# Patent

## Title
Biosynthetic system that produces immunogenic polysaccharides in prokaryotic cells

## Abstract

The invention is directed to bioconjugate vaccines comprising N-glycosylated proteins. Further, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising nucleic acids encoding an epimerase that synthesizes an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus. The invention is further directed to N-glycosylated proteins containing an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus and an expression system and methods for producing such N-glycosylated proteins.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/272,931, filed Nov. 19, 2009, herein incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to the use of a biosynthetic system and proteins for preparing a vaccine. In addition, the invention relates to a recombinant prokaryotic biosynthetic system having an epimerase that initiates the synthesis of an oligo- or polysaccharide with a specified monosaccharide at the reducing terminus. The invention further relates to N-glycosylated proteins produced with glycans in an expression system and bioconjugate vaccines made from said N-glycosylated proteins comprising immunogenic glycans, and provides methods for producing N-glycosylated proteins.
BACKGROUND OF THE INVENTION
Glycoproteins are proteins that have one or more covalently attached sugar polymers. N-linked protein glycosylation is an essential and conserved process occurring in the endoplasmic reticulum of eukaryotic organisms. It is important for protein folding, oligomerization, stability, quality control, sorting and transport of secretory and membrane proteins (Helenius. A., and Aebi, M. (2004). Roles of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019-1049).
Protein glycosylation has a profound influence on the immunogenicity, the stability and the half-life of a protein. In addition, glycosylation can assist the purification of proteins by chromatography, e.g. affinity chromatography with lectin ligands bound to a solid phase interacting with glycosylated moieties of the protein. It is therefore established practice to produce many glycosylated proteins recombinantly in eukaryotic cells to provide biologically and pharmaceutically useful glycosylation patterns.
WO 200307467 (Aebi et al.) demonstrated that the food-borne pathogen Campylobacter jejuni, which is a bacterium, could N-glycosylate its proteins, which was a unique feature among known prokaryotic organisms except for certain species of archaea. The machinery required for glycosylation is encoded by 12 genes that are clustered in the so-called pgl locus. Disruption of N-glycosylation affects invasion and pathogenesis of C. jejuni but is not lethal as in most eukaryotic organisms (Burda P. and M. Aebi, (1999). The dolichol pathway of N-linked glycosylation. Biochem Biophys Acta 1426(2):239-57). It is possible to reconstitute the N-glycosylation of C. jejuni proteins by recombinantly expressing the pgl locus and acceptor glycoprotein in E. coli the same time (Wacker et al. (2002). N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790-1793).
N-glycans have a glycan attached to a consensus sequence in a protein. The known N-glycosylation consensus sequence in a protein allows for the N-glycosylation of recombinant target proteins in prokaryotic organisms. Such organisms comprise an oligosaccharyl transferase (“OT”; “OTase”), such as, for example, an oligosaccharyl transferase of C. jejuni, which is an enzyme that transfers the glycan to the consensus sequence of the protein.
WO 200307467 (Aebi et al.) teaches a prokaryotic organism into which is introduced a nucleic acid encoding for (i) specific glycosyltransferases for the assembly of an oligosaccharide on a lipid carrier, (ii) a recombinant target protein comprising a consensus sequence “N-X-S/T”, wherein X can be any amino acid except proline, and (iii) an oligosaccharyl transferase, such as, for example, an oligosaccharyl transferase of C. jejuni that covalently links said oligosaccharide to the consensus sequence of the target protein. Said prokaryotic Organism produces N-glycans with a specific structure which is defined by the type of the specific glycosyltransferases.
WO 2006/119987 (Aebi et al.) describes proteins, as well as means and methods for producing proteins, with efficiency for N-glycosylation in prokaryotic organisms in vivo. It further describes an efficient introduction of N-glycans into recombinant proteins for modifying immunogenicity, stability, biological, prophylactic and/or therapeutic activity of said proteins, and the provision of a host cell that efficiently displays recombinant N-glycosylated proteins of the present invention on its surface. In addition, it describes a recombinant N-glycosylated protein comprising one or more of the following N-glycosylated optimized amino acid sequence(s):
D/E-X-N-Z-S/T (optimized consensus sequence),
wherein X and Z may be any natural amino acid except Pro, and wherein at least one of said N-glycosylated partial amino acid sequence(s) is introduced. The introduction of specific partial amino acid sequence(s) (optimized consensus sequence(s)) into proteins leads to proteins that are efficiently N-glycosylated by an oligosaccharyl transferase in these introduced positions.
The biosynthesis of different polysaccharides is conserved in bacterial cells. The polysaccharides are assembled on carrier lipids from common precursors (activated sugar nucleotides) at the cytoplasmic membrane by different glycosyltransferases with defined specificity. Lipopolysaccharides (“LPS”) are provided in gram-negative bacteria only, e.g. Shigella spp., Pseudomonas spp. and E. coli (ExPEC, EHEC).
The synthesis of LPS starts with the addition of a monosaccharide to the carrier lipid undecaprenyl phosphate (“Und-P-P”) at the cytoplasmic side of the membrane. The antigen is built up by sequential addition of monosaccharides from activated sugar nucleotides by different glycosyltransferases, and the lipid-linked polysaccharide is flipped through the membrane by a flippase. The antigen-repeating unit is polymerized by an enzymatic reaction. The polysaccharide is then transferred to the Lipid A by the Ligase WaaL forming the LPS that is exported to the surface, whereas the capsular polysaccharide is released from the carrier lipid after polymerization and exported to the surface. The biosynthetic pathway of these polysaccharides enables the production of LPS bioconjugates in vivo, capturing the polysaccharides in the periplasm to a protein carrier.
Such synthesized complexes of oligo- or polysaccharides (i.e., sugar residues) and proteins (i.e., protein carriers) can be used as conjugate vaccines to protect against a number of bacterial infections. Conjugate vaccines have been successfully used to protect against bacterial infections. The conjugation of an antigenic polysaccharide to a protein carrier is required for protective memory response, as polysaccharides are T-cell independent immunogens. Polysaccharides have been conjugated to protein carriers by different chemical methods, using activation reactive groups in the polysaccharide as well as the protein carrier.
Conjugate vaccines can be administered to children to protect against bacterial infections and also can provide a long lasting immune response to adults. Constructs of WO 2009/04074 (Fernandez, et al.) have been found to generate an IgG response in animals. It has been found that an IgG response to a Shigella O-specific polysaccharide-protein conjugate vaccine in humans correlates with immune protection in humans. (Passwell, J. H. et al., “Safety and Immunogenicity of Improved Shigella O-Specific Polysaccharide-Protein Conjugate Vaccines in Adults in Israel” Infection and Immunity, 69(3):1351-1357 (March 2001).) It is believed that the polysaccharide (i.e. sugar residues) triggers a short-term immune response that is sugar-specific. Indeed, the human immune system generates a strong response to specific polysaccharide surface structures of bacteria, such as O-antigens and capsular polysaccharides. However, since the immune response to polysaccharides is IgM dependent, the immune system develops no memory. The protein carrier that carries the polysaccharide triggers an IgG response that is T-cell dependent and that provides long lasting protection since the immune system develops memory.
E. coli O157 is an enterohemorrhagic strain responsible for approximately two-thirds of all recent cases of hemolytic-uremic syndrome and poses serious human health concerns (Law, D. (2000) J. App. Microbiol., 88, 729-745; Wang, L., and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551).
Escherichia coli strain O157 produces an O-antigen containing the repeating tetrasaccharide unit (4-N-acetyl perosamine→fucose→glucose→GalNAc) (α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc) (Perry, M. B., MacLean, L. and Griffith, D. W. (1986) Biochem. Cell. Biol., 64, 21-28). The tetrasaccharide is preassembled on undecaprenyl pyrophosphate. The E. coli cell envelope contains an inner plasma membrane, a stress-hearing peptidoglycan layer and an asymmetric outer membrane consisting of a phospholipid inner monolayer and an outer monolayer composed of bacterial LPS. LPS contains three components, the lipid A anchor, the 3-deoxy-D-manno-oct-2-ulosonic acid-containing core, and the O-antigen region (see: Raetz, C. R. H. and Whitfield, C. (2002) Annu. Rev. Biochem., 71, 635-700; Whitfield, C. (2006) Ann. Rev. Biochem. 75, 39-68; Samuel, G. and Reeves, P. R. (2003) Carbohydrate Research, 338, 2503-2519; and refs, therein for reviews on the assembly of O-antigens of bacterial LPS).
The O-antigen components of bacterial LPS are large, extremely diverse polysaccharides that can be either homopolymeric, composed of a single repeating monosaccharide, or heteropolymeric, containing 10-30 repeats of 3-6 sugar units (Reeves, P. R., Hobbs, M., Valvano, M. A., Skurnik, M., Whitfield, C., Coplin, D., Kido, N., Klena, J., Maskell, D., Raetz, C. R. H., and Rick, P. D. (1996) Trends Microbial., 4, 495-503). O-Antigens are, Thus, the Dominant Feature of the bacterial cell surface and constitute important determinants of virulence and pathogenicity (Law, D. (2000) J. App. Microbiol., 88, 729-745; Spears, K. J., Roe, A. J. and Golly, D. L. (2006) FEMS Microbiol. Lett., 255, 187-202; Liu, B., Knirel, Y. A., Feng, L., Perepelov, A. V., Senchenkova, S. N., Wang, Q., Reeves, P. R. and Wang, L (2008) FEMS Microbiol. Rev. 32, 627-653; Stenutz, R., Weintraub, A. and Widmalm, G. (2006) FEMS Microbiol. Rev. 30, 382-403). E. coli strains with more than 180 individual O-serotypes, attributed to unique O-antigen structures, have been identified (Stenutz, R., Weintraub, A. and Widmalm, G. (2006) FEMS Microbiol. Rev. 30, 382-403).
