtcatcact atgactactc gttcggctat acagggagga ttacctgaag 2220aagaagcttt tttgatgagt gatttatata ttcaagagct tgaagaatta acggaattag 2280aagaaattag aacgcttgcc tataatgtga tgatcgattt tgcagataaa gtgaaacagc 2340atcgatattg tcaggtttct tataaaatat tatcttgtca aaagtatatt gttaatcatt 2400tatacgaaaa actaagtgtg agtgaaattg cagaagagct acacatgaat atttcttatt 2460tatcttcaca attcaaaaaa gagacagggc aaacaattac aaactttatt caggagaagc 2520gaatagaaga agctagagaa ttaatccttt tctcagacta tcctttttca agaatttata 2580ccttgttggt tttactgcca aagtcatttt ataaaaatat ttaaaaaata tactggaata 2640actcccaaaa agtttcaaga tcagtatatt tatcatgcct ctacatcaat atatgattga 2700aattaaaaaa agacctagaa tttcaaaatt gataaaatac atacctaaaa tattaattct 2760gtactattac gggtggagta tctactgtat aatgagggta taaattatgg aagaagggag 2820taaaactaaa tttattgatg gttttacgaa ttaattagga tatttttttt aaaaaccaaa 2880gaaaacgctt acaaacgtta aaggagtgaa tctaaagatg gacaaatttg aaaaatggct 2940aaataagacc ttgatgccac ttgcctcaaa aatgaataaa aatcatttca tttcggcatt 3000aagtgaagca tttatgagat gtatgccctt aacattaggg attgcattat tgacaattat 3060aggatacttt ccagttcctg cctgggtaga tttcttaaac tctattggac tggctcagca 3120tttttcagca gttattggtg cagttaccag tgcgctagca atttatgtaa cttataattt 3180tgcttattct tatgtaaatc gtcatgaata taatggccat acggccggtt tattatcaat 3240cgcaagtttg ttaatgctaa tgccacaaat tattactgtc cctgtagtaa aaaacattcc 3300aaccgaattt ccgaaatccg cggtagttga cagtgtgtca aatgttgaag catttcaaac 3360ggtatacacg ggtagcacag gattaattgt agcaatcata attggtttta ttgtttcatt 3420agtctatata caattgagca aaagaaattt agttattaaa ttaccagctg gagttcctcc 3480aatggttgta gattcactaa gtccagcaat tatttcaatg gtgattttct gtttgatgtt 3540cgggattcgt gtgggattct cttatacgcc attccatgat attttcaatt tctcaacaca 3600actaattcaa gcaccgttga ctggtgctgt ggcaaatcca tgggttctta tgggcatctt 3660tacctttggt aatttcttat ggttctttgg tatccaccct aatttaattg ggggaatttt 3720aaatccattg ttattaacaa tgtcatatgc taatattgat gcctatgctg ccggaaaacc 3780tgtaccatac ttacaaatga tgattgtgtt tgctgtgggt gcgaacgcat ggggcggaag 3840tggaaatact tatgggttag