O-antigen repeat units are pre-assembled on the cytosolic face of the inner membrane attached to undecaprenyl pyrophosphate. The lipid-linked repeat units diffuse transversely (flip-flop) to the periplasmic surface of the inner membrane and are polymerized before transport to the outer membrane and ligation to LPS. Most heteropolymeric O-antigen repeat units have either N-acetylglucosamine (“GlcNAc”) or N-acetylgalactosamine (“GalNAc”) at the reducing terminus.
It had been assumed that the biosynthesis of the lipid intermediates is initiated by the transfer of GlcNAc-9 or GalNAc-P from their respective sugar nucleotide derivatives to undecaprenyl monophosphate (“Und-P”) catalyzed by WecA (Samuel, G. and Reeves, P. R. (2003) Carbohydrate Research, 338, 2503-2519; Alexander, D. C. and Valvano, M. A. (1994) J. Bacteriol., 176, 7079-7084; Zhang, L., Radziejewska-Lebrecht, J., Krajewska-Pietrasik, D., Tolvanen, P. and Skurkik. M. (1997) Mol. Microbiol. 23, 63-76; Amor, P. A. and Whitfield, C. (1997) Mol. Microbiol. 26 (145-161); Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). Although the properties and specificity of the GlcNAc-phosphotransferase activity of WecA have been characterized (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322), the conclusion that WecA catalyzes the synthesis of GalNAc-P-P-Und was based on genetic studies (Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). Such earlier genetic studies indicated that the biosynthesis of the lipid-linked tetrasaccharide intermediate was initiated by the enzymatic transfer of GalNAc-P from UDP-GalNAc to Und-P catalyzed by WecA (Wang, L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). However, there was no direct enzymological evidence demonstrating that WecA utilizes UDP-GalNAc as a GalNAc-P donor.
Furthermore, the E. coli O55 gne and gne1 genes were previously proposed to encode a UDP-GlcNAc 4-epimerase (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625; Guo, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609). Previous reports identified two genes from E. coli O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) and E. coli O86 (Guo, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609), E. coli O55 gne and E. coli O86 gne1, respectively, that are 100% identical to a Z3206 gene within the same gene family.
Accordingly, one of skill would have been led to believe that the Z3206 gene also encodes a UDP-GlcNAc/UDP-GalNAc epimerase.
BRIEF SUMMARY OF THE INVENTION
It has now been surprisingly discovered that an epimerase encoded by the 3206 gene in E. coli O157 catalyzes a reaction that synthesizes N-acetylgalactosamine (“GalNAc”) undecaprenyl pyrophosphate, which initiates the formation of an oligo- or polysaccharide.
In one aspect, the present invention relates to a recombinant prokaryotic biosynthetic system that produces all or a portion of a polysaccharide comprising an epimerase that synthesizes GalNAc on undecaprenyl pyrophosphate. The invention further includes glycosyltransferases that synthesize all or a portion of a polysaccharide having GalNAc at the reducing terminus, and still further includes glycosyltransferases that synthesize all or a portion of an antigenic polysaccharide having GalNAc at the reducing terminus.
In another aspect, the invention is directed to an epimerase to produce GalNAc on undecaprenyl pyrophosphate, and, in a further aspect, the epimerase is encoded by the Z3206 gene.
In an additional aspect, the present invention is directed to an expression system for producing an N-glycosylated protein comprising: a nucleotide sequence encoding an oligosaccharyl transferase; a nucleotide sequence encoding a protein carrier; at least one oligo- or polysaccharide gene cluster from at least one bacterium, wherein the polysaccharide contains GalNAc at the reducing terminus; and a nucleic acid sequence encoding an epimerase.
In a still further aspect, the instant invention is directed to a recombinant prokaryotic biosynthetic system comprising Z3206 gene which encodes an epimerase that converts GlcNAc-P-P-Und to GalNAc-P-P-Und.
In yet an additional aspect, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising E. coli O55 gne gene or E. coli O86 gne1 gene which encodes an epimerase that converts GlcNAc-P-P-Und to GalNAc-P-P-Und.
In yet another aspect, the present invention relates to an N-glycosylated protein comprising at least one introduced consensus sequence, D/E-X-N-Z-S/T, wherein X and Z can be any natural amino acid except proline, and a glycan having N-acetylgalactosamine at the reducing terminus.
In still another aspect, the present invention is directed to a bioconjugate vaccine comprising an N-glycosylated protein having at least one introduced consensus sequence, D/E-X-N-Z-S/T, wherein X and Z can be any natural amino acid except proline: an immunogenic glycan having N-acetylgalactosamine at the reducing terminus; and an adjuvant.
In an addition aspect, the invention relates to method for producing an N-linked glycosylated protein in a host cell comprising nucleic acids encoding: glycosyltransferases that assemble at least one oligo- or polysaccharide from at least one bacterium containing GalNAc at the reducing terminus; a protein carrier; an oligosaccharyl transferase; and an epimerase.
In a further aspect, the present invention relates to the use of a biosynthetic system and proteins for preparing a bioconjugate vaccine.
In an additional aspect, the present invention is directed to methods for producing mono-, oligo- and polysaccharides, and in a still further aspect the invention directed to methods for producing antigenic glycans and N-glycosylated proteins.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the time course of [3H]GlcNAc/GalNAc-P-P-Und synthesis by membrane fractions from E. coli O157. The membrane fraction from E. coli strain O157 was incubated with UDP-[3H]GlcNAc for the indicated times at 37° C. The [3H]lipid products were extracted and the incorporation of [3H]GlcNAc into [3H]GlcNAc-P-P-Und (O) and [3H]GalNAc-P-P-Und (•) was assayed as described in Example 2.
FIG. 2 shows the proposed biosynthetic pathway for the formation of GalNAc-P-P-Und from GlcNAc-P-P-Und.
FIGS. 3A, 3B, 3C, and 3D shows purification and characterization of [3H]GalNAc-P-P-Und synthesized by membrane fractions from E. coli strain O157. Membrane fractions from E. coli O157 were incubated with UDP-[3H]GlcNAc, and the [3H]GalNAc lipids were purified as described in Example 3. FIG. 3A, preparative thin layer chromatogram of [3H]HexNAc lipids on borate-impregnated silica gel G (Quantum 1) after purification on DEAE-cellulose is shown. FIG. 3B, thin layer chromatography of purified [3H]GalNAc-P-P-Und on borate-impregnated silica gel G (Baker, Si250) after recovery from the preparative plate in panel A is shown. FIG. 3C. descending paper chromatogram (borate-impregnated Whatman No. 1 paper) of the [3H]-amino sugar recovered after mild acid hydrolysis of [3H]GalNAc-P-P-Und purified in FIG. 3B is shown. FIG. 3D, descending paper chromatogram (Whatman No. 3MM) of the [3H]HexNAc-alditol produced by reduction of the [3H] amino sugar from FIG. 3C with NaBH4.
FIGS. 4A and 4B shows metabolic labeling of E. coli 21546 cells and E. coli 21546 cells after transformation with pMLBAD:Z3206. E. coli 21546 (FIG. 4A) and E. coli 21546:pMLBAD/Z3206 (FIG. 4B) were labeled metabolically with [3H]GlcNAc for 5 min at 37° C. [3H]GlcNAc/GalNAc-P-P-Und were extracted, freed of water soluble contaminants and separated by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) as described in Example 3. Radioactive lipids were detected using a Bioscan chromatoscanner. The chromatographic positions of GalNAc-P-P-Und and GlcNAc-P-P-Und are indicated by arrows.
FIGS. 5A, 5B, 5C, and 5D shows thin layer chromatography of [3H]GlcNAc/GalNAc-P-P-Und formed by incubation of membrane fractions from E. coli strains with UDP-[3H]GlcNAc. Membrane fractions from E. coli strains K12 (FIG. 5A), O157 (FIG. 5B), 21546 (FIG. 5C), and 21546:pMLBAD/Z3206 (FIG. 5D) were incubated with UDP-[3H]GlcNAc for 10 min at 37° C., and the [3H]lipid products were extracted, freed of water-soluble contaminants by partitioning, and separated by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) as described in Example 3. The chromatographic positions of GalNAc-P-P-Und and GlcNAc-P-P-Und are indicated by arrows.
FIGS. 6A, 6B, and 6C shows discharge of GlcNAc-P by incubation with UMP. Membrane fractions from E. coli 21546:Z3206 were preincubated with UDP-[3H]GlcNAc to enzymatically label GlcNAc-P-P-Und for 10 min (FIG. 6A) at 37° C. followed by a second incubation period with 1 mM UMP included for either 1 min (FIG. 6B) or 2 min (FIG. 6C). After the indicated incubation periods [3H]GlcNAc/GalNAc-P-P-Und were extracted and resolved by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) as described in Example 3. The chromatographic positions of GalNAc-P-P-Und and GlcNAc-P-P-Und are indicated by arrows.
FIGS. 7A, 7B, 7C, 7D, 7E, and 7F shows conversion of exogenous [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und to the pertinent [3H]HexNAc-P-P-Und product catalyzed by membranes from strain 21546 expressing Z3206. Membrane fractions from E. coli strain 21546 (FIG. 7B and FIG. 7E) and 215461:pMLBAD/Z3206 (FIG. 7C and FIG. 7F) were incubated with purified [3H]GlcNAc-P-P-Und (FIG. 7A, FIG. 7B, and FIG. 7C) or [3H]GalNAc-P-P-Und (panels at FIG. 7D, FIG. 7E, and FIG. 7F) (dispersed ultrasonically in 1% Triton X-100) for 1 min at 37° C. [3H]GlcNAc/GalNAc-P-P-Und were extracted, resolved by thin layer chromatography on borate-impregnated silica gel plates (Baker Si250) and detected with a Bioscan AR2000 radiochromatoscanner as described in Example 3.
FIG. 8 shows SDS-PAGE analysis of unglycosylated and glycosylated AcrA protein. Periplasmic extracts prepared from E. coli DH5α cells carrying the AcrA expression plasmid and the pgl operon Agile complemented with pMLBAD:Z3206 (lane 1), pMLBAD:gne (lane 2) or the vector control pMLBAD (lane 3) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. AcrA and its glycosylated forms were detected with anti AcrA antisera. The position of bands corresponding to unglycosylated (AcrA) and glycosylated AcrA (gAcrA) is indicated.
FIG. 9 shows the genes that have been identified by Liu B et al. (Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27).
FIG. 10 is a scheme showing the DNA region containing the genes required to synthesize the S. flexneri 6 O antigen.
FIG. 11 shows expression of the S. flexneri 6 O antigen in E. coli. LPS was visualized by either silver staining or by transfer to nitrocellulose membranes and detection by antibodies directed against S. flexneri 6.
FIG. 12 shows HPLC of O antigen. LLO analysis of E. coli cells (SCM3) containing S. flexneri—Z3206, E. coli cells (SCM3) containing S. flexneri+Z3206 or empty E. coli (SCM3) cells.
FIG. 13 shows Western blot of Nickel purified proteins from E. coli cells expressing EPA, pglB and S. flexneri 6 O-antigen+/−Z3206.