ttatttcaat gtttacggca aaatctgaac gctataaaca 3900attattaaaa ttaggtgcaa ttcctagtat tttcaatatc agtgaaccat tactttttgg 3960tcttccaatg atgttaaatc ctcttttctt tattcctttg gttttccaac cagcaatttt 4020aggaactgta gcattgggct tggcaaagat attatatatt acaaatctga atccaatgac 4080ggcacttctt ccttggacga caccagcacc tgtgagaatg gccatttcag gtggacttcc 4140atttttgatt atttttgcaa tctgtttagt cttgaatgtt cttatttact acccattctt 4200taaggtggcg tataataaag ctttagaaga agaaaaagca gctgttgaat tagagggttc 4260agaaactgcc tgatggatat tttttataaa tctggtttga acaaattata ttgacatctc 4320tttttctatc ctgataattc tgagaggtta ttttgggaaa tactattgaa ccatatcgag 4380gtggtgtggt ataatgaagg gaattaaaaa agataggaaa atttcatgga ttaagtcatc 4440ttacctcttt tattagtttt ttcttataat ctaatgataa catttttata attaatctat 4500aaaccatatc cctctttgga atcaaaattt attatctact cctttgtaga tatgttataa 4560tacaagtatc agatctggga gaccacaacg gtttcccact agaaataatt ttgtttaact 4620ttagaaagga gatatacgca tgaaaaaaaa gattatctca gctattttaa tgtctacagt 4680catactttct gctgcagccc cgttgtcagg tgtttacgcc tcagctggtc aaggtactca 4740atcagaaaac tcatgtactc actttccagg taacttgcca aacatgcttc gtgatttgcg 4800tgatgctttt tcacgtgtta aaactttttt tcaaatgaaa gatcaacttg ataacttgct 4860tttgaaagaa tcacttttgg aagattttaa aggttacctt ggttgtcaag ctttgtcaga 4920aatgatccaa ttttaccttg aagaagttat gccacaagct gaaaaccaag atccagatat 4980caaagctcac gttaactcat tgggtgaaaa ccttaaaact ttgcgtcttc gtttgcgtcg 5040ttgtcaccgt tttcttccat gtgaaaacaa atcaaaagct gttgaacaag ttaaaaacgc 5100ttttaacaaa ttgcaagaaa aaggtatcta caaagctatg tcagaatttg atatctttat 5160caactacatc gaagcttaca tgactatgaa aatccgtaac taactagaat taatctataa 5220gttactgaca aaactgtcag taactttttt tgtgggaaaa atgtattttt atgaccgtaa 5280agaatctgtc agtagaagtc tgaaattcgt ttaaaaatcg actagaatag gctttaacga 5340caagatgttt taaagagtac gctctaaatg tatttttgta tttttgtttg attacgaagt 5400ttaaatttaa ttgacaaatg ttttaaaatg agtataatag gacttgtaac cgattttatt 5460tttataaagg agaaagaaag atgaacaaac ttttacttgg aacagccttt ataggggcta 5520gcttactgat tggtgggggt gctcatgcag atcaaatgtt tatcgtttgt ataatcataa 5580tactggtgag cactctatac aactagtggg acaccaaaag aatgctaatg taagtgcggg 5640ttggacttat gaaggtgtcg gttggatcgc accaacaaca agttcaagcc cagtttaccg 5700tgtgtacaat ccaaatgcat tattacacaa aaagcaagta tgaagcccaa agtttagtaa 5760ataagggttg gaaatgggat aataacggaa aggcggtctt ctattctgga ggttctcaag 5820ccgtatatgt cgcttataat cccaatgcac aatctggcgc tcacaattac acggaaagta 5880gctttgagca aaatagctta ttgaatactg gttggaaata tggggcagta gcttggtacg 5940ggattggagt aaaaaacgaa atgttaaaca ttgctcaaat tgttagtggt aatttttcta 6000gtattgttgg aacttggaaa gatacttctg gaaatatgct tgaaattaat gcaatgggaa 6060atcttacttt aatatggaaa ggggcaaaga atcaaacctt tgaacttggc gcaggtcaac 6120aatttaatgg aactgcagat attgccttaa aaaatggaga gatttcccct ggtagtccac 6180ttaacatttt tgttgtacca acagaagttg ctttccctaa taataaaaaa gtagacgatt 6240caactgggca acaacgaatt tttgtgaatt attctggtac aagccctcaa atggcgaata 6300gtatggcagc ggtggctttt tttagagtta ttccatgatt atattaaagt tagaattgaa 6360taaaatgtat tattaaaaag ataatattat atcacgacaa ggcgacatct atcaacttta 6420ccactggtat ggaagtgacc attattacat caggaaacgc taaaacggtt gtttttacac 6480ccgtaaaata aataataaaa taatgtgaaa ttactgacag cattttgtca gtaatttttt 6540ttatcaaaat cacacaaaaa tgttcgttga cgaacaaaaa aaactatgtt ataataattc 6600gtatgcgaac taaaaaagaa gcgattggcc gacttttaaa agtagccagc aaccaaatgt 6660ctcgagaatt tgataatttt gcagctcaac ttgatttgac aggtcagcaa atgtcaattt 6720tagattttct tggaaatcaa agcgaagaag gttcaggaaa agaaattagt cagacgatga 6780ttgaattaga atttaatatc cgacgttcaa caacgacgga aattttacag cgcatggaaa 6840agcggctttt aattaatcga agaacaagcc tgaccgatgc ccgccaaaaa tcagttgaat 6900taactgaaga agggaaaaga tatttacctg aaatcagggc ttatatccaa gcacataata 6960aaaaagc 6967206753DNALactococcus lactismisc_featureThy12 20attaacagcc ttttgagcag ctagctcatt attttgaaat aaatcataaa tttctttccc 60actatctgat ttatgattgc tagcatattt gttgtataat cgaacgagtc cattttgaac 120agatccatat agattgagtg aactataaaa tacatctata tcatagttga gtttgttcac 180aatcatgaga ccaaattctc cagcatttcg tgtagaacca cgataaagct gtttatttag 240caaaatggca cctccgacac ctgtacctaa agtcatgcaa ataaaatttt ggctttcttg 300tccattccct agccaaagtt cagctagacc tgcacaattg gcatcatttt caacataaac 360cggaagattt aaatgttttt gtagttctgt ccccaatgga tagccataaa gatcagttag 420agctcctgcc agtaataatg ttcccttttt gtcagaagtt ccgggaacac ttacaccaat 480tgcagatact gaatgatgag cttttaactg atgaatattt gtgagcaagc tatccataat 540tttttctttt tttaatgggg ttggaacttg taaatgttgt atgatcgttc catcactagt 600tacaagacca aattttataa atgtaccacc gatatcaatt cctattgaat aatgcatctt 660ttattacctc tttctctaat ttgttttagt atagcaaaat caaaaaatta attatggtat 720gcattataga tatgttgtat aattttcaca aaaacggaga aaactatgaa aacaatagaa 780cagctcatga tagattcagc agatttaatg tcagatttta ttcaattgac aatttttata 840ttccgcaagg aggattttca acttttttat aggagtgatg aagaagagca agctttttca 900aggtaatgac tccaacttat tgatagtgtt ttatgttcag ataatgcccg atgactttgt 960catgcagctc caccgatttt gagaacgaca gcgacttccg tcccagccgt gccaggtgct 1020gcctcagatt caggttatgc cgctcaattc gctgcgtata tcgcttgctg attacgtgca 1080gctttccctt caggcgggat tcatacagcg gccagccatc cgtcatccat atcaccacgt 1140caaagggtga cagcaggctc ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata 1200cgtgcgcaac aaccgtcttc cggagactgt catacgcgta aaacagccag cgctggcgcg 1260atttagcccc gacatagccc cactgttcgt ccatttccgc gcagacgatg acgtcactgc 1320ccggctgtat gcgcgaggtt accgactgcg gcctgagttt tttaagtgac gtaaaatcgt 1380gttgaggcca acgcccataa tgcgggctgt tgcccggcat ccaacgccat tcatggccat 1440atcaatgatt ttctggtgcg taccgggttg agaagcggtg taagtgaact gcagttgcca 1500tgttttacgg cagtgagagc agagatagcg ctgatgtccg gcggtgcttt tgccgttacg 1560caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag ccactggagc 1620acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc tttttcaaaa 1680gaaatcatcg ctcatttatc tcagttgccc ttgaaggaag aggtgaattt attttatatg 1740cctaagataa aaggatatat tacttatttt tctgtatttg gtaaagagga gtatcttcta 1800cttattttta aaggacaaga aaaacttgca aataatcctt tccccgttga agtaaaacaa 1860ttattaaaaa gtggtatttt actctatcaa atgatttttc aagaaaaatt agattatgaa 1920gaattatttg agaaaaatca gcatattatt tctccattgc ttgctgctaa accaattgaa 1980tggaatgatt ccaatacgtg aggaaagtaa attcccataa aacatatctt tttgaaaaat 2040atttggggga atgtgttatt cgtggagatg ttgcagagtt aaaaaaagct ttttcaaatt 2100atatgaataa aggaactgct ggaaaattat ctaataattc aatgcgacat aagaaaaaca 2160ttttgatttc agtcatcact atgactactc gttcggctat acagggagga ttacctgaag 2220aagaagcttt tttgatgagt gatttatata ttcaagagct tgaagaatta acggaattag 2280aagaaattag aacgcttgcc tataatgtga tgatcgattt tgcagataaa gtgaaacagc 2340atcgatattg tcaggtttct tataaaatat tatcttgtca aaagtatatt gttaatcatt 2400tatacgaaaa actaagtgtg agtgaaattg cagaagagct acacatgaat atttcttatt 2460tatcttcaca attcaaaaaa gagacagggc aaacaattac aaactttatt caggagaagc 2520gaatagaaga agctagagaa ttaatccttt tctcagacta tcctttttca agaatttata 2580ccttgttggt tttactgcca aagtcatttt ataaaaatat ttaaaaaata tactggaata 2640actcccaaaa agtttcaaga tcagtatatt tatcatgcct ctacatcaat atatgattga 2700aattaaaaaa agacctagaa tttcaaaatt gataaaatac atacctaaaa tattaattct 2760gtactattac gggtggagta tctactgtat aatgagggta taaattatgg aagaagggag 2820taaaactaaa tttattgatg gttttacgaa ttaattagga tatttttttt aaaaaccaaa 2880gaaaacgctt acaaacgtta aaggagtgaa tctaaagatg gacaaatttg aaaaatggct 2940aaataagacc ttgatgccac ttgcctcaaa aatgaataaa aatcatttca tttcggcatt 3000aagtgaagca tttatgagat gtatgccctt aacattaggg attgcattat tgacaattat 3060aggatacttt ccagttcctg cctgggtaga tttcttaaac tctattggac tggctcagca 3120tttttcagca gttattggtg cagttaccag tgcgctagca atttatgtaa cttataattt 3180tgcttattct tatgtaaatc gtcatgaata taatggccat acggccggtt tattatcaat 3240cgcaagtttg ttaatgctaa tgccacaaat tattactgtc cctgtagtaa aaaacattcc 3300aaccgaattt ccgaaatccg cggtagttga cagtgtgtca aatgttgaag catttcaaac 3360ggtatacacg ggtagcacag gattaattgt agcaatcata attggtttta ttgtttcatt 3420agtctatata caattgagca aaagaaattt agttattaaa ttaccagctg gagttcctcc 3480aatggttgta gattcactaa gtccagcaat tatttcaatg gtgattttct gtttgatgtt 3540cgggattcgt gtgggattct cttatacgcc attccatgat attttcaatt tctcaacaca 3600actaattcaa gcaccgttga ctggtgctgt ggcaaatcca tgggttctta tgggcatctt 3660tacctttggt aatttcttat ggttctttgg tatccaccct aatttaattg ggggaatttt 3720aaatccattg ttattaacaa tgtcatatgc taatattgat gcctatgctg ccggaaaacc 3780tgtaccatac ttacaaatga tgattgtgtt tgctgtgggt gcgaacgcat ggggcggaag 3840tggaaatact tatgggttag ttatttcaat gtttacggca aaatctgaac gctataaaca 3900attattaaaa ttaggtgcaa ttcctagtat tttcaatatc agtgaaccat tactttttgg 3960tcttccaatg atgttaaatc ctcttttctt tattcctttg gttttccaac cagcaatttt 4020aggaactgta gcattgggct tggcaaagat attatatatt acaaatctga atccaatgac 4080ggcacttctt ccttggacga caccagcacc tgtgagaatg gccatttcag gtggacttcc 4140atttttgatt atttttgcaa tctgtttagt cttgaatgtt cttatttact acccattctt 4200taaggtggcg tataataaag ctttagaaga agaaaaagca gctgttgaat tagagggttc 4260agaaactgcc tgatggatat tttttataaa tctggtttga acaaattata ttgacatctc 4320tttttctatc ctgataattc