DETAILED DESCRIPTION OF THE INVENTION
The present invention encompasses a recombinant prokaryotic biosynthetic system comprising nucleic acids encoding an epimerase that synthesizes an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus, and N-glycosylated proteins having N-acetylgalactosamine at the reducing terminus of the glycan.
The term “partial amino acid sequence(s)” is also referred to as “optimized consensus sequence(s)” or “consensus sequence(s).” The optimized consensus sequence is N-glycosylated by an oligosaccharyl transferase (“OST,” “OTase”), much more efficiently than the regular consensus sequence “N-X-ST.”
In general, the term “recombinant N-glycosylated protein” refers to any poly- or oligopeptide produced in a host cell that does not naturally comprise the nucleic acid encoding said protein. In the context of the present invention, this term refers to a protein produced recombinantly in a prokaryotic host cell, for example, Escherichia spp., Campylobacter spp., Salmonella spp., Shigella spp., Helicobacter spp., Pseudomonas spp., Bacillus spp., and in further embodiments Escherichia cell, Campylobacter jejuni, Salmonella typhimurium etc., wherein the nucleic acid encoding said protein has been introduced into said host cell and wherein the encoded protein is N-glycosylated by the OTase, said transferase enzyme naturally occurring in or being introduced recombinantly into said host cell.
In accordance with the internationally accepted one letter code for amino acids the abbreviations D, E, N, S and T denote aspartic acid, glutamic acid, asparagine, serine, and threonine, respectively.
Proteins according to the invention comprise one or more of an optimized consensus sequence(s) D/E-X-N-Z-S/T that is/are introduced into the protein and N-glycosylated. Hence, the proteins of the present invention differ from the naturally occurring C. jejuni N-glycoproteins which also contain the optimized consensus sequence but do not comprise any additional (introduced) optimized consensus sequences.
The introduction of the optimized consensus sequence can be accomplished by the addition, deletion and/or substitution of one or more amino acids. The addition, deletion and/or substitution of one or more amino acids for the purpose of introducing the optimized consensus sequence can be accomplished by chemical synthetic Strategies, which, in view of the instant invention, would be well known to those skilled in the art such as solid phase-assisted chemical peptide synthesis. Alternatively, and preferred for larger polypeptides, the proteins of the present invention can be prepared by recombinant techniques that would be art-standard techniques in light of the invention.
The proteins of the present invention have the advantage that they may be produced with high efficiency and in any host. In one embodiment of the invention, the host comprises a functional pgl operon from Campylobacter spp., for example, from C. jejuni. In further embodiments, oligosaccharyl transferases from Campylobacter spp. for practicing the invention are from Campylobacter coli or Campylobacter lari. In view of the invention, oligosaccharyl transferases would be apparent to one of skill in the art. For example, oligosaccharyl transferases are disclosed in references such as Szymanski, C. M. and Wren, B. W. (2005) Protein glycosylation in bacterial mucosal pathogens, Nat. Rev. Microbiol. 3:225-237. The functional pgl operon may be present naturally when said prokaryotic host is Campylobacter spp., or, for example, C. jejuni. However, as demonstrated before in the art and mentioned above, the pgl operon can be transferred into cells and remain functional in said new cellular environment.
The term “functional pgl operon from Campylobacter spp., preferably C. jejuni” is meant to refer to the cluster of nucleic acids encoding the functional oligosaccharyl transferase (OTase) of Campylobacter spp., for example, C. jejuni, and one or more specific glycosyltransferases capable of assembling an oligosaccharide on a lipid carrier, and wherein said oligosaccharide can be transferred from the lipid carrier to the target protein having one or more optimized amino acid sequence(s): D/E-X-N-Z-S/T by the OTase. It to be understood that the term “functional pgl operon from Campylobacter spp., preferably C. jejuni” in the context of this invention does not necessarily refer to an operon as a singular transcriptional unit. The term merely requires the presence of the functional components for N-glycosylation of the recombinant protein in one host cell. These components may be transcribed as one or more separate mRNAs and may be regulated together or separately. For example, the term also encompasses functional components positioned in genomic DNA and plasmid(s) in one host cell. For the purpose of efficiency, in one embodiment all components of the functional pgl operon are regulated and expressed simultaneously.
The oligosaccharyl transferase can originate, in some embodiments, from Campylobacter spp., and in other embodiments, from C. jejuni. In additional embodiments, the oligosaccharyl transferase can originate from other organisms which are known to those of skill in the art as having an oligosaccharyl transferase, such as, for example, Wolinella spp. and eukaryotic organisms.
The one or more specific glycosyltransferases capable of assembling an oligosaccharide on a lipid carrier may originate from the host cell or be introduced recombinantly into said host cell, the only functional limitation being that the oligosaccharide assembled by said glycosyltransferases can be transferred from the lipid carrier to the target protein having one or more optimized consensus sequences by the OTase. Hence, the selection of the host cell comprising specific glycosyltransferases naturally and/or replacing specific glycosyltransferases naturally present in said host as well as the introduction of heterologous specific glycosyltransferases will enable those skilled in the art to vary the N-glycans bound to the optimized N-glycosylation consensus site in the proteins of the present invention.
As a result of the above, the present invention provides for the individual design of N-glycan-patterns on the proteins of the present invention. The proteins can therefore be individualized in their N-glycan pattern to suit biological, pharmaceutical and purification needs.
In embodiments of the present invention, the proteins may comprise one but also more than one, such as at least two, at least 3 or at least 5 of said N-glycosylated optimized amino acid sequences.
The presence of one or more N-glycosylated optimized amino acid sequence(s) in the proteins of the present invention can be of advantage for increasing their immunogenicity, increasing their stability, affecting their biological activity, prolonging their biological half-life and/or simplifying their purification.
The optimized consensus sequence may include any amino acid except proline in position(s) X and Z. The term “any amino acids” is meant to encompass common and rare natural amino acids as well as synthetic amino acid derivatives and analogs that will still allow the optimized consensus sequence to be N-glycosylated by the OTase. Naturally occurring common and rare amino acids are preferred for X and Z. X and Z may be the same or different.
It is noted that X and Z may differ for each optimized consensus sequence in a protein according to the present invention.
The N-glycan hound to the optimized consensus sequence will be determined by the specific glycosyltransferases and their interaction when assembling the oligosaccharide on a lipid carrier for transfer by the OTase. In view of the instant invention, those skilled in the art would be able to design the N-glycan by varying the type(s) and amount of the specific glycosyltransferases present in the desired host cell.
“Monosaccharide” as used herein refers to one sugar residue. “Oligo- and polysaccharide” refer to two or more sugar residues. The term “glycans” as used herein refers to mono-, oligo- or polysaccharides. “N-glycans” are defined herein as mono-, oligo- or polysaccharides of variable compositions that are linked to an ε-amide nitrogen of an asparagine residue in a protein via an N-glycosidic linkage. In an embodiment, the N-glycans transferred by the OTase are assembled on an undecaprenol pyrophosphate (“Und-P-P”) lipid-anchor that is present in the cytoplasmic membrane of gram-negative or positive bacteria. They are involved in the synthesis of O antigen, O polysaccharide and peptidoglycan (Bugg, T. D., and Brandish, P. E. (1994). From peptidoglycan to glycoproteins: common features of lipid-linked oligosaccharide biosynthesis. FEMS Microbiol Lett 119, 255-262; Valvano, M. A. (2003). Export of O-specific lipopolysaccharide. Front Biosci 8, s452-471).
Studies were conducted to determine whether the biosynthesis of a lipid-linked repeating tetrasaccharide (4-N-acetyl perosamine→fucose→glucose→GalNAc) was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, 0157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[3H]GalNAc, neither the enzymatic synthesis of [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[3H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und, confirming that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of an epimerase was also confirmed by showing that exogenous [3H]GlcNAc-P-P-Und was converted to [3H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [3H]GlcNAc, both [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [3H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [3H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, it was confirmed that GalNAc-P-P-Und is synthesized reversibly by a GlcNAc-P-P-Und epimerase following the formation of GlcNAc-P-P-Und by WecA in E. coli O157.
The initiating reaction of E. coli O157 O-antigen subunit assembly was investigated to confirm that GalNAc-P-P-Und synthesis is catalyzed by some previously unknown mechanism rather than by WecA. The evidence presented herein shows that GalNAc-P-P-Und is not synthesized by GalNAc-P transfer from UDP-GalNAc catalyzed by WecA but rather by the reversible epimerization of the 4-OH of GlcNAc-P-P-Und catalyzed by an epimerase encoded by the Z3206 gene in E. coli O157.
Accordingly, the invention encompasses a novel biosynthetic pathway for the assembly of an important bacterial cell surface component as well as a new biosynthetic route for the synthesis of GalNAc-P-P-Und. A further embodiment of the invention includes the bacterial epimerase as a new target for antimicrobial agents.
E. coli O157 synthesizes an O-antigen with the repeating tetrasaccharide structure (4-N-acetyl perosamine→fucose→glucose→GalNAc). It is shown herein that the biosynthesis of the lipid-linked tetrasaccharide intermediate was not initiated by the enzymatic transfer of GalNAc-P from UDP-GalNAc to Und-P catalyzed by WecA, contrary to earlier genetic studies (Wang. L. and Reeves, P. R. (1998) Infect. Immun. 66, 3545-3551). The invention described herein, obtained by homology searches and then confirmed by results from genetic, enzymology, and metabolic labeling experiments, demonstrates that WecA does not utilize UDP-GalNAc as a substrate, but that WecA is required to synthesize GlcNAc-P-P-Und which is then reversibly converted to GalNAc-P-P-Und by an epimerase encoded by the Z3206 gene in strain O157.
The Z3206 gene of the present invention belongs to a family of genes present in several strains that produce surface O-antigen repeat units containing GalNAc residues at their reducing termini (Table 1). The Z3206 gene sequence is shown in SEQ ID NO: 1. Previous reports identified two genes from E. coli O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) and E. coli O86 (Gun, H., Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Comm., 356, 604-609), E. coli O55 gne and E. coli O86 gne1, respectively, that are 100% identical to a Z3206 gene (Table 1). The E. coli O55 gne gene sequence is shown as SEQ ID NO: 3, and E. coli O86 gne1 gene sequence is shown as SEQ ID NO: 5.







TABLE 1







Correlation of Z3206 gene in bacterial strains expressing O-antigen


chains with GalNAc at the reducing termini.











GalNAc



% Identity
at the reducing



with
terminus of O-antigen



Z3206
repeat unit













E. coli O55 gne (SEQ ID NO: 3)
100
Yes


E. coli O86 gnel (SEQ ID NO: 5)
100
Yes


Shigella boydii O18 gne (SEQ ID
88
Yes


NO: 7)


Salmonella enterica O30 gne
94
Yes


(SEQ ID NO: 9)


C. jejuni gne (SEQ ID NO: 11)
21
No


E. coli K12 galE (SEQ ID NO: 13)
27
No


E. coli O86 gne2 (SEQ ID NO: 15)
18
Yes









Accordingly, we conclude that E. coli O55 gne and E. coli O86 gne1 also encode epimerases capable of converting GlcNAc-P-P-Und to GalNAc-P-P-Und in strains O55 and O86, respectively, which also produce O-antigen repeat units with GalNAc at the reducing termini (Table 1).
Two experimental approaches in this study indicate that the Z3206 protein does not catalyze the epimerization of UDP-GlcNAc to UDP-GalNAc in strain O157. First, when membranes from strain O157 were incubated with [3H]UDP-GalNAc, neither [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected (Table 3). If Z3206 catalyzed the conversion of [3H]UDP-GalNAc to [3H]UDP-GlcNAc, it would be expected that [3H]GlcNAc-P-P-Und should be observed. Second, we have shown that hemagglutinin-tagged Z3206 was incapable of complementing the UDP-GalNAc-dependent C. jejuni N-glycosylation reporter system (FIG. 8).
E. coli O55 gne gene from strain O55 (Wang, L., Huskic, S., Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625) was also assayed for epimerase activity by incubating crude extracts with UDP-GalNAc and indirectly assaying the conversion to UDP-GlcNAc by measuring an increase in reactivity with p-dimethylaminobenzaldehyde after acid hydrolysis. In both studies, the formation of the product was based on changes in reactivity with p-dimethylaminobenzaldehyde, and not a definitive characterization of the sugar nucleotide end product. A 90% pure polyhistidine-tagged E. coli O86 gne1 was also shown to have a low level of UDP-glucose epimerase activity relative to Gne2 in a coupled assay.
Accordingly, an embodiment of the invention is directed to a recombinant prokaryotic biosynthetic system containing Z3206 gene, E. coli O55 gne gene or E. coli O86 gne1 gene that converts GlcNAc-P-P-Und to GalNAc-P-P-Und.
It is significant that E. coli O86, which synthesizes an O-antigen containing two GalNAc residues, which would presumably require UDP-GalNAc as the glycosyl donor for the additional, non-reducing terminal GalNAc, also possesses an additional GlcNAc 4-epimerase gene, termed gne2, within the O-antigen gene cluster (Guo. B, Yi, W., Li, L. and Wang, P. G. (2007) Biochem. Biophys. Res. Commun., 356, 604-609). This additional epimerase gene has high homology with the galE gene of the colanic acid gene cluster and appears to be a UDP-GlcNAc 4-epimerase capable of synthesizing UDP-GalNAc.
The Z3206 gene appears to be highly conserved in E. coli O-serotypes initiated with GalNAc. In a recent study, 62 E. coli strains, with established O-antigen repeat unit structures, were screened for expression of Z3206 by a polymerase chain reaction based method using nucleotide primers designed to specifically detect the E. coli O157 Z3206 gene (Wang, L., Huskic, Cisterne, A., Rothemund, D. and Reeves, P. R. (2002) J. Bacteriol. 184, 2620-2625). In this study Z3206 was detected in 16 of the 22 E. coli strains that were known to contain GalNAc, and in only 4 of the 40 strains lacking GalNAc. Moreover, a similar screen of the 22 GalNAc-containing strains with primers designed to detect an alternative epimerase with UDP-GlcNAc 4-epimerase activity (the GalE gene of E. coli O113) detected no strains carrying this gene, indicating that Z3206 is the GlcNAc 4-epimerase gene most commonly associated with the presence of a reducing-terminal GalNAc in O-antigen repeat units of E. coli. 
Analysis of the Z3206 protein sequence by a variety of web-based topological prediction algorithms indicates that the Z3206 protein is not highly hydrophobic. The majority of the topological prediction algorithms indicate that Z3206 is a soluble 37 kDa protein, although TMPred (Hofmann, K., and Stoffel, W. (1993) Biol. Chem. Hoppe-Seyler 374, 166 (abstr.)) predicted a single weak N-terminal transmembrane helix. However, Western blotting after SDS-PAGE of cellular fractions from E. coli cells expressing hemagglutinin-tagged Z3206 clearly shows that the tagged protein is associated with the particulate fraction following hypotonic lysis of the cells. Preliminary experiments show that the protein remains associated with the particulate fraction following incubation of the membrane fraction with 1 M KCl, but is solubilized in an active form by incubation with 0.1% Triton X-100.
E. coli O157 Z3206 has significant sequence homology with the short-chain dehydrogenase/reductase family of oxido-reductases including the GXXGXXG motif (Rossman fold), consistent with the NAD(P) binding pocket (Allard, S. T. M., Giraud, M. F., and Naismith, J. H. (2001) Cell. Mol. Life Sci. 58, 1650-1655) and the conserved SX24YX3K sequence, involved in proton abstraction and donation (Field, R. A. and Naismith, J. H. (2003) Biochemistry 42, 7637-7647). Molecular modeling based on crystal structures of UDP-Glc 4-epimerase, another member of the short-chain dehydrogenase/reductase family, suggests that, after hydride abstraction, the 4-keto intermediate rotates around the β phosphate of UDP to present the opposite face of the keto intermediate and allow re-insertion of hydride from the opposite side, thus inverting the configuration of the hydroxyl at carbon 4. The presence of these conserved sequences suggests that Z3206 likely functions via a similar mechanism. Although the equilibrium distribution of the epimerase products, seen in FIG. 7, seems to favor the formation of GlcNAc-P-P-Und, the utilization of GalNAc-P-P-Und for O-antigen repeat unit assembly would drive the epimerization reaction in the direction of GalNAc-P-P-Und by mass action.
Epimerization of the glycosyl moieties of polyisoprenoid lipid intermediates has not been widely reported in nature. In one previous study the 2-epimerization of ribosyl-P-decaprenol to form arabinosyl-P-decaprenol, an arabinosyl donor in arabinogalactan biosynthesis in mycobacteria, was reported (Mikusová, K., Huang, H., Yagi, T., Holsters, M., Vereecke, D., D'Haeze, W., Scherman, M. S., Brennan, P. J., McNeil, M. R., and Crick, D. C. (2005) J. Bacterial. 187, 8020-8025). Arabinosyl-P-decaprenol is formed via a two-step oxidation/reduction reaction requiring two mycobacterial proteins, Rv3790 and Rv3791. Although epimerization was modestly stimulated by the addition of NAD and NADP, neither Rv3790 nor Rv3791 contain either the Rossman fold or the SX24YXXXK motif, characteristic of the short-chain dehydrogenase/reductase family (Allard, S. T. M., Giraud, M.-F. and Naismith, J. H. (2001) Cell. Mal. Life Sci. 58, 1650-1655; Field, R. A. and Naismith, J. H. (2003) Biochemistry 42, 7637-7647).
In summary, a novel biosynthetic pathway for the formation of GalNAc-P-P-Und by the epimerization of GlcNAc-P-P-Und, is described.
Several antibiotics have been shown to inhibit the synthesis of GlcNAc-P-P-Und, but are limited in their utility because they also block the synthesis of GlcNAc-P-P-dolichol, the initiating dolichol-linked intermediate of the protein N-glycosylation pathway. Although GlcNAc-P-P-dolichol is a structurally related mammalian counterpart of the bacterial glycolipid intermediate, GlcNAc-P-P-Und, there is no evidence for a similar epimerization reaction converting GlcNAc-P-P-dolichol to GalNAc-P-P-dolichol in eukaryotic cells. Thus, this raises the possibility that in strains where the surface O-antigen containing GalNAc at the reducing termini are involved in a pathological process, O-antigen synthesis could potentially be blocked by inhibiting the bacterial epimerases.
An embodiment of the present invention involves an epimerase that converts GlcNAc-P-P-Und (N-acetylglucosaminylpyrophosphorylundecaprenol) to GalNAc-P-P-Und (N-acetylgalactosaminylpyrophosphorylundecaprenol) in E. coli O157. A still further exemplary aspect of the invention involves the initiation of synthesis of lipid-bound repeating tetrasaccharide having GalNAc at the reducing terminus.
The basis of another aspect of the invention includes the discovery that Campylobacter jejuni contains a general N-linked protein glycosylation system. Various proteins of C. jejuni have been shown to be modified by a heptasaccharide. This heptasaccharide is assembled on undecaprenyl pyrophosphate, the carrier lipid, at the cytoplasmic side of the inner membrane by the stepwise addition of nucleotide activated monosaccharides catalyzed by specific glycosyltransferases. The lipid-linked oligosaccharide then flip-flops (diffuses transversely) into the periplasmic space by a flippase, e.g., PglK. In the final step of N-linked protein glycosylation, the oligosaccharyltransferase (e.g., PglB) catalyzes the transfer of the oligosaccharide from the carrier lipid to asparagine (Asn) residues within the consensus sequence D/E-X-N-Z-S/T, where the X and Z can be any amino acid except Pro. The glycosylation cluster for the heptasaccharide had been successfully transferred into E. coli and N-linked glycoproteins of Campylobacter had been produced.
It had been demonstrated that PglB does not have a strict specificity for the lipid-linked sugar substrate. The antigenic polysaccharides assembled on undecaprenyl pyrophosphate are captured by PglB in the periplasm and transferred to a protein carrier (Feldman, 2005; Wacker, M., et al., Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems. Proc Natl. Acad Sci USA. 2006. 103(18): p. 7088-93.) The enzyme will also transfer a diverse array of undecaprenyl pyrophosphate (UPP) linked oligosaccharides if they contain an N-acetylated hexosamine at the reducing terminus. The nucleotide sequence for pglB and the amino acid sequence for pglB are published at WO2009/04074.
Accordingly, one embodiment of the invention involves a recombinant N-glycosylated protein comprising: one or more of an introduced consensus sequence. D/E-X-N-Z-S/T, wherein X and Z can be any natural amino acid except proline; and an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus and N-linked to each of said one or more introduced consensus sequences by an N-glycosidic linkage.
In a further embodiment, the present invention is directed to a recombinant prokaryotic biosynthetic system for producing all or a portion of a polysaccharide comprising an epimerase that synthesizes N-acetylgalactosamine (“GalNAc”) on undecaprenyl pyrophosphate. In a further embodiment, all or a portion of the polysaccharide is antigenic.
In another embodiment, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising: an epimerase that synthesizes GalNAc on undecaprenyl pyrophosphate; and glycosyltransferases that synthesize a polysaccharide having GalNAc at the reducing terminus.
An embodiment of the invention further comprises a recombinant prokaryotic biosynthetic system comprising an epimerase that synthesizes GalNAc on undecaprenyl pyrophosphate and glycosyltransferases that synthesize a polysaccharide, wherein said polysaccharide has the following structure: α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc; and wherein GalNAc is at the reducing terminus of said polysaccharide.
The recombinant prokaryotic biosynthetic system can produce mono-, oligo- or polysaccharides of various origins. Embodiments of the invention are directed to oligo- and polysaccharides of various origins. Such oligo- and polysaccharides can be of prokaryotic or eukaryotic origin. Oligo- or polysaccharides of prokaryotic origin may be from gram-negative or gram-positive bacteria. In one embodiment of the invention, the oligo- or polysaccharide is from E. coli. In a further aspect of the invention, said oligo- or polysaccharide is from E. coli O157. In another embodiment, said oligo- or polysaccharide comprises the following structure: α-D-PerNAc-α-L-Fuc-P-D-Glc-α-D-GalNAc. In a further embodiment of the invention, the oligo- or polysaccharide is from Shigella flexneri. In a still further embodiment, the oligo- or polysaccharide is from Shigella flexneri 6. In a still further aspect, said oligo- or polysaccharide comprises the following structure:




Embodiments of the invention further include proteins of various origins. Such proteins include proteins native to prokaryotic and eukaryotic organisms. The protein carrier can be, for example, AcrA or a protein carrier that has been modified to contain the consensus sequence for protein glycosylation, i.e., D/E-X-N-Z-S/T, wherein X and Z can be any amino acid except proline (e.g., a modified Exotoxin Pseudomonas aeruginosa (“EPA”)). In one embodiment of the invention, the protein is Pseudomonas aeruginosa EPA.
A further aspect of the invention involves novel bioconjugate vaccines having GalNAc at the reducing terminus of the N-glycan. An additional embodiment of the invention involves a novel approach for producing such bioconjugate vaccines that uses recombinant bacterial cells that contain an epimerase which produces GalNAc on undecaprenyl pyrophosphate. In one embodiment, bioconjugate vaccines can be used to treat or prevent bacterial diseases. In further embodiments, bioconjugate vaccines may have therapeutic and/or prophylactic potential for cancer or other diseases.
A typical vaccination dosage for humans is about 1 to 25 μg, preferably about 1 μg to about 10 μg, most preferably about 10 μg. Optionally, a vaccine, such as a bioconjugate vaccine of the present invention, includes an adjuvant.
In an additional embodiment, the present invention is directed to an expression system for producing a bioconjugate vaccine against at least one bacterium comprising: a nucleotide sequence encoding an oligosaccharyl transferase; a nucleotide sequence encoding a protein carrier; at least one polysaccharide gene cluster from the at least one bacterium, wherein the polysaccharide contains GalNAc at the reducing terminus; and a nucleic acid sequence encoding an epimerase. In a further embodiment, the polysaccharide gene cluster encodes an antigenic polysaccharide.
In still a further embodiment, the present invention is directed to an expression system for producing a bioconjugate vaccine against at least one bacterium comprising: a nucleotide sequence encoding an oligosaccharyl transferase; a nucleotide sequence encoding a protein carrier comprising at least one inserted consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; at least one polysaccharide gene cluster from the at least one bacterium, wherein the polysaccharide contains GalNAc at the reducing terminus; and the Z3206 gene. In a further embodiment, the polysaccharide gene cluster encodes an antigenic polysaccharide.
In yet another embodiment, the present invention is directed to a bioconjugate vaccine comprising: a protein carrier; at least one immunogenic polysaccharide chain linked to the protein carrier, wherein said polysaccharide has GalNAc at the reducing terminus, and further wherein said GalNAc is directly linked to the protein carrier; and an adjuvant.
In yet an additional embodiment, the present invention is directed to a bioconjugate vaccine comprising: a protein carrier comprising at least one inserted consensus sequence, D/E-X-N-Z-S/T, wherein X and Z may be any natural amino acid except proline; least one immunogenic polysaccharide from at least one bacterium, linked to the protein carrier, wherein the at least one immunogenic polysaccharide contains GalNAc at the reducing terminus directly linked to the protein carrier; and, optionally, an adjuvant.
Another embodiment of the invention is directed to a method of producing a bioconjugate vaccine, said method comprising: assembling a polysaccharide having GalNAc at the reducing terminus in a recombinant organism through the use of glycosyltransferases; linking said GalNAc to an asparagine residue of one or more target proteins in said recombinant organism, wherein said one or more target proteins contain one or more T-cell epitopes.
In a further embodiment, the present invention is directed to a method of producing a bioconjugate vaccine, said method comprising: introducing genetic information encoding for a metabolic apparatus that carries out N-glycosylation of a target protein into a prokaryotic organism to produce a modified prokaryotic organism; wherein the genetic information required for the expression of one or more recombinant target proteins is introduced into said prokaryotic organism; wherein the genetic information required for the expression of E. coli strain O157 epimerase is introduced into said prokaryotic organism; and wherein the metabolic apparatus comprises glycosyltransferases of a type that assembles a polysaccharide having GalNAc at the reducing terminus on a lipid carrier, and an oligosaccharyltransferase, the oligosaccharyltransferase covalently linking GalNAc of the polysaccharide to an asparagine residue of the target protein, and the target protein containing at least one T-cell epitope; producing a culture of the modified prokaryotic organism; and obtaining glycosylated proteins from the culture medium.
A further aspect of the present invention relates to a pharmaceutical composition. An additional aspect of the invention involves a pharmaceutical composition comprising at least one N-glycosylated protein according to the invention. In light of the disclosure herein, the preparation of medicaments comprising proteins would be well known in the art. A still further aspect of the invention relates to a pharmaceutical composition comprising an antibiotic that inhibits an epimerase that converts GlcNAc-P-P-Und to GalNAc-P-P-Und. In a preferred embodiment, the pharmaceutical composition of the invention comprises a pharmaceutically acceptable excipient, diluent and/or adjuvant.
Suitable excipients, diluents and/or adjuvants are well-known in the art. An excipient or diluent may be a solid, semi-solid or liquid material which may serve as a vehicle or medium for the active ingredient. One of ordinary skill in the art in the field of preparing compositions can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition, and other relevant circumstances (Remington's Pharmaceutical Sciences, Mack Publishing Co. (1990)). The proportion and nature of the pharmaceutically acceptable diluent or excipient are determined by the solubility and chemical properties of the pharmaceutically active compound selected, the chosen route of administration, and standard pharmaceutical practice. The pharmaceutical preparation may be adapted for oral, parenteral or topical use and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions, or the like. The pharmaceutically active compounds of the present invention, while effective themselves, can be formulated and administered in the form of their pharmaceutically acceptable salts, such as acid addition salts or base addition salts, for purposes of stability, convenience of crystallization, increased solubility, and the like.
In instances where specific nucleotide or amino acid sequences are noted, it will be understood that the present invention encompasses homologous sequences that still embody the same functionality as the noted sequences. In an embodiment of the invention, such sequences are at least 85% homologous. In another embodiment, such sequences are at least 90% homologous. In still further embodiments, such sequences are at least 95% homologous.
The determination of percent identity between two nucleotide or amino acid sequences is known to one of skill in the art.
Nucleic acid sequences described herein, such as those described in the sequence listing below, are examples only, and it will be apparent to one of skill in the art that the sequences can be combined in different ways. Additional embodiments of the invention include variants of nucleic acids. A variant of a nucleic acid (e.g., a codon-optimized nucleic acid) can be substantially identical, that is, at least 80% identical, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical, to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29. Nucleic acid variants of a sequence that contains SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29 include nucleic acids with a substitution, variation, modification, replacement, deletion, and/or addition of one or more nucleotides (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 nucleotides) from a sequence that contains SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29, or parts thereof.
For example, in an embodiment of the instant invention, such variants include nucleic acids that encode an epimerase which converts GlcNAc-P-P-Und to GalNAc-P-P-Und and that i) are expressed in a host cell, such as, for example, E. coli and ii) are substantially identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, or parts thereof.
Nucleic acids described herein include recombinant DNA and synthetic (e.g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. In the case of single-stranded nucleic acids, the nucleic acid can be a sense strand or antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives.
Plasmids that include a nucleic acid described herein can be transfected or transformed into host cells for expression. Techniques for transfection and transformation are known to those of skill in the art.
All publications mentioned herein are incorporated by reference in their entirety. It is to be understood that the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination. As used herein, unless the context clearly dictates otherwise, references to the singular, such as the singular forms “a,” an,” and “the,” include the plural, and references to the plural include the singular.
The invention is further defined by reference to the following examples that further describe the compositions and methods of the present invention, as well as its utility. It will be apparent to those skilled in the art that modifications, both to compositions and methods, may be practiced which are within the scope of the invention.
EXAMPLES
Bacterial Strains and Plasmids—
E. coli strains PR4019 (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322) and PR21546 (Meier-Dieter, U., Starman, R., Barr, K., Mayer, H. and Rick, P. I). (1990) J. Biol. Chem., 265, 13490-13497) were generous gifts from Dr. Paul Rick, Bethesda, Md., and E. coli O157:H45 (Stephan, R., Borel, N., Zweifel, C., Blanco, M, and Blanco, J. E. (2004) BMC Microbiol 4:10) was a gift from Dr. Claudio Zweifel, Veterinary Institute, University of Zurich, E. coli DH5α (Invitrogen) was used as the host for cloning experiments and for protein glycosylation analysis. Plasmids used are listed in Table 2.