tgagaggtta ttttgggaaa tactattgaa ccatatcgag 4380gtggtgtggt ataatgaagg gaattaaaaa agataggaaa atttcatgaa aaaaaagatt 4440atctcagcta ttttaatgtc tacagtcata ctttctgctg cagccccgtt gtcaggtgtt 4500tacgcctcag ctggtcaagg tactcaatca gaaaactcat gtactcactt tccaggtaac 4560ttgccaaaca tgcttcgtga tttgcgtgat gctttttcac gtgttaaaac tttttttcaa 4620atgaaagatc aacttgataa cttgcttttg aaagaatcac ttttggaaga ttttaaaggt 4680taccttggtt gtcaagcttt gtcagaaatg atccaatttt accttgaaga agttatgcca 4740caagctgaaa accaagatcc agatatcaaa gctcacgtta actcattggg tgaaaacctt 4800aaaactttgc gtcttcgttt gcgtcgttgt caccgttttc ttccatgtga aaacaaatca 4860aaagctgttg aacaagttaa aaacgctttt aacaaattgc aagaaaaagg tatctacaaa 4920gctatgtcag aatttgatat ctttatcaac tacatcgaag cttacatgac tatgaaaatc 4980cgtaactaac tagaattaat ctataagtta ctgacaaaac tgtcagtaac tttttttgtg 5040ggaaaaatgt atttttatga ccgtaaagaa tctgtcagta gaagtctgaa attcgtttaa 5100aaatcgacta gaataggctt taacgacaag atgttttaaa gagtacgctc taaatgtatt 5160tttgtatttt tgtttgatta cgaagtttaa atttaattga caaatgtttt aaaatgagta 5220taataggact tgtaaccgat tttattttta taaaggagaa agaaagatga acaaactttt 5280acttggaaca gcctttatag gggctagctt actgattggt gggggtgctc atgcagatca 5340aatgtttatc gtttgtataa tcataatact ggtgagcact ctatacaact agtgggacac 5400caaaagaatg ctaatgtaag tgcgggttgg acttatgaag gtgtcggttg gatcgcacca 5460acaacaagtt caagcccagt ttaccgtgtg tacaatccaa atgcattatt acacaaaaag 5520caagtatgaa gcccaaagtt tagtaaataa gggttggaaa tgggataata acggaaaggc 5580ggtcttctat tctggaggtt ctcaagccgt atatgtcgct tataatccca atgcacaatc 5640tggcgctcac aattacacgg aaagtagctt tgagcaaaat agcttattga atactggttg 5700gaaatatggg gcagtagctt ggtacgggat tggagtaaaa aacgaaatgt taaacattgc 5760tcaaattgtt agtggtaatt tttctagtat tgttggaact tggaaagata cttctggaaa 5820tatgcttgaa attaatgcaa tgggaaatct tactttaata tggaaagggg caaagaatca 5880aacctttgaa cttggcgcag gtcaacaatt taatggaact gcagatattg ccttaaaaaa 5940tggagagatt tcccctggta gtccacttaa catttttgtt gtaccaacag aagttgcttt 6000ccctaataat aaaaaagtag acgattcaac tgggcaacaa cgaatttttg tgaattattc 6060tggtacaagc cctcaaatgg cgaatagtat ggcagcggtg gcttttttta gagttattcc 6120atgattatat taaagttaga attgaataaa atgtattatt aaaaagataa tattatatca 6180cgacaaggcg acatctatca actttaccac tggtatggaa gtgaccatta ttacatcagg 6240aaacgctaaa acggttgttt ttacacccgt aaaataaata ataaaataat gtgaaattac 6300tgacagcatt ttgtcagtaa ttttttttat caaaatcaca caaaaatgtt cgttgacgaa 6360caaaaaaaac tatgttataa taattcgtat gcgaactaaa aaagaagcga ttggccgact 6420tttaaaagta gccagcaacc aaatgtctcg agaatttgat aattttgcag ctcaacttga 6480tttgacaggt cagcaaatgt caattttaga ttttcttgga aatcaaagcg aagaaggttc 6540aggaaaagaa attagtcaga cgatgattga attagaattt aatatccgac gttcaacaac 6600gacggaaatt ttacagcgca tggaaaagcg gcttttaatt aatcgaagaa caagcctgac 6660cgatgcccgc caaaaatcag ttgaattaac tgaagaaggg aaaagatatt tacctgaaat 6720cagggcttat atccaagcac ataataaaaa agc 6753216904DNALactococcus lactismisc_featureThy15 21attaacagcc ttttgagcag ctagctcatt attttgaaat aaatcataaa tttctttccc 60actatctgat ttatgattgc tagcatattt gttgtataat cgaacgagtc cattttgaac 120agatccatat agattgagtg aactataaaa tacatctata tcatagttga gtttgttcac 180aatcatgaga ccaaattctc cagcatttcg tgtagaacca cgataaagct gtttatttag 240caaaatggca cctccgacac ctgtacctaa agtcatgcaa ataaaatttt ggctttcttg 300tccattccct agccaaagtt cagctagacc tgcacaattg gcatcatttt caacataaac 360cggaagattt aaatgttttt gtagttctgt ccccaatgga tagccataaa gatcagttag 420agctcctgcc agtaataatg ttcccttttt gtcagaagtt ccgggaacac ttacaccaat 480tgcagatact gaatgatgag cttttaactg atgaatattt gtgagcaagc tatccataat 540tttttctttt tttaatgggg ttggaacttg taaatgttgt atgatcgttc catcactagt 600tacaagacca aattttataa atgtaccacc gatatcaatt cctattgaat aatgcatctt 660ttattacctc tttctctaat ttgttttagt atagcaaaat caaaaaatta attatggtat 720gcattataga tatgttgtat aattttcaca aaaacggaga aaactatgaa aacaatagaa 780cagctcatga tagattcagc agatttaatg tcagatttta ttcaattgac aatttttata 840ttccgcaagg aggattttca acttttttat aggagtgatg aagaagagca agctttttca 900aggtaatgac tccaacttat tgatagtgtt ttatgttcag ataatgcccg atgactttgt 960catgcagctc caccgatttt gagaacgaca gcgacttccg tcccagccgt gccaggtgct 1020gcctcagatt caggttatgc cgctcaattc gctgcgtata tcgcttgctg attacgtgca 1080gctttccctt caggcgggat tcatacagcg gccagccatc cgtcatccat atcaccacgt 1140caaagggtga cagcaggctc ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata 1200cgtgcgcaac aaccgtcttc cggagactgt catacgcgta aaacagccag cgctggcgcg 1260atttagcccc gacatagccc cactgttcgt ccatttccgc gcagacgatg acgtcactgc 1320ccggctgtat gcgcgaggtt accgactgcg gcctgagttt tttaagtgac gtaaaatcgt 1380gttgaggcca acgcccataa tgcgggctgt tgcccggcat ccaacgccat tcatggccat 1440atcaatgatt ttctggtgcg taccgggttg agaagcggtg taagtgaact gcagttgcca 1500tgttttacgg cagtgagagc agagatagcg ctgatgtccg gcggtgcttt tgccgttacg 1560caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag ccactggagc 1620acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc tttttcaaaa 1680gaaatcatcg ctcatttatc tcagttgccc ttgaaggaag aggtgaattt attttatatg 1740cctaagataa aaggatatat tacttatttt tctgtatttg gtaaagagga gtatcttcta 1800cttattttta aaggacaaga aaaacttgca aataatcctt tccccgttga agtaaaacaa 1860ttattaaaaa gtggtatttt actctatcaa atgatttttc aagaaaaatt agattatgaa 1920gaattatttg agaaaaatca gcatattatt tctccattgc ttgctgctaa accaattgaa 1980tggaatgatt ccaatacgtg aggaaagtaa attcccataa aacatatctt tttgaaaaat 2040atttggggga atgtgttatt cgtggagatg ttgcagagtt aaaaaaagct ttttcaaatt 2100atatgaataa aggaactgct ggaaaattat ctaataattc aatgcgacat aagaaaaaca 2160ttttgatttc agtcatcact atgactactc gttcggctat acagggagga ttacctgaag 2220aagaagcttt tttgatgagt gatttatata ttcaagagct tgaagaatta acggaattag 2280aagaaattag aacgcttgcc tataatgtga tgatcgattt tgcagataaa gtgaaacagc 2340atcgatattg tcaggtttct tataaaatat tatcttgtca aaagtatatt gttaatcatt 2400tatacgaaaa actaagtgtg agtgaaattg cagaagagct acacatgaat atttcttatt 2460tatcttcaca attcaaaaaa gagacagggc aaacaattac aaactttatt caggagaagc 2520gaatagaaga agctagagaa ttaatccttt tctcagacta tcctttttca agaatttata 2580ccttgttggt tttactgcca aagtcatttt ataaaaatat ttaaaaaata tactggaata 2640actcccaaaa agtttcaaga tcagtatatt tatcatgcct ctacatcaat atatgattga 2700aattaaaaaa agacctagaa tttcaaaatt gataaaatac atacctaaaa tattaattct 2760gtactattac gggtggagta tctactgtat aatgagggta taaattatgg aagaagggag 2820taaaactaaa tttattgatg gttttacgaa ttaattagga tatttttttt aaaaaccaaa 2880gaaaacgctt acaaacgtta aaggagtgaa tctaaagatg gacaaatttg aaaaatggct 2940aaataagacc ttgatgccac ttgcctcaaa aatgaataaa aatcatttca tttcggcatt 3000aagtgaagca tttatgagat gtatgccctt aacattaggg attgcattat tgacaattat 3060aggatacttt ccagttcctg cctgggtaga tttcttaaac tctattggac tggctcagca 3120tttttcagca gttattggtg cagttaccag tgcgctagca atttatgtaa cttataattt 3180tgcttattct tatgtaaatc gtcatgaata taatggccat acggccggtt tattatcaat 3240cgcaagtttg ttaatgctaa tgccacaaat tattactgtc cctgtagtaa aaaacattcc 3300aaccgaattt ccgaaatccg cggtagttga cagtgtgtca aatgttgaag catttcaaac 3360ggtatacacg ggtagcacag gattaattgt agcaatcata attggtttta ttgtttcatt 3420agtctatata caattgagca aaagaaattt agttattaaa ttaccagctg gagttcctcc 3480aatggttgta gattcactaa gtccagcaat tatttcaatg gtgattttct gtttgatgtt 3540cgggattcgt gtgggattct cttatacgcc attccatgat attttcaatt tctcaacaca 3600actaattcaa gcaccgttga ctggtgctgt ggcaaatcca tgggttctta tgggcatctt 3660tacctttggt aatttcttat ggttctttgg tatccaccct aatttaattg ggggaatttt 3720aaatccattg ttattaacaa tgtcatatgc taatattgat gcctatgctg ccggaaaacc 3780tgtaccatac ttacaaatga tgattgtgtt tgctgtgggt gcgaacgcat ggggcggaag 3840tggaaatact tatgggttag ttatttcaat gtttacggca aaatctgaac gctataaaca 3900attattaaaa ttaggtgcaa ttcctagtat tttcaatatc agtgaaccat tactttttgg 3960tcttccaatg atgttaaatc ctcttttctt tattcctttg gttttccaac cagcaatttt 4020aggaactgta gcattgggct tggcaaagat attatatatt acaaatctga atccaatgac 4080ggcacttctt ccttggacga caccagcacc tgtgagaatg gccatttcag gtggacttcc 4140atttttgatt atttttgcaa tctgtttagt cttgaatgtt cttatttact acccattctt 4200taaggtggcg tataataaag ctttagaaga agaaaaagca gctgttgaat tagagggttc 4260agaaactgcc tgatggatat tttttataaa tctggtttga acaaattata ttgacatctc 4320tttttctatc ctgataattc tgagaggtta ttttgggaaa tactagatta agtcatctta 4380cctcttttat tagttttttc ttataatcta atgataacat ttttataatt aatctataaa 4440ccatatccct ctttggaatc aaaatttatt atctactcct ttgtagatat gttataatac 4500aagtatcaga tctgggagac cacaacggtt tcccactaga aataattttg tttaacttta 4560gaaaggagat atacgcatga aaaaaaagat tatctcagct attttaatgt ctacagtcat 4620actttctgct gcagccccgt tgtcaggtgt ttacgcctca gctggtcaag gtactcaatc 4680agaaaactca tgtactcact ttccaggtaa cttgccaaac atgcttcgtg atttgcgtga 4740tgctttttca cgtgttaaaa ctttttttca aatgaaagat caacttgata acttgctttt 4800gaaagaatca cttttggaag attttaaagg ttaccttggt tgtcaagctt tgtcagaaat 4860gatccaattt taccttgaag aagttatgcc acaagctgaa aaccaagatc cagatatcaa 4920agctcacgtt aactcattgg gtgaaaacct taaaactttg cgtcttcgtt tgcgtcgttg 4980tcaccgtttt cttccatgtg aaaacaaatc aaaagctgtt gaacaagtta aaaacgcttt

5040taacaaattg caagaaaaag gtatctacaa agctatgtca gaatttgata tctttatcaa 5100ctacatcgaa gcttacatga ctatgaaaat ccgtaactaa ctagaattaa tctataagtt 5160actgacaaaa ctgtcagtaa ctttttttgt gggaaaaatg tatttttatg accgtaaaga 5220atctgtcagt agaagtctga aattcgttta aaaatcgact agaataggct ttaacgacaa 5280gatgttttaa agagtacgct ctaaatgtat ttttgtattt ttgtttgatt acgaagttta 5340aatttaattg acaaatgttt taaaatgagt ataataggac ttgtaaccga ttttattttt 5400ataaaggaga aagaaagatg aacaaacttt tacttggaac agcctttata ggggctagct 5460tactgattgg tgggggtgct catgcagatc aaatgtttat cgtttgtata atcataatac 5520tggtgagcac tctatacaac tagtgggaca ccaaaagaat gctaatgtaa gtgcgggttg 5580gacttatgaa ggtgtcggtt ggatcgcacc aacaacaagt tcaagcccag tttaccgtgt 5640gtacaatcca aatgcattat tacacaaaaa gcaagtatga agcccaaagt ttagtaaata 5700agggttggaa atgggataat aacggaaagg cggtcttcta ttctggaggt tctcaagccg 5760tatatgtcgc ttataatccc aatgcacaat ctggcgctca caattacacg gaaagtagct 5820ttgagcaaaa tagcttattg aatactggtt ggaaatatgg ggcagtagct tggtacggga 5880ttggagtaaa aaacgaaatg ttaaacattg ctcaaattgt tagtggtaat ttttctagta 5940ttgttggaac ttggaaagat acttctggaa atatgcttga aattaatgca atgggaaatc 6000ttactttaat atggaaaggg gcaaagaatc aaacctttga acttggcgca ggtcaacaat 6060ttaatggaac tgcagatatt gccttaaaaa atggagagat ttcccctggt agtccactta 6120acatttttgt tgtaccaaca gaagttgctt tccctaataa taaaaaagta gacgattcaa 6180ctgggcaaca acgaattttt gtgaattatt ctggtacaag ccctcaaatg gcgaatagta 6240tggcagcggt ggcttttttt agagttattc catgattata ttaaagttag aattgaataa 6300aatgtattat taaaaagata atattatatc acgacaaggc gacatctatc aactttacca 6360ctggtatgga agtgaccatt attacatcag gaaacgctaa aacggttgtt tttacacccg 6420taaaataaat aataaaataa tgtgaaatta ctgacagcat tttgtcagta atttttttta 6480tcaaaatcac acaaaaatgt tcgttgacga acaaaaaaaa ctatgttata ataattcgta 6540tgcgaactaa aaaagaagcg attggccgac ttttaaaagt agccagcaac caaatgtctc 6600gagaatttga taattttgca gctcaacttg atttgacagg tcagcaaatg tcaattttag 6660attttcttgg aaatcaaagc gaagaaggtt caggaaaaga aattagtcag acgatgattg 6720aattagaatt taatatccga cgttcaacaa cgacggaaat tttacagcgc atggaaaagc 6780ggcttttaat taatcgaaga acaagcctga ccgatgcccg ccaaaaatca gttgaattaa 6840ctgaagaagg gaaaagatat ttacctgaaa tcagggctta tatccaagca cataataaaa 6900aagc 6904226964DNALactococcus lactismisc_featureThy16 22attaacagcc ttttgagcag ctagctcatt attttgaaat aaatcataaa tttctttccc 60actatctgat ttatgattgc tagcatattt gttgtataat cgaacgagtc cattttgaac 120agatccatat agattgagtg aactataaaa tacatctata tcatagttga gtttgttcac 180aatcatgaga ccaaattctc cagcatttcg tgtagaacca cgataaagct gtttatttag 240caaaatggca cctccgacac ctgtacctaa agtcatgcaa ataaaatttt ggctttcttg 300tccattccct agccaaagtt cagctagacc tgcacaattg gcatcatttt caacataaac 360cggaagattt aaatgttttt gtagttctgt ccccaatgga tagccataaa gatcagttag 420agctcctgcc agtaataatg ttcccttttt gtcagaagtt ccgggaacac ttacaccaat 480tgcagatact gaatgatgag cttttaactg atgaatattt gtgagcaagc tatccataat 540tttttctttt tttaatgggg ttggaacttg taaatgttgt atgatcgttc catcactagt 600tacaagacca aattttataa atgtaccacc gatatcaatt cctattgaat aatgcatctt 660ttattacctc tttctctaat ttgttttagt atagcaaaat caaaaaatta attatggtat 720gcattataga tatgttgtat aattttcaca aaaacggaga aaactatgaa aacaatagaa 780cagctcatga tagattcagc agatttaatg tcagatttta ttcaattgac aatttttata 840ttccgcaagg aggattttca acttttttat aggagtgatg aagaagagca agctttttca 900aggtaatgac tccaacttat tgatagtgtt ttatgttcag ataatgcccg atgactttgt 960catgcagctc caccgatttt gagaacgaca gcgacttccg tcccagccgt gccaggtgct 1020gcctcagatt caggttatgc cgctcaattc gctgcgtata tcgcttgctg attacgtgca 1080gctttccctt caggcgggat tcatacagcg gccagccatc cgtcatccat atcaccacgt 1140caaagggtga cagcaggctc ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata 1200cgtgcgcaac aaccgtcttc cggagactgt catacgcgta aaacagccag cgctggcgcg 1260atttagcccc gacatagccc cactgttcgt ccatttccgc gcagacgatg acgtcactgc 1320ccggctgtat gcgcgaggtt accgactgcg gcctgagttt tttaagtgac gtaaaatcgt 1380gttgaggcca acgcccataa tgcgggctgt tgcccggcat ccaacgccat tcatggccat 1440atcaatgatt ttctggtgcg taccgggttg agaagcggtg taagtgaact gcagttgcca 1500tgttttacgg cagtgagagc agagatagcg ctgatgtccg gcggtgcttt tgccgttacg 1560caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag ccactggagc 1620acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc tttttcaaaa 1680gaaatcatcg ctcatttatc tcagttgccc ttgaaggaag aggtgaattt attttatatg 1740cctaagataa aaggatatat tacttatttt tctgtatttg gtaaagagga gtatcttcta 1800cttattttta aaggacaaga aaaacttgca aataatcctt tccccgttga agtaaaacaa 1860ttattaaaaa gtggtatttt actctatcaa atgatttttc aagaaaaatt agattatgaa 1920gaattatttg agaaaaatca gcatattatt tctccattgc ttgctgctaa accaattgaa 1980tggaatgatt ccaatacgtg aggaaagtaa attcccataa aacatatctt tttgaaaaat 2040atttggggga atgtgttatt cgtggagatg ttgcagagtt aaaaaaagct ttttcaaatt 2100atatgaataa aggaactgct ggaaaattat ctaataattc aatgcgacat aagaaaaaca 2160ttttgatttc agtcatcact atgactactc gttcggctat acagggagga ttacctgaag 2220aagaagcttt tttgatgagt gatttatata ttcaagagct tgaagaatta acggaattag 2280aagaaattag aacgcttgcc tataatgtga tgatcgattt tgcagataaa gtgaaacagc 2340atcgatattg tcaggtttct tataaaatat tatcttgtca aaagtatatt gttaatcatt 2400tatacgaaaa actaagtgtg agtgaaattg cagaagagct acacatgaat atttcttatt 2460tatcttcaca attcaaaaaa gagacagggc aaacaattac aaactttatt caggagaagc 2520gaatagaaga agctagagaa ttaatccttt tctcagacta tcctttttca agaatttata 2580ccttgttggt tttactgcca aagtcatttt ataaaaatat ttaaaaaata tactggaata 2640actcccaaaa agtttcaaga tcagtatatt tatcatgcct ctacatcaat atatgattga 2700aattaaaaaa agacctagaa tttcaaaatt gataaaatac atacctaaaa tattaattct 2760gtactattac gggtggagta tctactgtat aatgagggta taaattatgg aagaagggag 2820taaaactaaa tttattgatg gttttacgaa ttaattagga tatttttttt aaaaaccaaa 2880gaaaacgctt acaaacgtta aaggagtgaa tctaaagatg gacaaatttg aaaaatggct 2940aaataagacc ttgatgccac ttgcctcaaa aatgaataaa aatcatttca tttcggcatt 3000aagtgaagca tttatgagat gtatgccctt aacattaggg attgcattat tgacaattat 3060aggatacttt ccagttcctg cctgggtaga tttcttaaac tctattggac tggctcagca 3120tttttcagca gttattggtg cagttaccag tgcgctagca atttatgtaa cttataattt 3180tgcttattct tatgtaaatc gtcatgaata taatggccat acggccggtt tattatcaat 3240cgcaagtttg ttaatgctaa tgccacaaat tattactgtc cctgtagtaa aaaacattcc 3300aaccgaattt ccgaaatccg cggtagttga cagtgtgtca aatgttgaag catttcaaac 3360ggtatacacg ggtagcacag gattaattgt agcaatcata attggtttta ttgtttcatt 3420agtctatata caattgagca aaagaaattt agttattaaa ttaccagctg gagttcctcc 3480aatggttgta gattcactaa gtccagcaat tatttcaatg gtgattttct gtttgatgtt 3540cgggattcgt gtgggattct cttatacgcc attccatgat attttcaatt tctcaacaca 3600actaattcaa gcaccgttga ctggtgctgt ggcaaatcca tgggttctta tgggcatctt 3660tacctttggt aatttcttat ggttctttgg tatccaccct aatttaattg ggggaatttt 3720aaatccattg ttattaacaa tgtcatatgc taatattgat gcctatgctg ccggaaaacc 3780tgtaccatac ttacaaatga tgattgtgtt tgctgtgggt gcgaacgcat ggggcggaag 3840tggaaatact tatgggttag ttatttcaat gtttacggca aaatctgaac gctataaaca 3900attattaaaa ttaggtgcaa ttcctagtat tttcaatatc agtgaaccat tactttttgg 3960tcttccaatg atgttaaatc ctcttttctt tattcctttg gttttccaac cagcaatttt 4020aggaactgta gcattgggct tggcaaagat attatatatt acaaatctga atccaatgac 4080ggcacttctt ccttggacga caccagcacc tgtgagaatg gccatttcag gtggacttcc 4140atttttgatt atttttgcaa tctgtttagt cttgaatgtt cttatttact acccattctt 4200taaggtggcg tataataaag ctttagaaga agaaaaagca gctgttgaat tagagggttc 4260agaaactgcc tgatggatat tttttataaa tctggtttga acaaattata ttgacatctc 4320tttttctatc ctgataattc tgagaggtta ttttgggaaa tactattgaa ccatatcgag 4380gtggtgtggt ataatgaagg gaattaaaaa agataggaaa atttcgatta agtcatctta 4440cctcttttat tagttttttc ttataatcta atgataacat ttttataatt aatctataaa 4500ccatatccct ctttggaatc aaaatttatt atctactcct ttgtagatat gttataatac 4560aagtatcaga tctgggagac cacaacggtt tcccactaga aataattttg tttaacttta 4620gaaaggagat atacgcatga aaaaaaagat tatctcagct attttaatgt ctacagtcat 4680actttctgct gcagccccgt tgtcaggtgt ttacgcctca gctggtcaag gtactcaatc 4740agaaaactca tgtactcact ttccaggtaa cttgccaaac atgcttcgtg atttgcgtga 4800tgctttttca cgtgttaaaa ctttttttca aatgaaagat caacttgata acttgctttt 4860gaaagaatca cttttggaag attttaaagg ttaccttggt tgtcaagctt tgtcagaaat 4920gatccaattt taccttgaag aagttatgcc acaagctgaa aaccaagatc cagatatcaa 4980agctcacgtt aactcattgg gtgaaaacct taaaactttg cgtcttcgtt tgcgtcgttg 5040tcaccgtttt cttccatgtg aaaacaaatc aaaagctgtt gaacaagtta aaaacgcttt 5100taacaaattg caagaaaaag gtatctacaa agctatgtca gaatttgata tctttatcaa 5160ctacatcgaa gcttacatga ctatgaaaat ccgtaactaa ctagaattaa tctataagtt 5220actgacaaaa ctgtcagtaa ctttttttgt gggaaaaatg tatttttatg accgtaaaga 5280atctgtcagt agaagtctga aattcgttta aaaatcgact agaataggct ttaacgacaa 5340gatgttttaa agagtacgct ctaaatgtat ttttgtattt ttgtttgatt acgaagttta 5400aatttaattg acaaatgttt taaaatgagt ataataggac ttgtaaccga ttttattttt 5460ataaaggaga aagaaagatg aacaaacttt tacttggaac