TABLE 2







Plasmids used in Examples









Plasmid
Description
Ref





pMLBAD
Cloning vector, TmpR
Lefebre &




Valvano




(2002)


pMLBAD:Z3206
Z3206 in pMLBAD, TmpR, expression
Examples


(SEQ ID NO: 23)
controlled by arabinose-inducible
herein



promoter


pMLBAD:gne
gne in pMLBAD, TmpR, expression
Examples


(SEQ ID NO: 24)
controlled by arabinose-inducible
herein



promoter


pACYCpgl
C. jejuni pgl cluster CmR
Wacker,




et al.




(2002)


pACYCgne::kan
C. jejuni pgl cluster containing a kan
Linton, et



cassette in gne, CmR, KanR
al. (2005)


pWA2
Soluble periplasmic hexa-His-tagged
Feldman,



AcrA under control of Tet promoter in
et al.



pBR322, AmpR
(2005)









Materials—
[1,6-3H]GlcNAc (30 Ci/mmol), UDP-[1-3H]GlcNAc (20 Ci/mmol) and UDP-[6-3H]GalNAc (20 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, Mo.). Quantum 1 silica gel G thin layer plates are a product of Quantum Industries (Fairfield, N.J.), and Baker Si250 Silica Gel G plates are manufactured by Mallinekrodt Chemical Works. Yeast extract and Bacto-peptone were products of BD Biosciences. All other chemicals were obtained from standard commercial sources. Trimethoprim (50 μg/ml), chloramphenicol (20 μg/ml), ampicillin (100 μg/ml), and kanamycin (50 μg/ml) were added to the media as needed.
Construction of Recombinant Plasmids—
E. coli strain DH5α was used for DNA cloning experiments and constructed plasmids were verified by DNA sequencing. The Z3206 gene was amplified from E. coli O157:H45 by PCR with oligonucleotides Z3206-Fw and Z3206-RvHA (AAACCCGGGATGAACGATAACG TTTTGCTC (SEQ ID NO: 17) and AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTACTCAGAAACAA ACGTTATGTC (SEQ ID NO: 18): restriction sites are underlined). The PCR fragment was digested with SmaI and XbaI and ligated into SmaI-XbaI cleaved pMLBAD vector (Lefebre, M. D. and Valvano M. A. (2002) Appl Environ Microbiol 68: 5956-5964). This resulted in plasmid pMLBAD:Z3206 (SEQ ID NO: 23) encoding Z3206 with a C-terminal hemagglutinin tag.
The gne gene was amplified from pACYCpgl (Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wrenn, B. W., Aebi, M. (2002) Science 298, 1790-1793), encoding Campylobacter jejuni pgl cluster, with oligonucleotides gne-Fw and gne-RV (AAACCATGGATGAAAATTCTTATTAGCGG (SEQ ID NO: 19) and AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTAGCACTGTTTTTC CCAATC (SEQ ID NO: 20); restriction sites are underlined). The PCR product was digested with NcoI and XbaI and ligated into the same sites of pMLBAD to generate plasmid pMLBAD:gne (SEQ ID NO: 24) which encodes One with a C-terminal hemagglutinin tag (Table 2).
Growth Conditions, Protein Expression and Immunodetection—
E. coli strains were cultured in Luria-Bertani medium (1% yeast extract, 2% Bacto-peptone, 0.6% NaCl) at 37° C. with vigorous shaking. Arabinose inducible expression was achieved by adding arabinose at a final concentration of 0.02-0.2% (w/v) to E. coli cells grown up to an A600 of 0.05-0.4. The same amount of arabinose was added again 5 h post-induction, and incubation continued for 4-15 h.
Analytical Procedures—
Protein concentrations were determined using the BCA protein assay (Pierce) after precipitation of membrane proteins with deoxycholate and trichloroacetic acid according to the Pierce Biotechnology bulletin “Eliminate Interfering Substances from Samples for BCA Protein Assay.” Samples were analyzed for radioactivity by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation spectrometer after the addition of 0.5 ml of 1% SDS and 4 ml of Econosafe Economical Biodegradable Counting Mixture (Research Products International, Corp., Mount Prospect, Ill.).
Example 1: Identification of an E. coli O157 Gene Encoding GlcNAc-P-P-Und 4-Epimerase
We describe herein the surprising discovery of a new biosynthetic pathway in which GalNAc-P-P-Und is formed by the epimerization of the 4-OH of GlcNAc-P-P-Und catalyzed by the previously unknown action of a 4-epimerase. In this pathway, GlcNAc-P-P-Und is formed by the transfer of GlcNAc-P from UDP-GlcNAc, catalyzed by WecA, and then GlcNAc-P-P-Und is epimerized to GalNAc-P-P-Und by GlcNAc-P-P-Und-4-epimerase, which was a previously unknown pathway (FIG. 2.
The gene encoding a candidate for the GlcNAc-P-P-Und 4-epimerase was identified by DNA homology searches. Homology searches were performed using the U.S. National Library of Medicine databases found at http:blast.ncbi.nlm.nih.govBlast.cgi. Genomic sequences of different bacteria encoding O antigen repeating units having a GalNAc at the reducing terminus were screened. One group with a repeating unit containing a GalNAc at the reducing terminus, and a second group lacking a terminal GalNAc in the repeating unit were compared to identify potential epimerases. Using these criteria, Z3206 was identified as a candidate GlcNAc-P-P-Und 4-epimerase (Table 1).
The GlcNAc 4-epimerase genes present in E. coli strains with O-antigen repeat units containing GalNAc can be separated into two homology groups as shown in Table 1. It was surprisingly discovered that one homology group (containing grid) clearly was correlated with the presence of GalNAc as the initiating sugar on the O-antigen repeat unit. It was further surprisingly discovered that the second group (containing gne2) exhibits a high degree of similarity to the UDP-Glc epimerase, GalE, and is found in E. coli strains that do not initiate O-antigen repeat unit synthesis with GalNAc. Z3206 in E. coli O157, a gene with a high degree of homology to gne1, was identified as a candidate GlcNAc-P-P-Und 4-epimerase. The genomic location of the Z3206 gene is consistent with a role in this pathway, as it resides between galF of the O-antigen cluster and wcaM which belongs to the colanic acid cluster.
The research described in Examples 2-11 further confirms the above discoveries, including identifying the GlcNAc 4-epimerase (E. coli O157 Z3206) as catalyzing the formation of GalNAc-P-P-Und.
Example 2: UDP-GalNAc is not a Substrate for E. coli WecA (GlcNAc-phosphotransferase)
To determine if E. coli WecA will utilize UDP-GalNAc as a GalNAc-P donor to form GalNAc-P-P-Und, membrane fractions from E. coli strains K12, PR4019, a WecA-overexpressing strain, and O157, which synthesize a tetrasaccharide O-antigen repeat unit with GalNAc at the reducing terminus presumably initiated by the synthesis of GalNAc-P-P-Und, were incubated with UDP-[3H]GalNAc.
Preparation of E. coli Membranes—
Bacterial cells were collected by centrifugation at 1,000×g for 10 min, washed once in ice-cold phosphate-buffered saline, once with cold water, and once with 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose. The cells were resuspended to a density of  ̃200 A600 units/ml in 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 10 mM EDTA containing 0.2 mg/ml lysozyme, and incubated at 30° C. for 30 min. Bacterial cells were recovered by centrifugation at 1,000×g for 10 min, quickly resuspended in 40 volumes of ice-cold 10 mM Tris-HCl, pH 7.4, and placed on ice. After 10 min the cells were homogenized with 15 strokes with a tight-fitting Dounce homogenizer and supplemented with 0.1 mM phenylmethylsulfonyl fluoride and sucrose to a final concentration of 0.25 M. Unbroken cells were removed by centrifugation at 1,000×g for 10 min, and cell envelopes were recovered by centrifugation at 40,000×g for 20 min. The membrane fraction was resuspended in 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA and again sedimented at 40,000×g and resuspended in the same buffer to a protein concentration of  ̃20 mg/ml. Membrane fractions were stored at −20° C. until needed.
Assay for the Biosynthesis of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und in E. coli Membranes In Vitro—
Reaction mixtures for the synthesis of GlcNAc-P-P-Und and GalNAc-P-P-Und contained 50 mM Tris-HCl, pH 8, 40 mM MgCl2, 5 mM dithiothreitol, 5 mM 5′ AMP. E. coli membrane fraction (50-200 μg membrane protein, and either 5 μm UDP-[3H]GlcNAc/GalNAc (500-2500 dpm/pmol) in a total volume of 0.05 ml. After incubation at 37° C., reactions were terminated by the addition of 40 volumes of CHCl3/CH3OH (2:1), and the total lipid extract containing [3H]HexNAc-P-P-undecaprcnols was prepared as described previously (Waechter. C. J., Kennedy, J. L. and Harford, J. B. (1976) Arch. Biochem, Biophys. 174, 726-737). After partitioning, the organic phase was dried under a stream of nitrogen and redissolved in 1 ml CHCl3/CH3OH (2:1), and an aliquot (0.2 ml) was removed, dried in a scintillation vial, and analyzed for radioactivity by liquid scintillation spectrometry in a Packard Tri-Carb 2100 TR liquid scintillation spectrometer. To determine the rate of synthesis of [3H]GlcNAc-P-P-Und or [3H]GalNAc-P-P-Und, the lipid extract was dried under a stream of nitrogen, redissolved in a small volume of CHCl3/CH3OH (2:1), and spotted on a 10×20-cm borate-impregnated Baker Si250 silica gel plate, and the plate was developed with CHCl3, CH3OH, H2O, 0.2 M sodium borate (65:25:2:2). Individual glycolipids were detected with a Bioscan AR2000 Imaging Scanner (Bioscan, Washington, D.C.). The biosynthetic rates for each glycolipid were calculated by multiplying the total amount of radioactivity in [3H]GlcNAc/GalNAc-P-P-Und by the percentage of the individual [3H] glycolipids.
Membrane fractions from different E. coli strains (K12, PR4019 and O157) were incubated with either UDP-[3H]GlcNAc or UDP-[3H]GalNAc and the incorporation into [3H]GlcNAc/GalNAc-P-P-Und was determined as described above. As seen in Table 3, no labeled glycolipids were detected after the incubation with UDP-[3H]GalNAc, only GlcNAc-P-P-Und was detectable when membrane fractions were incubated with UDP-[3H]GlcNAc