agcctttata ggggctagct 5520tactgattgg tgggggtgct catgcagatc aaatgtttat cgtttgtata atcataatac 5580tggtgagcac tctatacaac tagtgggaca ccaaaagaat gctaatgtaa gtgcgggttg 5640gacttatgaa ggtgtcggtt ggatcgcacc aacaacaagt tcaagcccag tttaccgtgt 5700gtacaatcca aatgcattat tacacaaaaa gcaagtatga agcccaaagt ttagtaaata 5760agggttggaa atgggataat aacggaaagg cggtcttcta ttctggaggt tctcaagccg 5820tatatgtcgc ttataatccc aatgcacaat ctggcgctca caattacacg gaaagtagct 5880ttgagcaaaa tagcttattg aatactggtt ggaaatatgg ggcagtagct tggtacggga 5940ttggagtaaa aaacgaaatg ttaaacattg ctcaaattgt tagtggtaat ttttctagta 6000ttgttggaac ttggaaagat acttctggaa atatgcttga aattaatgca atgggaaatc 6060ttactttaat atggaaaggg gcaaagaatc aaacctttga acttggcgca ggtcaacaat 6120ttaatggaac tgcagatatt gccttaaaaa atggagagat ttcccctggt agtccactta 6180acatttttgt tgtaccaaca gaagttgctt tccctaataa taaaaaagta gacgattcaa 6240ctgggcaaca acgaattttt gtgaattatt ctggtacaag ccctcaaatg gcgaatagta 6300tggcagcggt ggcttttttt agagttattc catgattata ttaaagttag aattgaataa 6360aatgtattat taaaaagata atattatatc acgacaaggc gacatctatc aactttacca 6420ctggtatgga agtgaccatt attacatcag gaaacgctaa aacggttgtt tttacacccg 6480taaaataaat aataaaataa tgtgaaatta ctgacagcat tttgtcagta atttttttta 6540tcaaaatcac acaaaaatgt tcgttgacga acaaaaaaaa ctatgttata ataattcgta 6600tgcgaactaa aaaagaagcg attggccgac ttttaaaagt agccagcaac caaatgtctc 6660gagaatttga taattttgca gctcaacttg atttgacagg tcagcaaatg tcaattttag 6720attttcttgg aaatcaaagc gaagaaggtt caggaaaaga aattagtcag acgatgattg 6780aattagaatt taatatccga cgttcaacaa cgacggaaat tttacagcgc atggaaaagc 6840ggcttttaat taatcgaaga acaagcctga ccgatgcccg ccaaaaatca gttgaattaa 6900ctgaagaagg gaaaagatat ttacctgaaa tcagggctta tatccaagca cataataaaa 6960aagc 6964234998DNAArtificial SequencepOThy11 23aatttcatgg attaagtcat cttacctctt ttattagttt tttcttataa tctaatgata 60acatttttat aattaatcta taaaccatat ccctctttgg aatcaaaatt tattatctac 120tcctttgtag atatgttata atacaagtat cagatctggg agaccacaac ggtttcccac 180tagaaataat tttgtttaac tttagaaagg agatatacgc atgaaaaaaa agattatctc 240agctatttta atgtctacag tcatactttc tgctgcagcc ccgttgtcag gtgtttacgc 300ctcagctggt caaggtactc aatcagaaaa ctcatgtact cactttccag gtaacttgcc 360aaacatgctt cgtgatttgc gtgatgcttt ttcacgtgtt aaaacttttt ttcaaatgaa 420agatcaactt gataacttgc ttttgaaaga atcacttttg gaagatttta aaggttacct 480tggttgtcaa gctttgtcag aaatgatcca attttacctt gaagaagtta tgccacaagc 540tgaaaaccaa gatccagata tcaaagctca cgttaactca ttgggtgaaa accttaaaac 600tttgcgtctt cgtttgcgtc gttgtcaccg ttttcttcca tgtgaaaaca aatcaaaagc 660tgttgaacaa gttaaaaacg cttttaacaa attgcaagaa aaaggtatct acaaagctat 720gtcagaattt gatatcttta tcaactacat cgaagcttac atgactatga aaatccgtaa 780ctaactagaa ttaatctata agttactgac aaaactgtca gtaacttttt ttgtgggaaa 840aatgtatttt tatgaccgta aagaatctgt cagtagaagt ctgaaattcg tttaaaaatc 900gactagaata ggctttaacg acaagatgtt ttaaagagta cgctctaaat gtatttttgt 960atttttgttt gattacgaag tttaaattta attgacaaat gttttaaaat gagtataata 1020ggacttgtaa ccgattttat ttttataaag gagaaagaaa gatgaacaaa cttttacttg 1080gaacagcctt tataggggct agcttactga ttggtggggg tgctcatgca gatcaaatgt 1140ttatcgtttg tataatcata atactggtga gcactctata caactagtgg gacaccaaaa 1200gaatgctaat gtaagtgcgg gttggactta tgaaggtgtc ggttggatcg caccaacaac 1260aagttcaagc ccagtttacc gtgtgtacaa tccaaatgca ttattacaca aaaagcaagt 1320atgaagccca aagtttagta aataagggtt ggaaatggga taataacgga aaggcggtct 1380tctattctgg aggttctcaa gccgtatatg tcgcttataa tcccaatgca caatctggcg 1440ctcacaatta cacggaaagt agctttgagc aaaatagctt attgaatact ggttggaaat 1500atggggcagt agcttggtac gggattggag taaaaaacga aatgttaaac attgctcaaa 1560ttgttagtgg taatttttct agtattgttg gaacttggaa agatacttct ggaaatatgc 1620ttgaaattaa tgcaatggga aatcttactt taatatggaa aggggcaaag aatcaaacct 1680ttgaacttgg cgcaggtcaa caatttaatg gaactgcaga tattgcctta aaaaatggag 1740agatttcccc tggtagtcca cttaacattt ttgttgtacc aacagaagtg aattcactgg 1800ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 1860cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 1920cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc 1980atctgtgcgg tatttcacac cgcatatggt gcactagaac tagcgattct gaaatcacca 2040tttaaaaaac tccaatcaaa taattttata aagttagtgt atcactttgt aatcataaaa 2100acaacaataa agctacttaa atatagattt ataaaaaacg ttggcgaaaa cgttggcgat 2160tcgttggcga ttgaaaaacc ccttaaaccc ttgagccagt tgggatagag cgtttttggc 2220acaaaaattg gcactcggca cttaatgggg ggtcgtagta cggaagcaaa attcgcttcc 2280tttcccccca tttttttcca aattccaaat ttttttcaaa aattttccag cgctaccgct 2340cggcaaaatt gcaagcaatt tttaaaatca aacccatgag ggaatttcat tccctcatac 2400tcccttgagc ctcctccaac cgaaatagaa gggcgctgcg cttattattt cattcagtca 2460tcggctttca taatctaaca gacaacatct tcgctgcaaa gccacgctac gctcaagggc 2520ttttacgcta cgataacgcc tgttttaacg attatgccga taactaaacg aaataaacgc 2580taaaacgtct cagaaacgat tttgagacgt tttaataaaa aatcgctagt ccgaggcctc 2640gacccgattc acaaaaaata ggcacacgaa aaacaagtta agggatgcag tttatgcatc 2700ccttaactta cttattaaat aatttatagc tattgaaaag agataagaat tgttcaaagc 2760taatattgtt taaatcgtca attcctgcat gttttaagga attgttaaat tgattttttg 2820taaatatttt cttgtattct ttgttaaccc atttcataac gaaataatta tacttttgtt 2880tatctttgtg tgatattctt gatttttttc tacttaatct gataagtgag ctattcactt 2940taggtttagg atgaaaatat tctcttggaa ccatacttaa tatagaaata tcaacttctg 3000ccattaaaag taatgccaat gagcgttttg tatttaataa tcttttagca aacccgtatt 3060ccacgattaa ataaatctca ttagctatac tatcaaaaac aattttgcgt attatatccg 3120tacttatgtt ataaggtata ttaccatata ttttatagga ttggttttta ggaaatttaa 3180actgcaatat atccttgttt aaaacttgga aattatcgtg atcaacaagt ttattttctg 3240tagttttgca taatttatgg tctatttcaa tggcagttac gaaattacac ctctttacta 3300attcaagggt aaaatggcct tttcctgagc cgatttcaaa gatattatca tgttcattta 3360atcttatatt tgtcattatt ttatctatat tatgttttga agtaataaag ttttgactgt 3420gttttatatt tttctcgttc attataaccc tctttaattt ggttatatga attttgctta 3480ttaacgattc attataacca cttatttttt gtttggttga taatgaactg tgctgattac 3540aaaaatacta aaaatgccca tattttttcc tccttataaa attagtataa ttatagcacg 3600ggtcgagatc catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 3660cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 3720gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 3780attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 3840ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 3900gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 3960attacgccaa gcttgcatgc ctgcaggtcg actctagagg atcctatata caattgagca 4020aaagaaattt agttattaaa ttaccagctg gagttcctcc aatggttgta gattcactaa 4080gtccagcaat tatttcaatg gtgattttct gtttgatgtt cgggattcgt gtgggattct 4140cttatacgcc attccatgat attttcaatt tctcaacaca actaattcaa gcaccgttga 4200ctggtgctgt ggcaaatcca tgggttctta tgggcatctt tacctttggt aatttcttat 4260ggttctttgg tatccaccct aatttaattg ggggaatttt aaatccattg ttattaacaa 4320tgtcatatgc taatattgat gcctatgctg ccggaaaacc tgtaccatac ttacaaatga 4380tgattgtgtt tgctgtgggt gcgaacgcat ggggcggaag tggaaatact tatgggttag 4440ttatttcaat gtttacggca aaatctgaac gctataaaca attattaaaa ttaggtgcaa 4500ttcctagtat tttcaatatc agtgaaccat tactttttgg tcttccaatg atgttaaatc 4560ctcttttctt tattcctttg gttttccaac cagcaatttt aggaactgta gcattgggct 4620tggcaaagat attatatatt acaaatctga atccaatgac ggcacttctt ccttggacga 4680caccagcacc tgtgagaatg gccatttcag gtggacttcc atttttgatt atttttgcaa 4740tctgtttagt cttgaatgtt cttatttact acccattctt taaggtggcg tataataaag 4800ctttagaaga agaaaaagca gctgttgaat tagagggttc agaaactgcc tgatggatat 4860tttttataaa tctggtttga acaaattata ttgacatctc tttttctatc ctgataattc 4920tgagaggtta ttttgggaaa tactattgaa ccatatcgag gtgtgtggta taatgaaggg 4980aattaaaaaa gataggaa 4998244784DNAArtificial SequencepOThy12 24gggttcagaa actgcctgat ggatattttt tataaatctg gtttgaacaa attatattga 60catctctttt tctatcctga taattctgag aggttatttt gggaaatact attgaaccat 120atcgaggtgt gtggtataat gaagggaatt aaaaaagata ggaaaatttc atgaaaaaaa 180agattatctc agctatttta atgtctacag tcatactttc tgctgcagcc ccgttgtcag 240gtgtttacgc ctcagctggt caaggtactc aatcagaaaa ctcatgtact cactttccag 300gtaacttgcc aaacatgctt cgtgatttgc gtgatgcttt ttcacgtgtt aaaacttttt 360ttcaaatgaa agatcaactt gataacttgc ttttgaaaga atcacttttg gaagatttta 420aaggttacct tggttgtcaa gctttgtcag aaatgatcca attttacctt gaagaagtta 480tgccacaagc tgaaaaccaa gatccagata tcaaagctca cgttaactca ttgggtgaaa 540accttaaaac tttgcgtctt cgtttgcgtc gttgtcaccg ttttcttcca tgtgaaaaca 600aatcaaaagc tgttgaacaa gttaaaaacg cttttaacaa attgcaagaa aaaggtatct 660acaaagctat gtcagaattt gatatcttta tcaactacat cgaagcttac atgactatga 720aaatccgtaa ctaactagaa ttaatctata agttactgac aaaactgtca gtaacttttt 780ttgtgggaaa aatgtatttt tatgaccgta aagaatctgt cagtagaagt ctgaaattcg 840tttaaaaatc gactagaata ggctttaacg acaagatgtt ttaaagagta cgctctaaat 900gtatttttgt atttttgttt gattacgaag tttaaattta attgacaaat gttttaaaat 960gagtataata