TABLE 3







Synthesis of [3H]GlcNAc/GalNAc-P-P-undecaprenol in E. coli membrane


fractions using either UDP-[3H]GlcNAc or UDP-[3H]GalNAc as substrate









[3H]Glycolipid formed










Source of
Sugar nucleotide
GlcNAc-P-P-Und
GalNAc-P-P-Und


membranes
added
(pmol/mg)
(pmol/mg)













K12
UDP-[3H]GlcNAc
6.4
<0.01


K12
UDP-[3H]GalNAc
<0.01
<0.01


PR4019
UDP-[3H]GlcNAc
44
<0.01


PR4019
UDP-[3H]GalNAc
<0.01
<0.01


O157
UDP-[3H]GlcNAc
1.5
0.5


O157
UDP-[3H]GalNAc
<0.01
<0.01









Moreover, neither the addition of exogenous Und-P to incubations with membranes from PR4019, the WecA-overexpressing strain, or the addition of cytosolic fractions from O157 cells resulted in the formation of GalNAc-P-P-Und from UDP-GalNAc. These results demonstrate that UDP-GalNAc is not a substrate for WecA and suggest that GalNAc-P-P-Und is formed by an alternative mechanism.
When membranes from strain K12 were incubated with UDP-[3H]GlcNAc, [3H]GlcNAc-P-P-Und was synthesized as expected (Rush, J. S., Rick, P. D. and Waechter, C. J. (1997) Glycobiology, 7, 315-322). However, when membranes from strain O157 were incubated with UDP-[3H]GlcNAc, in addition to [3H]GlcNAc-P-P-Und, a second labeled lipid shown to be [3H]GalNAc-P-P-Und (see below) was observed. When the time course for the formation of the two glycolipids was examined, the incorporation of radioactivity into [3H]GlcNAc-P-P-Und (FIG. 1, O) occurred more quickly and to a higher extent than into [3H]GalNAc-P-P-Und (FIG. 1, ●), compatible with a precursor-product relationship (FIG. 2).
The observation that E. coli O157 membranes do not utilize UDP-GalNAc as a GalNAc-P donor for the synthesis of GalNAc-P-P-Und is one example which confirms the biosynthetic pathway for the formation of GalNAc-P-P-Und illustrated in FIG. 2. In this scheme, GlcNAc-P-P-Und is formed by the transfer of GlcNAc-P from UDP-GlcNAc, catalyzed by WecA, and then GlcNAc-P-P-Und is epimerized by the action of a previously unknown 4-epimerase to produce GalNAc-P-P-Und.
Example 3: Characterization of [3H]GalNAc-P-P-Und Formed In Vitro with Membrane Fractions from E. coli Strain O157
Consistent with the additional O157-specific glycolipid product detected in FIG. 1, as GalNAc-P-P-Und, it was stable to mild alkaline methanolysis (toluene/methanol 1:3, containing 0.1 N KOH, 0° C., 60 min), retained by DEAE-cellulose equilibrated in CHCl3/CH3OH/H2O (10:10:3), and eluted with CHCl3/CH3OH/H2O (10:10:3) containing 20 mM ammonium acetate as reported previously for [3H]GlcNAc1-2-P-P-Dol (Waechter, J. and Harford, B. (1977) Arch. Biochem. Biophys. 181, 185-198).
[3H]GalNAc-P-P-Und was clearly resolved from [3H]GalNAc-P-P-Und by thin layer chromatography on borate-impregnated silica gel G (Kean, E. L. (1966) J. Lipid Res. 7, 149-452) and purified by preparative TLC as shown in FIG. 3A and FIG. 3B.
Preparation of Borate-Impregnated Thin Layer Plates and Whatman No. 1 Paper—
Silica gel thin layer plates were impregnated with sodium borate by briefly immersing the plates in 2.5% Na2B4O7.10 H2O in 95% methanol as described by Kean (Kean, E. L. (1966) J. Lipid Res. 7.449-452). The borate-impregnated TLC plates were dried overnight at room temperature and stored in a vacuum dessicator over Drierite until use. Immediately before chromatography, the plates were activated by heating briefly ( ̃10-15 min) to 100° C. Whatman No. 1 paper was impregnated with sodium borate by dipping 20×30-cm sheets of Whatman 1 paper in 0.2 M Na2B4O7.10H2O. The Whatman No. 1 paper sheets were pressed firmly between two sheets of Whatman No. 3MM paper and allowed to dry at room temperature for several days, as described by Cardini and Leloir (Cardini, C. E. and Leloir, L. F. (1957) J. Biol. Chem. 225, 317-324).
Characterization of Glycan Products Formed in In Vitro Reactions—
The glycans of the individual glycolipids ([3H]GalNAc-P-P-Und and [3H]GlcNAc-P-P-Und) were characterized by descending paper chromatography after release by mild acid hydrolysis. The GlcNAc/GalNAc lipids were dried under a stream of nitrogen in a conical screw-cap tube and heated to 100° C., 15 min in 0.2 ml 0.01 M HCl. After hydrolysis the samples were applied to a 0.8-ml mixed-bed ion-exchange column containing 0.4 ml of AG50WX8 (H+) and 0.4 ml AG1X8 (acetate form) and eluted with 1.5 ml water. The eluate was dried under a stream of nitrogen, redissolved in a small volume of H2O (0.02 ml), spotted on a 30-cm strip of borate-impregnated Whatman No. 1 paper, and developed in descending mode with butanol/pyridine/water (6:4:3) for 40-50 h. After drying, the paper strips were cut into 1-cm zones and analyzed for radioactivity by scintillation spectrometry. GlcNAc and GalNAc standards were detected using an aniline-diphenylamine dip reagent (Schwimmer, S. and Benvenue, A. (1956) Science 123, 543-544).
Glycan products were converted to their corresponding alditols by reduction with 0.1 M NaBH4 in 0.1 M NaOH (final volume ml) following mild acid hydrolysis as described above. After incubation at room temperature overnight, the reactions were quenched with several drops of glacial acetic acid and dried under a stream of nitrogen out of methanol containing 1 drop of acetic acid, several times. The alditols were dissolved in water, desalted by passage over 0.5 ml columns of AG50WX8 (H+) and AG1X8 (acetate), dried under nitrogen, and spotted on 30-cm strips of Whatman No. 3MM paper. The Whatman No. 3 MM strips were developed overnight in descending mode with ethyl acetate, pyridine, 0.1 M boric acid (65:25:20), dried, cut into 1-cm zones, and analyzed for radioactivity by scintillation spectrometry. GlcNAcitol and GalNAcitol standards were visualized using a modification of the periodate-benzidine dip procedure (Gordon, H. T., Thornburg, W. and Werum, L. N. (1956) Anal. Chem. 28, 849-855). The paper strips were dipped in acetone, 0.1 M NaIO4 (95:5), allowed to air dry for 3 min, and then dipped in acetone/acetic acid/H2O/o-tolidine (96:0.6:4.4:0.2 gm). Alditols containing cis-diols stain as yellow spots on a blue background.
Mass Spectrometry (“MS”) of Glycolipids—
Purified glycolipids were analyzed using an ABI/MDS Sciex 4000 Q-Trap hybrid triple quadrupole linear ion trap mass spectrometer with an ABI Turbo V electrospray ionsource (ABIMDS-Sciex, Toronto, Canada). In brief, samples were infused at 10 μl/min with ion source settings determined empirically, and MS/MS (mass spectroscopy in a second dimension) information was obtained by fragmentation of the molecular ion in linear ion trap mode.
When the glycolipid was treated with mild acid (0.01 N HCl, 100° C., 15 min), the water-soluble product co-chromatographed with [3H]GalNAc on descending paper chromatography with borate-impregnated Whatman No. 1 paper (FIG. 3C). In addition, when the labeled sugar was reduced, it was converted to [3H]alditol, GalNAc-OH (FIG. 3D). Moreover, negative-ion MS analysis yielded the [M-H]-ion of m/z=1128, expected for GalNAc-P-P-Und, and the MS/MS daughter ion spectrum showed a prominent ion at m/z=907, expected for a glycolipid containing P-P-Und (Guan, Z., Breazeale, S. D. and Raetz, C. R. (2005) Anal. Biochem. 345, 336-339). The identification of the glycolipid product formed by strain O157 as GalNAc-P-P-Und is also supported by its formation from exogenous GlcNAc-P-P-Und (see Example 7).
Example 4: Metabolic Labeling of [3H]GalNAc-P-P-Und (In Vivo) with [3H]GlcNAc in E. coli Cells Expressing the Z3206 Gene
To investigate whether expression of the E. coli O157 Z3206 gene enabled cells to synthesize GalNAc-P-P-Und, E. coli strain 21546 (Meier-Dieter, U., Starman, R., Barr, K., Mayer, H. and Rick, P. D. (1990) J. Biol. Chem., 265, 13490-13497) expressing the Z3206 gene was labeled metabolically with [3H]GlcNAc and analyzed for [3H]GlcNAc/GalNAc-P-P-Und formation.
Metabolic Labeling of Bacterial Cells—
E. coli cells were cultured with vigorous shaking in Luria-Bertani medium at 37° C. to an A600 of 0.5-1. [3H]GlcNAc was added to a final concentration of 1 μCi/ml and the incubation was continued for 5 min at 37° C. The incorporation of radiolabel into glycolipids was terminated by the addition of 0.5 gm/ml crushed ice, and the cultures were thoroughly mixed. The bacterial cells were recovered by centrifugation at 4000×g for 10 min, and the supernatant was discarded. The cells were washed with ice-cold phosphate-buffered saline two times, resuspended by vigorous vortex mixing in 10 volumes (cell pellet) of methanol, and sonicated briefly with a probe sonicator at 40% full power. After sonication, 20 volumes of chloroform were added, and the extracts were mixed vigorously and allowed to stand at room temperature for 15 min. The insoluble material was sedimented by centrifugation, and the pellet was re-extracted with a small volume of CHCl3/CH3OH (2:1) twice. The combined organic extracts were then processed as described below.
Purification of GlcNAc-P-P-Und and GalNAc-P-P-Und—
GlcNAc/GalNAc-P-P-Und was extracted with CHCl3/CH3OH (2:1) and freed of water-soluble material by partitioning as described elsewhere (Waechter, C. J., Kennedy, J. L. and Harford, J. B. (1976) Arch. Biochem. Biophys. 174, 726-737). The organic extract was then dried under a stream of nitrogen, and the bulk glycerophospholipids were destroyed by deacylation in toluene/methanol (1:3) containing 0.1 N KOH at 0° C. for 60 min. The deacylation reaction was neutralized with acetic acid, diluted with 4 volumes of CHCl3/CH3OH (2:1), and washed with 15 volume of 0.9% NaCl. The organic (lower) phase was washed with 13 volume of CHCl3, CH3OH, 0.9% NaCl (3:48:47), and the aqueous phase was discarded. The organic phase was diluted with sufficient methanol to accommodate the residual aqueous phase in the organic phase and applied to a DEAE-cellulose column (5 ml) equilibrated with CHCl3/CH3OH (2:1). The column was washed with 20 column volumes of CHCl3/CH3OH/H2O (10:10:3) and then eluted with CHCl3/CH3OH/H2O (10:10:3) containing 20 mM ammonium acetate. Fractions (2 ml) were collected and monitored for either radioactivity, or GlcNAc/GalNAc-P-P-Und using an anisaldehyde spray reagent (Dunphy, P. J., Kerr, J. D., Pennock, J. F., Whittle, K. J., and Feeney, J. (1967) Biochim. Biophys. Acta 136, 136-147) after resolution by thin layer chromatography on borate-impregnated silica plates (as described earlier).
E. coli strain 21546 was selected as the host for the Z3206 expression studies because a mutation in UDP-ManNAcA synthesis results in a block in the utilization of GlcNAc-P-P-Und for the synthesis of the enterobacterial common antigen. Because E. coli 21546 is derived from E. coli K12 it does not synthesize an O-antigen repeat as well (Stevenson, G., Neal, B., Liu, D., Hobbs, M., Packer, N. H., Batley, M., Redmond, J. W., Lindquist, L. and Reeves, P. (1994) J. Bacterial., 176, 4144-4156), and thus, larger amounts of GlcNAc-P-P-Und accumulate for the conversion to GalNAc-P-P-Und. When strain 21546 and the transformant expressing the Z3206 gene were labeled with [3H]GlcNAc and the radiolabeled lipids were analyzed by thin layer chromatography on borate-impregnated silica gel plates, the parental strain (FIG. 4A) synthesized only one labeled lipid, GlcNAc-P-P-Und. However, 21546 cells expressing the Z3206 gene (FIG. 4B) also synthesized an additional labeled lipid shown to be GalNAc-P-P-Und.
Example 5: Membrane Fractions from E. coli Cells Expressing the Z3206 Gene Synthesize GalNAc-P-P-Und In Vitro
To corroborate that the protein encoded by the E. coli O157 Z3206 gene catalyzed the synthesis of GalNAc-P-P-Und, membrane fractions from E. coli cells expressing the Z3206 gene were incubated with [3H]UDP-GlcNAc and the [3H]glycolipid products were analyzed by thin layer chromatography (chromatographic preparation and characterization methods are described in Example 3) on borate-impregnated silica gel plates as shown in FIG. 5. When membrane fractions from E. coli K12 or the host strain E. coli 21546 cells were incubated with UDP-[3H]GlcNAc, only [3H]GlcNAc-P-P-Und was observed (FIG. 5A and FIG. 5C). However, membrane fractions from E. Coli O157 and E. coli 21546 expressing Z3206 formed GalNAc-P-P-Und as well (FIG. 5B and FIG. 5D).
Example 6: Formation of GlcNAc-P-P-Und, but not GalNAc-P-P-Und, is Reversed in the Presence of UMP
To provide additional evidence that GalNAc-P-P-Und is synthesized from GlcNAc-P-P-Und, and not by the action of WecA using UDP-GalNAc as a glycosyl donor, the effect of discharging endogenous, pre-labeled [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und with UMP was examined. The GlcNAc-phosphotransferase reaction catalyzed by WecA is freely reversible by the addition of excess UMP re-synthesizing UDP-GlcNAc and releasing Und-P.
In this experiment membrane fractions from E. coli strain 21546 expressing Z3206 were pre-labeled for 10 min with UDP-[3H]GlcNAc followed by the addition of 1 mM UMP, and the amount of each labeled glycolipid remaining was determined. The results illustrated in FIG. 6A show the relative amounts of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und at the end of the 10 min labeling period. After incubation with 1 mM UMP for 1 min it can be seen that there is a substantial loss of [3H]GalNAc-P-P-Und, whereas the [3H]GalNAc-P-P-Und peak is relatively unchanged (FIG. 6B) (chromatographic preparation and characterization methods are described in Example 5), This observation is consistent with the results in Table 3 indicating that WecA does not catalyze the transfer of GalNAc-P into GalNAc-P-P-Und from UDP-GalNAc. It is noteworthy that during the second minute of incubation with UMP (FIG. 6C), the loss of GlcNAc-P-P-Und slows, and there is a slight reduction in the peak of [3H]GalNAc-P-P-Und, suggesting that [3H]GalNAc-P-P-Und is re-equilibrating with the [3H]GlcNAc-P-P-Und pool by reversal of the epimerase reaction (see Example 7).