ggacttgtaa ccgattttat ttttataaag gagaaagaaa gatgaacaaa 1020cttttacttg gaacagcctt tataggggct agcttactga ttggtggggg tgctcatgca 1080gatcaaatgt ttatcgtttg tataatcata atactggtga gcactctata caactagtgg 1140gacaccaaaa gaatgctaat gtaagtgcgg gttggactta tgaaggtgtc ggttggatcg 1200caccaacaac aagttcaagc ccagtttacc gtgtgtacaa tccaaatgca ttattacaca 1260aaaagcaagt atgaagccca aagtttagta aataagggtt ggaaatggga taataacgga 1320aaggcggtct tctattctgg aggttctcaa gccgtatatg tcgcttataa tcccaatgca 1380caatctggcg ctcacaatta cacggaaagt agctttgagc aaaatagctt attgaatact 1440ggttggaaat atggggcagt agcttggtac gggattggag taaaaaacga aatgttaaac 1500attgctcaaa ttgttagtgg taatttttct agtattgttg gaacttggaa agatacttct 1560ggaaatatgc ttgaaattaa tgcaatggga aatcttactt taatatggaa aggggcaaag 1620aatcaaacct ttgaacttgg cgcaggtcaa caatttaatg gaactgcaga tattgcctta 1680aaaaatggag agatttcccc tggtagtcca cttaacattt ttgttgtacc aacagaagtg 1740aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 1800aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 1860gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt 1920ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactagaac tagcgattct 1980gaaatcacca tttaaaaaac tccaatcaaa taattttata aagttagtgt atcactttgt 2040aatcataaaa acaacaataa agctacttaa atatagattt ataaaaaacg ttggcgaaaa 2100cgttggcgat tcgttggcga ttgaaaaacc ccttaaaccc ttgagccagt tgggatagag 2160cgtttttggc acaaaaattg gcactcggca cttaatgggg ggtcgtagta cggaagcaaa 2220attcgcttcc tttcccccca tttttttcca aattccaaat ttttttcaaa aattttccag 2280cgctaccgct cggcaaaatt gcaagcaatt tttaaaatca aacccatgag ggaatttcat 2340tccctcatac tcccttgagc ctcctccaac cgaaatagaa gggcgctgcg cttattattt 2400cattcagtca tcggctttca taatctaaca gacaacatct tcgctgcaaa gccacgctac 2460gctcaagggc ttttacgcta cgataacgcc tgttttaacg attatgccga taactaaacg 2520aaataaacgc taaaacgtct cagaaacgat tttgagacgt tttaataaaa aatcgctagt 2580ccgaggcctc gacccgattc acaaaaaata ggcacacgaa aaacaagtta agggatgcag 2640tttatgcatc ccttaactta cttattaaat aatttatagc tattgaaaag agataagaat 2700tgttcaaagc taatattgtt taaatcgtca attcctgcat gttttaagga attgttaaat 2760tgattttttg taaatatttt cttgtattct ttgttaaccc atttcataac gaaataatta 2820tacttttgtt tatctttgtg tgatattctt gatttttttc tacttaatct gataagtgag 2880ctattcactt taggtttagg atgaaaatat tctcttggaa ccatacttaa tatagaaata 2940tcaacttctg ccattaaaag taatgccaat gagcgttttg tatttaataa tcttttagca 3000aacccgtatt ccacgattaa ataaatctca ttagctatac tatcaaaaac aattttgcgt 3060attatatccg tacttatgtt ataaggtata ttaccatata ttttatagga ttggttttta 3120ggaaatttaa actgcaatat atccttgttt aaaacttgga aattatcgtg atcaacaagt 3180ttattttctg tagttttgca taatttatgg tctatttcaa tggcagttac gaaattacac 3240ctctttacta attcaagggt aaaatggcct tttcctgagc cgatttcaaa gatattatca 3300tgttcattta atcttatatt tgtcattatt ttatctatat tatgttttga agtaataaag 3360ttttgactgt gttttatatt tttctcgttc attataaccc tctttaattt ggttatatga 3420attttgctta ttaacgattc attataacca cttatttttt gtttggttga taatgaactg 3480tgctgattac aaaaatacta aaaatgccca tattttttcc tccttataaa attagtataa 3540ttatagcacg ggtcgagatc catgttcttt cctgcgttat cccctgattc tgtggataac 3600cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 3660gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 3720tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 3780cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 3840cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 3900tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg actctagagg atcctatata 3960caattgagca aaagaaattt agttattaaa ttaccagctg gagttcctcc aatggttgta 4020gattcactaa gtccagcaat tatttcaatg gtgattttct gtttgatgtt cgggattcgt 4080gtgggattct cttatacgcc attccatgat attttcaatt tctcaacaca actaattcaa 4140gcaccgttga ctggtgctgt ggcaaatcca tgggttctta tgggcatctt tacctttggt 4200aatttcttat ggttctttgg tatccaccct aatttaattg ggggaatttt aaatccattg 4260ttattaacaa tgtcatatgc taatattgat gcctatgctg ccggaaaacc tgtaccatac 4320ttacaaatga tgattgtgtt tgctgtgggt gcgaacgcat ggggcggaag tggaaatact 4380tatgggttag ttatttcaat gtttacggca aaatctgaac gctataaaca attattaaaa 4440ttaggtgcaa ttcctagtat tttcaatatc agtgaaccat tactttttgg tcttccaatg 4500atgttaaatc ctcttttctt tattcctttg gttttccaac cagcaatttt aggaactgta 4560gcattgggct tggcaaagat attatatatt acaaatctga atccaatgac ggcacttctt 4620ccttggacga caccagcacc tgtgagaatg gccatttcag gtggacttcc atttttgatt 4680atttttgcaa tctgtttagt cttgaatgtt cttatttact acccattctt taaggtggcg 4740tataataaag ctttagaaga agaaaaagca gctgttgaat taga 4784254936DNAArtificial SequencepOThy15 25gattaagtca tcttacctct tttattagtt ttttcttata atctaatgat aacattttta 60taattaatct ataaaccata tccctctttg gaatcaaaat ttattatcta ctcctttgta 120gatatgttat aatacaagta tcagatctgg gagaccacaa cggtttccca ctagaaataa 180ttttgtttaa ctttagaaag gagatatacg catgaaaaaa aagattatct cagctatttt 240aatgtctaca gtcatacttt ctgctgcagc cccgttgtca ggtgtttacg cctcagctgg 300tcaaggtact caatcagaaa actcatgtac tcactttcca ggtaacttgc caaacatgct 360tcgtgatttg cgtgatgctt tttcacgtgt taaaactttt tttcaaatga aagatcaact 420tgataacttg cttttgaaag aatcactttt ggaagatttt aaaggttacc ttggttgtca 480agctttgtca gaaatgatcc aattttacct tgaagaagtt atgccacaag ctgaaaacca 540agatccagat atcaaagctc acgttaactc attgggtgaa aaccttaaaa ctttgcgtct 600tcgtttgcgt cgttgtcacc gttttcttcc atgtgaaaac aaatcaaaag ctgttgaaca 660agttaaaaac gcttttaaca aattgcaaga aaaaggtatc tacaaagcta tgtcagaatt 720tgatatcttt atcaactaca tcgaagctta catgactatg aaaatccgta actaactaga 780attaatctat aagttactga caaaactgtc agtaactttt tttgtgggaa aaatgtattt 840ttatgaccgt aaagaatctg tcagtagaag tctgaaattc gtttaaaaat cgactagaat 900aggctttaac gacaagatgt tttaaagagt acgctctaaa tgtatttttg tatttttgtt 960tgattacgaa gtttaaattt aattgacaaa tgttttaaaa tgagtataat aggacttgta 1020accgatttta tttttataaa ggagaaagaa agatgaacaa acttttactt ggaacagcct 1080ttataggggc tagcttactg attggtgggg gtgctcatgc agatcaaatg tttatcgttt 1140gtataatcat aatactggtg agcactctat acaactagtg ggacaccaaa agaatgctaa 1200tgtaagtgcg ggttggactt atgaaggtgt cggttggatc gcaccaacaa caagttcaag 1260cccagtttac cgtgtgtaca atccaaatgc attattacac aaaaagcaag tatgaagccc 1320aaagtttagt aaataagggt tggaaatggg ataataacgg aaaggcggtc ttctattctg 1380gaggttctca agccgtatat gtcgcttata atcccaatgc acaatctggc gctcacaatt 1440acacggaaag tagctttgag caaaatagct tattgaatac tggttggaaa tatggggcag 1500tagcttggta cgggattgga gtaaaaaacg aaatgttaaa cattgctcaa attgttagtg 1560gtaatttttc tagtattgtt ggaacttgga aagatacttc tggaaatatg cttgaaatta 1620atgcaatggg aaatcttact ttaatatgga aaggggcaaa gaatcaaacc tttgaacttg 1680gcgcaggtca acaatttaat ggaactgcag atattgcctt aaaaaatgga gagatttccc 1740ctggtagtcc acttaacatt tttgttgtac caacagaagt gaattcactg gccgtcgttt 1800tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 1860cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 1920tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 1980gtatttcaca ccgcatatgg tgcactagaa ctagcgattc tgaaatcacc atttaaaaaa 2040ctccaatcaa ataattttat aaagttagtg tatcactttg taatcataaa aacaacaata 2100aagctactta aatatagatt tataaaaaac gttggcgaaa acgttggcga ttcgttggcg 2160attgaaaaac cccttaaacc cttgagccag ttgggataga gcgtttttgg cacaaaaatt 2220ggcactcggc acttaatggg gggtcgtagt acggaagcaa aattcgcttc ctttcccccc 2280atttttttcc aaattccaaa tttttttcaa aaattttcca gcgctaccgc tcggcaaaat 2340tgcaagcaat ttttaaaatc aaacccatga gggaatttca ttccctcata ctcccttgag 2400cctcctccaa ccgaaataga agggcgctgc gcttattatt tcattcagtc atcggctttc 2460ataatctaac agacaacatc ttcgctgcaa agccacgcta cgctcaaggg cttttacgct 2520acgataacgc ctgttttaac gattatgccg ataactaaac gaaataaacg ctaaaacgtc 2580tcagaaacga ttttgagacg ttttaataaa aaatcgctag tccgaggcct cgacccgatt 2640cacaaaaaat aggcacacga aaaacaagtt aagggatgca gtttatgcat cccttaactt 2700acttattaaa taatttatag ctattgaaaa gagataagaa ttgttcaaag ctaatattgt 2760ttaaatcgtc aattcctgca tgttttaagg aattgttaaa ttgatttttt gtaaatattt 2820tcttgtattc tttgttaacc catttcataa cgaaataatt atacttttgt ttatctttgt 2880gtgatattct tgattttttt ctacttaatc tgataagtga gctattcact ttaggtttag 2940gatgaaaata ttctcttgga accatactta atatagaaat atcaacttct gccattaaaa 3000gtaatgccaa tgagcgtttt gtatttaata atcttttagc aaacccgtat tccacgatta 3060aataaatctc attagctata ctatcaaaaa caattttgcg tattatatcc gtacttatgt 3120tataaggtat attaccatat attttatagg attggttttt aggaaattta aactgcaata 3180tatccttgtt taaaacttgg aaattatcgt gatcaacaag tttattttct gtagttttgc 3240ataatttatg gtctatttca atggcagtta cgaaattaca cctctttact aattcaaggg 3300taaaatggcc ttttcctgag