Example 7: Interconversion of Exogenous, Purified [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und Catalyzed by Membranes from E. Coli Cells Expressing Z3206
To provide direct evidence that GlcNAc-P-P-Und and GalNAc-P-P-Und can be directly interconverted by membrane fractions from E. coli cells expressing Z3260, purified [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were tested as exogenous substrates.
Purified [3H]GlcNAc-P-P-Und/[3H]GalNAc-P-P-Und were prepared as in Example 4 (Metabolic Labeling of Bacterial Cells and Purification of GlcNAc-P-P-Und and GalNAc-P-P-Und). [3H]HexNAc-P-P-undecaprenols (2000 dpm/pmol, dispersed in 1% Triton X-100, final concentration 0.1%) were incubated with E. coli membranes as in Example 2 in Assay For the Biosynthesis of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und in E. coli Membranes In Vitro.
Preliminary experiments showed that the epimerase was active when exogenous [3H]GalNAc-P-P-Und was added to the reaction mixtures dispersed in Triton X-100, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid), Nonidet P-40, or octylglucoside and exhibited a pH optimum in the range 7-8.5. The chromatographic mobility of the purified [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und before incubation with membrane fractions is shown in FIG. 7A and FIG. 7D. As seen in FIG. 7B and FIG. 7E, the glycolipids are unaffected by incubation with membrane fractions from E. coli 21546. However, incubation of the purified glycolipids with membrane fractions from E. coli 21546 expressing Z3206 catalyzes the conversion of exogenous [3H]GlcNAc-P-P-Und to [3H]GalNAc-P-P-Und (FIG. 7C) and the conversion of [3H]GalNAc-P-P-Und to [3H]GlcNAc-P-P-Und (FIG. 7F). These results demonstrate directly that GlcNAc-P-P-Und and GalNAc-P-P-Und can be enzymatically interconverted in E. coli strains expressing the Z3206.
Example 8: E. coli Z3206 is not a UDP-GlcNAc 4-Epimerase
To determine if Z3206 can catalyze the formation of UDP-GalNAc, the N-glycosylation apparatus from C. jejuni was expressed in E. coli. In this reporter system, glycosylation of the target protein AcrA is dependent on the presence of the pgl locus (Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wrenn, B. W., Aebi, M. (2002) Science 298, 1790-1793), including a functional Gne UDP-Glc/UDP-GlcNAc epimerase (Bernatchez, S., Szymanski, C. M., Ishiyama, N., Li, J., Jarrell, H. C., Lau, P. C., Berghuis, A. M., Young, N. M., Wakarchuk, W. W. (2005) J. Biol. Chem. 280, 4792-4802). Glycosylation of AcrA is lost if the pgl cluster contains a deletion of gne (Linton, D., Dorrell, N., Hitchen, P. G., Amber, S., Karlyshev, A. V., Morris, H. R., Dell, A., Valvano, M. A., Aebi, M. and Wren, B. W. (2005) Mol Microbiol. 55, 1695-1703). The ability of Z3206 to restore AcrA-glycosylation in the presence of the pgl operon Δgne was investigated in vivo by expressing AcrA (pWA2) together with the pgl locus Δgne complemented by either Gne (pMLBAD:gne) or Z3206 (pMLBAD:Z3206).
Total E. coli cell extracts were prepared for immunodetection analysis using cells at a concentration equivalent to 1 A600 unit that were resuspended in 100 μl of SDS loading buffer (Laemmli, U. (1970) Nature 227, 680-685). Aliquots of 10 μl were loaded on 10% SDS-PAGE. Periplasmic extracts of E. coli cells were prepared by lysozyme treatment (Feldman, M. F., Wacker, M., Hernandez, M., Hitchen, P. G., Marolda, C. L., Kowarik, M., Morris, H. R., Dell, A., Valvano, M. A., Aebi, M. (2005) Proc Natl Acad Sci USA 102, 3016-3021), and 10 μl of the final sample (corresponding to 0.2 A600 units of cells) was analyzed by SDS-PAGE. After being blotted on nitrocellulose membrane, sample was immunostained with the specific antiserum (Aebi, M., Gasscnhuber, J., Domdey, H., and te Heesen, S. (1996) Glycobiology 6, 439-444). Anti-AcrA (Wacker, M., Linton, D., Hitchen, P. G., Nita-Lazar, M., Haslam, S. M., North, S. J., Panico, M., Morris, H. R., Dell, A., Wrenn, B. W., Aebi, M. (2002) Science 298, 1790-1793) antibodies were used. Anti-rabbit IgG-HRP (Bio-Rad) was used as secondary antibody. Detection was carried out with ECLTM Western blotting detection reagents (Amersham Biosciences).
As shown in FIG. 8, the glycosylated protein, which migrates slower than the unglycosylated form, was formed only when cells expressing pgl locus Δgne were complemented by One (lane 2). Z3206 was unable to restore glycosylation of the reporter glycoprotein (FIG. 8, lane 1). Accordingly, Z3206 does not complement glycosylation of AcrA in a Gne dependent glycosylation system. Expression of Gne and membrane-associated Z3206 were confirmed by immunodctection.
Example 9: Analysis of S. flexneri 6+/− Z3206 LPS
In FIG. 9 are depicted some of the genes required for the biosynthesis of the Shigella flexneri 6 O-antigen: genes encoding enzymes for biosynthesis of nucleotide sugar precursors; genes encoding glycosyltransferases; genes encoding O antigen processing proteins; and genes encoding proteins responsible for the O-acetylation. The structure of the O antigen has been elucidated by Dmitriev, B. A. et al (Dmitriev. B. A., et al Somatic Antigens of Shigella Eur J Biochem, 1979. 98: p. 8; Liu B et al Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27).
To identify all the genes required for the biosynthesis of the Shigella flexneri 6 O-antigen a genomic library was constructed.
Cloning of S. flexneri 6 genomic DNA—
S. flexneri 6 genomic DNA was isolated using a Macherey-Nagel NucleoSpin® Tissue Kit following the protocol for DNA isolation from bacteria. DNA was isolated from five S. flexneri 6 overnight cultures at 2 ml each and final elution was done with 100 μl elution buffer (5 mM Tris/HCl, pH 8.5). The eluted fractions were pooled, precipitated by isopropanol and the final pellet was resuspended in 52 μl TE buffer of which the total volume was subjected to end-repair according to the protocol given by CopyControlTM Fosmid Library Production Kit (EPICENTRE). End-repaired DNA was purified on a 1% low melting point agarose gel run with 1×TAE buffer, recovered and precipitated by ethanol as described in the kit protocol. Resuspension of the precipitated DNA was done in 7 μl TE buffer of which 0.15 μl DNA was ligated into pCC1FOS (SEQ ID NO: 27) according to the EPICENTRE protocol. Packaging of the ligation product into phage was performed according to protocol and the packaged phage was diluted 1:1 in phage dilution buffer of which 10 μl were used to infect 100 μl EPI300-T1 cells that were previous grown as described by EPICENTRE. Cells (110 μl) were plated six times with approximately 100 colonies per plate such that the six plates contain the entire S. flexneri 6 genomic library. Plates were developed by colony blotting and positive/negative colonies were western blotted and silver stained.
Colony Blotting—
For colony blots a nitrocellulose membrane was laid over the solid agar plate, removed, washed three times in 1×PBST and treated in the same manner. The membrane was first blocked in 10% milk for one hour at room temperature after which it was incubated for one hour at room temperature in 2 ml 1% milk (in PBST) with the anti-type VI antiserum (primary antibody). After three washes in PBST at 10 minutes each, the membrane was incubated for another hour at room temperature in the secondary antibody, 1:20000 peroxidase conjugated goat-anti-rabbit IgG (BioRad) in 2 ml 1% milk (in PBST). After a final three washes with PBST (10 minutes each) the membrane was developed in a UVP Chemi Doc Imaging System with a 1:1 mix of luminol and peroxide buffer provided by the SuperSignal® West Dura Extended Duration Substrate Kit (Thermo Scientific).
The clone reacting with S. flexneri 6 antiserum following production of a S. flexneri 6 genomic library was sequenced by primer walking out of the region previously sequenced by Liu et al. (Liu et al., 2008) reaching from rmlB to wtbZ (FIG. 9). Primers rmlB_rev and wfbZ_fwd (S. flexneri—Z3206) annealed in rmlB and wfbZ and were used to sequence the insert of the clone until wcaM and hisI/F were reached (S. flexneri+Z3206), respectively (FIG. 10).
In order to establish whether O antigen synthesis is maintained in clones lacking Z3206 (thus hindering epimerization of und-GlcNAc to und-GalNAc), two plasmids were constructed (SEQ ID NO. 28 and SEQ ID NO. 29) (FIG. 10), transformed into E. coli cells and analyzed by silver staining and western blot.
As shown in FIG. 11, LPS is produced in E. coli cells + or −Z3206. The O antigen can be produced without Z3206 however with lower production yield, which indicates that the efficiency of polysaccharide production without the epimerase (Z3206) is lower.
Example 10: Analysis of S. flexneri 6+/− Z3206 LLO
Purification of Undecaprenol-PP-O Antigen by C18 Column Chromatography—
E. coli cells expressing S. flexeneri antigen+/− Z3206 were pelleted, washed once in 50 ml 0.9% NaCl and the final pellets were lyophilized overnight. The pellets were washed once in 30 ml 85-95% methanol, reextracted with 10:10:3 chloroform-methanol-water (v/v/v) and the extracts were converted to a two-phase Bligh/Dyer system by addition of water, resulting in a final ratio of 10:10:9 (C:M:W). Phases were separated by centrifugation and the upper aqueous phases were loaded each on a C18 Sep-Pak cartridge conditioned with 10 ml methanol and equilibrated with 10 ml 3:48:47 (C:M:W). Following loading, the cartridges were washed with 10 ml 3:48:47 (C:M:W) and eluted with 5 ml 10:10:3 (C:M:W). 20 OD samples of the loads, flow-throughs, washes and elutions of the C18 column were dried in an Eppendorf Concentrator Plus, washed with 250 μl methanol, reevaporated and washed a further three times with 30 μl ddH2O.
Glycolipid Hydrolysis
The glycolipid samples from the wash of the C18 column were hydrolysed by dissolving the dried samples in 2 ml n-propanol:2 M trifluoroacetic acid (1:1), heating to 50° C. for 15 minutes and evaporating to dryness under N2.
Oligosaccharide Labeling with 2-Aminobenzoate and HPLC
Labeling was done according to Bigge et al. (Bigge, 1995) and glycan cleanup was performed using the paper disk method described in Merry et al. (2002) (Merry et al., 2002). Separation of 2-AB labeled glycans was performed by HPLC using a GlycoSep-N normal phase column according to Royle et al. (Royle, 2002) but modified to a three solvent system. Solvent A was 10 mM ammonium formate pH 4.4 in 80% acetonitrole. Solvent B was 30 mM ammonium formate pH 4.4. in 40% acetonitrile. Solvent C was 0.5% formic acid. The column temperature was 30° C. and 2-AB labeled glycans were detected by fluorescence (λex=330 nm, λem=420 nm). Gradient conditions were a linear gradient of 100% A to 100% B over 160 minutes at a flow rate of 0.4 ml/min, followed by 2 minutes 100% B to 100% C, increasing the flow rate to 1 ml/min. The column was washed for 5 minutes with 100% C, returning to 100% A over 2 minutes and running for 15 minutes at 100% A at a flow rate of 1 ml/min, then returning the flow rate to 0.4 ml/min for 5 minutes. All samples were injected in water.
The plasmids expressing the S. flexneri O-antigen with (SEQ ID NO: 29) or without (SEQ ID NO: 28) Z3206 were transformed into SCM3 cells (FIG. 10). Traces at late elution volumes shows a difference between the curves of the two samples containing the S. flexneri O antigen+/−Z3206 (FIG. 12). This difference in the elution pattern can be explained by a different oligosaccharide structure carrying a different monosaccharide at the reducing end: GlcNAc or GalNAc depending on the presence of the epimerase (Z3206).
Example 11: Analysis of pglB Specificity by Production and Characterization of Bioconjugate Produced from S. flexneri 6+/−Z3206
To assess whether pglB can transfer oligosaccharides having GlcNAc (S. flexneri 6 O-antigen) at the reducing end to the carrier protein EPA Nickel purified extracts from E. coli cells expressing EPA (SEQ ID NO: 25), PglB (SEQ ID NO: 26) and S. flexneri 6 O-antigen+/−Z3206 (SEQ ID NO: 29/SEQ ID NO: 28) were analyzed by western blot using anti EPA and anti type VI antibodies. The S. flexneri O6 antigen with and without GalNAc at the reducing end was transferred to EPA by PglB as detected by antiEPA and anti VI antisera (FIG. 13).
The O antigen is still produced and detected, but with lower production yield, which indicates that the efficiency of polysaccharide production without the epimerase is lower.
While this invention has been particularly shown and described with references to embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention encompassed by the claims. Such various changes that will be understood by those skilled in the art as covered within the scope of the invention include, in particular, N-glycosylated proteins and bioconjugates comprising a glycan other than those from E. coli and S. flexneri with GalNAc at the reducing terminus.