ccgatttcaa agatattatc atgttcattt aatcttatat 3360ttgtcattat tttatctata ttatgttttg aagtaataaa gttttgactg tgttttatat 3420ttttctcgtt cattataacc ctctttaatt tggttatatg aattttgctt attaacgatt 3480cattataacc acttattttt tgtttggttg ataatgaact gtgctgatta caaaaatact 3540aaaaatgccc atattttttc ctccttataa aattagtata attatagcac gggtcgagat 3600ccatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 3660gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 3720cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 3780gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 3840gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 3900gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 3960agcttgcatg cctgcaggtc gactctagag gatcctatat acaattgagc aaaagaaatt 4020tagttattaa attaccagct ggagttcctc caatggttgt agattcacta agtccagcaa 4080ttatttcaat ggtgattttc tgtttgatgt tcgggattcg tgtgggattc tcttatacgc 4140cattccatga tattttcaat ttctcaacac aactaattca agcaccgttg actggtgctg 4200tggcaaatcc atgggttctt atgggcatct ttacctttgg taatttctta tggttctttg 4260gtatccaccc taatttaatt gggggaattt taaatccatt gttattaaca atgtcatatg 4320ctaatattga tgcctatgct gccggaaaac ctgtaccata cttacaaatg atgattgtgt 4380ttgctgtggg tgcgaacgca tggggcggaa gtggaaatac ttatgggtta gttatttcaa 4440tgtttacggc aaaatctgaa cgctataaac aattattaaa attaggtgca attcctagta 4500ttttcaatat cagtgaacca ttactttttg gtcttccaat gatgttaaat cctcttttct 4560ttattccttt ggttttccaa ccagcaattt taggaactgt agcattgggc ttggcaaaga 4620tattatatat tacaaatctg aatccaatga cggcacttct tccttggacg acaccagcac 4680ctgtgagaat ggccatttca ggtggacttc catttttgat tatttttgca atctgtttag 4740tcttgaatgt tcttatttac tacccattct ttaaggtggc gtataataaa gctttagaag 4800aagaaaaagc agctgttgaa ttagagggtt cagaaactgc ctgatggata ttttttataa 4860atctggtttg aacaaattat attgacatct ctttttctat cctgataatt ctgagaggtt 4920attttgggaa atacta 4936264995DNAArtificial SequencepOThy16 26aatttcgatt aagtcatctt acctctttta ttagtttttt cttataatct aatgataaca 60tttttataat taatctataa accatatccc tctttggaat caaaatttat tatctactcc 120tttgtagata tgttataata caagtatcag atctgggaga ccacaacggt ttcccactag 180aaataatttt gtttaacttt agaaaggaga tatacgcatg aaaaaaaaga ttatctcagc 240tattttaatg tctacagtca tactttctgc tgcagccccg ttgtcaggtg tttacgcctc 300agctggtcaa ggtactcaat cagaaaactc atgtactcac tttccaggta acttgccaaa 360catgcttcgt gatttgcgtg atgctttttc acgtgttaaa actttttttc aaatgaaaga 420tcaacttgat aacttgcttt tgaaagaatc acttttggaa gattttaaag gttaccttgg 480ttgtcaagct ttgtcagaaa tgatccaatt ttaccttgaa gaagttatgc cacaagctga 540aaaccaagat ccagatatca aagctcacgt taactcattg ggtgaaaacc ttaaaacttt 600gcgtcttcgt ttgcgtcgtt gtcaccgttt tcttccatgt gaaaacaaat caaaagctgt 660tgaacaagtt aaaaacgctt ttaacaaatt gcaagaaaaa ggtatctaca aagctatgtc 720agaatttgat atctttatca actacatcga agcttacatg actatgaaaa tccgtaacta 780actagaatta atctataagt tactgacaaa actgtcagta actttttttg tgggaaaaat 840gtatttttat gaccgtaaag aatctgtcag tagaagtctg aaattcgttt aaaaatcgac 900tagaataggc tttaacgaca agatgtttta aagagtacgc tctaaatgta tttttgtatt 960tttgtttgat tacgaagttt aaatttaatt gacaaatgtt ttaaaatgag tataatagga 1020cttgtaaccg attttatttt tataaaggag aaagaaagat gaacaaactt ttacttggaa 1080cagcctttat aggggctagc ttactgattg gtgggggtgc tcatgcagat caaatgttta 1140tcgtttgtat aatcataata ctggtgagca ctctatacaa ctagtgggac accaaaagaa 1200tgctaatgta agtgcgggtt ggacttatga aggtgtcggt tggatcgcac caacaacaag 1260ttcaagccca gtttaccgtg tgtacaatcc aaatgcatta ttacacaaaa agcaagtatg 1320aagcccaaag tttagtaaat aagggttgga aatgggataa taacggaaag gcggtcttct 1380attctggagg ttctcaagcc gtatatgtcg cttataatcc caatgcacaa tctggcgctc 1440acaattacac ggaaagtagc tttgagcaaa atagcttatt gaatactggt tggaaatatg 1500gggcagtagc ttggtacggg attggagtaa aaaacgaaat gttaaacatt gctcaaattg 1560ttagtggtaa tttttctagt attgttggaa cttggaaaga tacttctgga aatatgcttg 1620aaattaatgc aatgggaaat cttactttaa tatggaaagg ggcaaagaat caaacctttg 1680aacttggcgc aggtcaacaa tttaatggaa ctgcagatat tgccttaaaa aatggagaga 1740tttcccctgg tagtccactt aacatttttg ttgtaccaac agaagtgaat tcactggccg 1800tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 1860cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 1920aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc 1980tgtgcggtat ttcacaccgc atatggtgca ctagaactag cgattctgaa atcaccattt 2040aaaaaactcc aatcaaataa ttttataaag ttagtgtatc actttgtaat cataaaaaca 2100acaataaagc tacttaaata tagatttata aaaaacgttg gcgaaaacgt tggcgattcg 2160ttggcgattg aaaaacccct taaacccttg agccagttgg gatagagcgt ttttggcaca 2220aaaattggca ctcggcactt aatggggggt cgtagtacgg aagcaaaatt cgcttccttt 2280ccccccattt ttttccaaat tccaaatttt tttcaaaaat tttccagcgc taccgctcgg 2340caaaattgca agcaattttt aaaatcaaac ccatgaggga atttcattcc ctcatactcc 2400cttgagcctc ctccaaccga aatagaaggg cgctgcgctt attatttcat tcagtcatcg 2460gctttcataa tctaacagac aacatcttcg ctgcaaagcc acgctacgct caagggcttt 2520tacgctacga taacgcctgt tttaacgatt atgccgataa ctaaacgaaa taaacgctaa 2580aacgtctcag aaacgatttt gagacgtttt aataaaaaat cgctagtccg aggcctcgac 2640ccgattcaca aaaaataggc acacgaaaaa caagttaagg gatgcagttt atgcatccct 2700taacttactt attaaataat ttatagctat tgaaaagaga taagaattgt tcaaagctaa 2760tattgtttaa atcgtcaatt cctgcatgtt ttaaggaatt gttaaattga ttttttgtaa 2820atattttctt gtattctttg ttaacccatt tcataacgaa ataattatac ttttgtttat 2880ctttgtgtga tattcttgat ttttttctac ttaatctgat aagtgagcta ttcactttag 2940gtttaggatg aaaatattct cttggaacca tacttaatat agaaatatca acttctgcca 3000ttaaaagtaa tgccaatgag cgttttgtat ttaataatct tttagcaaac ccgtattcca 3060cgattaaata aatctcatta gctatactat caaaaacaat tttgcgtatt atatccgtac 3120ttatgttata aggtatatta ccatatattt tataggattg gtttttagga aatttaaact 3180gcaatatatc cttgtttaaa acttggaaat tatcgtgatc aacaagttta ttttctgtag 3240ttttgcataa tttatggtct atttcaatgg cagttacgaa attacacctc tttactaatt 3300caagggtaaa atggcctttt cctgagccga tttcaaagat attatcatgt tcatttaatc 3360ttatatttgt cattatttta tctatattat gttttgaagt aataaagttt tgactgtgtt 3420ttatattttt ctcgttcatt ataaccctct ttaatttggt tatatgaatt ttgcttatta 3480acgattcatt ataaccactt attttttgtt tggttgataa tgaactgtgc tgattacaaa 3540aatactaaaa atgcccatat tttttcctcc ttataaaatt agtataatta tagcacgggt 3600cgagatccat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 3660ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 3720aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 3780aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 3840atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 3900tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 3960acgccaagct tgcatgcctg caggtcgact ctagaggatc ctatatacaa ttgagcaaaa 4020gaaatttagt tattaaatta ccagctggag ttcctccaat ggttgtagat tcactaagtc 4080cagcaattat ttcaatggtg attttctgtt tgatgttcgg gattcgtgtg ggattctctt 4140atacgccatt ccatgatatt ttcaatttct caacacaact aattcaagca ccgttgactg 4200gtgctgtggc aaatccatgg gttcttatgg gcatctttac ctttggtaat ttcttatggt 4260tctttggtat ccaccctaat ttaattgggg gaattttaaa tccattgtta ttaacaatgt 4320catatgctaa tattgatgcc tatgctgccg gaaaacctgt accatactta caaatgatga 4380ttgtgtttgc tgtgggtgcg aacgcatggg gcggaagtgg aaatacttat gggttagtta 4440tttcaatgtt tacggcaaaa tctgaacgct ataaacaatt attaaaatta ggtgcaattc 4500ctagtatttt caatatcagt gaaccattac tttttggtct tccaatgatg ttaaatcctc 4560ttttctttat tcctttggtt ttccaaccag caattttagg aactgtagca ttgggcttgg 4620caaagatatt atatattaca aatctgaatc caatgacggc acttcttcct tggacgacac 4680cagcacctgt gagaatggcc atttcaggtg gacttccatt tttgattatt tttgcaatct 4740gtttagtctt gaatgttctt atttactacc cattctttaa ggtggcgtat aataaagctt 4800tagaagaaga aaaagcagct gttgaattag agggttcaga aactgcctga tggatatttt 4860ttataaatct ggtttgaaca aattatattg acatctcttt ttctatcctg ataattctga 4920gaggttattt tgggaaatac tattgaacca tatcgaggtg tgtggtataa tgaagggaat 4980taaaaaagat aggaa 49952739DNAArtificial Sequencelactococcal P1 promoter expression unit 27agataggaaa atttcatgac ttacgcagat caagttttt 392836DNAArtificial Sequencelactococcal P1 promoter with no 5' ATG 28agataggaaa atttcactta cgcagatcaa gttttt 362989DNAArtificial SequencethyA promoter not included 29tctgagaggt tattttggga aatactattg aaccatatcg aggtgtgtgg tataatgaag 60ggaattaaaa aagataggaa aatttcatg 893020DNAArtificial Sequence3' SpeI restriction site 30aaaatccgta actaactagt 203135DNAArtificial Sequencestop codon of usp45-hIL-10 sequence 31gatttagcaa tttaaattaa attaatctat aagtt 353226DNAArtificial Sequenceengineered XbaI restriction site 32tctagaatta atctataagt tactga 26

Claims

1-20. (canceled)

21. An isolated thymidylate synthase (thyA) mutant of Lactococcus species comprising an inactive thymidylate synthase gene and a gene or expression unit encoding a heterologous prophylactic and/or therapeutic molecule.

22. The isolated thyA mutant of Lactococcus species according to claim 21, wherein the inactive thymidylate synthase gene has been inactivated by gene disruption.

23. The isolated thyA mutant of Lactococcus species according to claim 22, wherein the inactive thymidylate synthase gene has been disrupted by the gene or expression unit encoding the heterologous prophylactic and/or therapeutic molecule.

24. The isolated thyA mutant of Lactococcus species according to claim 21, wherein said Lactococcus species is Lactococcus lactis.

25. The isolated thyA mutant of Lactococcus species according to claim 21, wherein said isolated thyA mutant is transformed with a transforming plasmid, wherein said transforming plasmid does not comprise an intact thymidylate synthase gene.

26. The isolated thyA mutant of Lactococcus species according to claim 21, wherein the heterologous prophylactic and/or therapeutic molecule is interleukin-10.

27. A pharmaceutical composition comprising an isolated thymidylate synthase (thyA) mutant of Lactococcus species, said isolated thyA mutant comprising an inactive thymidylate synthase gene and a gene or expression unit encoding a heterologous prophylactic and/or therapeutic molecule.

28. The pharmaceutical composition according to claim 27, wherein the inactive thymidylate synthase gene has been inactivated by gene disruption.

29. The pharmaceutical composition according to claim 28, wherein the inactive thymidylate synthase gene has been disrupted by the gene or expression unit encoding the heterologous prophylactic and/or therapeutic molecule.

30. The pharmaceutical composition according to claim 27, wherein said Lactococcus species is Lactococcus lactis.

31. The pharmaceutical composition according to claim 27, wherein said isolated thyA mutant is transformed with a transforming plasmid, wherein said transforming plasmid does not comprise an intact thymidylate synthase gene.

32. The pharmaceutical composition according to claim 27, wherein the heterologous prophylactic and/or therapeutic molecule is interleukin-10.

33. A method for delivering a prophylactic and/or therapeutic molecule to a subject, the method comprising administering to the subject an isolated thymidylate synthase (thyA) mutant of Lactococcus species comprising an inactive thymidylate synthase gene and a gene or expression unit encoding the heterologous prophylactic and/or therapeutic molecule.

34. The method according to claim 33, wherein the inactive thymidylate synthase gene has been inactivated by gene disruption.

35. The method according to claim 34, wherein the inactive thymidylate synthase gene has been disrupted by the gene or expression unit encoding the heterologous prophylactic and/or therapeutic molecule.

36. The method according to claim 33, wherein said Lactococcus species is Lactococcus lactis.

37. The method according to claim 33, wherein said isolated thyA mutant is transformed with a transforming plasmid, wherein said transforming plasmid does not comprise an intact thymidylate synthase gene.

38. The method according to claim 33, wherein the heterologous prophylactic and/or therapeutic molecule is interleukin-10.

39. A method for treating inflammatory bowel disease in a subject, the method comprising administering to the subject an isolated thymidylate synthase (thyA) mutant of Lactococcus species comprising an inactive thymidylate synthase gene and a gene or expression unit encoding the heterologous prophylactic and/or therapeutic molecule.

40. The method according to claim 39, wherein the inactive thymidylate synthase gene has been inactivated by gene disruption.

41. The method according to claim 40, wherein the inactive thymidylate synthase gene has been disrupted by the gene or expression unit encoding the heterologous prophylactic and/or therapeutic molecule.

42. The method according to claim 39, wherein said Lactococcus species is Lactococcus lactis.

43. The method according to claim 39, wherein said isolated thyA mutant is transformed with a transforming plasmid, wherein said transforming plasmid does not comprise an intact thymidylate synthase gene.

44. The method according to claim 39, wherein the heterologous prophylactic and/or therapeutic molecule is interleukin-10.

Images