Sequence Listing















Applicant: GlycoVaxyn AG


Title: Biosynthetic System That Produces Immunogenic


Polysaccharides In Prokaryotic Cells


Number of SEQ ID NOs: 29


 


Nucleotide Sequence for E. coli O157 Z3206


Length: 993


Type: DNA


Organism: E. coli O157


Sequence:


SEQ ID NO: 1


ATGAACGATAACGTTTTGCTCATAGGAGCTTCCGGATTCGTAGGAACCCGACTACTTGAAACGG


 


CAATTGCTGACTTTAATATCAAGAAGCTGGACAAACAGCAGAGCCACTTTTATCCAGAAATCAC


 


ACAGATTGGCGATGTTCGCCATCAACAGGCACTGGACCAGGCGTTAGTCGGTTTTGACACTGTT


 


GTACTACTGGCAGCGGAACACCGCGATGACGTCAGCCCTACTTCTCTCTATTATGATGTCAACG


 


TTCAGGGTAGCCGCAATGTGCTGGCGGCCATGGAAAAAAATGGCGTTAAAAATATCATCTTTAC


 


CAGTTCCGTTGCTGTTTATGGTTTGAACAAACACAACCCTGACGAAAACCATCCACACGACCCT


 


TTGAACCACTACGGCAAAAGTAAGTGGCAGGCAGAGGAAGTGCTGCGTGAATGGTATAACAAAG


 


CACCAACAGAACGTTCATTAACCATCATCCGTGCTACCGTTATCTTCGGTGAACGCAACCGCGG


 


TAACGTCTATAACTTGCTGAAACAGATCGGTGGCGGCAAGTTTATGATGGTGGGCGCAGGGACT


 


AACTATAAGTCCATGGCTTATGTTGGAAACATTGTTGAGTTTATGAAGTACAAACTGAAGAATG


 


TTGCCGCAGGTTATGAGGTTTATAACTACGTTGATAAGCCAGACCTGAACATGAACCAGTTGGT


 


TGCTGAAGTTGAACAAAGCCTGAACAAAAAGATCCCTTCTATGCACTTGCCTTACCCACTAGGA


 


ATGCTGGGTGGATATTGCTTTGATATCCTGAGCAAAATTACGGGCAAAAAATACGCTGTCAGCT


 


CAGTGCGCGTGAAAAAATTCTGCGCAACAACACAGTTTGACGCAACGAAAGTGCATTCTTCAGG


 


TTTTGTGGCACCGTATACGCTGTCGCAAGGTCTGGATCGAAGACTGCAGTATGAATTCGTTCAT


 


GCCAAAAAAGACGACATAACGTTTGTTTCTGAG


 


Amino Acid Sequence for Z3206


Length: 331


Type: PRT


Organism: E coli O157


Sequence:


SEQ ID NO: 2


MNDNVLLIGASGFVGTRLLETAIADFNIKNLDKQQSHFYPEITQIGDVRDQQALDQALVGFDTV


 


VLLAAEHRDDVSPTSLYYDVNVQGTRNVLAAMEKNGVKNIIFTSSVAVYGLNKHNPDENHPHDP


 


FNHYGKSKWQAEEVLREWYNKAPTERSLTIIRPTVIFGERNRGNVYNLLKQIAGGKFMMVGAGT


 


NYKSMAYVGNIVEFIKYKLKNVAAGYEVYNYVDKPDLNMNQLVAEVEQSLNKKIPSMHLPYPLG


 


MLGGYCFDILSKITGKKYAVSSVRVKKFCATTQFDATKVHSSGFVAPYTLSQGLDRTLQYEFVH


 


AKKDDITFVSE


 


Nucleotide Sequence for E. coli O55 gne


Locus AF461121_1 BCT 2 May 2002


Definition (UDP-GlcNAc 4-epimerase Gne [Escherichia coil])


Accession AAL67550


Length: 993


Type: DNA


Organism: E. coli O55


Sequence:


SEQ ID NO: 3


ATGAACGATA ACGTTTTGCT CATAGGAGCT TCCGGATTCG TAGGAACCCG


 


ACTACTTGAA ACGGCAATTG CTGACTTTAA TATCAAGAAC CTGGACAAAC


 


AGCAGAGCCA CTTTTATCCA GAAATCACAC AGATTGGTGA TOTTCGTGAT


 


CAACAGGCAC TCGACCAGGC GTTAGCCGGT TTTGACACTG TTGTGCTACT


 


GGCAGCGGAA CACCGCGATG ACGTCAGCCC TACTTCTCTC TATTATGATG


 


TCAACGTTCA GGGTACCCGC AATGTGCTGG CGGCCATGGA AAAAAATGGC


 


GTTAAAAATA TCATCTTTAC CAGTTCCGTT GCTGTTTATG GTTTGAACAA


 


ACACAACCCT GACGAAAACC ATCCACACGA TCCTTTCAAC CACTACGGCA


 


AAAGTAAGTG GCAGGCAGAG GAAGTGCTGC GTGAATGGTA TAACAAAGCA


 


CCAACAGAAC GTTCATTAAC CATCATCCGT CCTACCGTTA TCTTCGGTGA


 


ACGGAACCGC GGTAACGTCT ATAACTTGCT GAAACAGATC GCTGGCGGCA


 


AGTTTATGAT GGTGGGCGCA GGGACTAACT ATAAGTCCAT GGCTTATGTT


 


GGAAACATTG TTGAGTTTAT CAAGTACAAA CTGAAGAATG TTGCCGCAGG


 


TTACGAGGTT TATAACTACG TTGATAAGCC AGACCTGAAC ATGAACCAGT


 


TGGTTGCTGA AGTTGAACAA AGCCTGAACA AAAAGATCCC TTCTATGCAC


 


TTGCCTTACC CACTAGGAAT GCTGGGTGGA TATTGCTTTG ATATCCTGAG


 


CAAAATTACG GGCAAAAAAT ACGCTGTCAG CTCTGTGCGC GTGAAAAAAT


 


TCTGCGCAAC AACACAGTTT GACGCAACGA NAGTGCATTC TTCAGGTTTT


 


GTGGCACCGT ATACGCTGTC GCAAGGTCTG GATCGAACTC TGCAGTATGA


 


ATTCGTCCAT GCCAAAAAAG ACGACATAAC GTTTGTTTCT GAG


 


Amino Acid Sequence for E. coli O55 UDP-GlcNAc 4-epimerase Gne


Locus AF461121_1


Definition (UDP-GlcNAc 4-epimerase Gne [Escherichia coli])


Accession AAL67550


Length: 331 aa linear


Type: PRT


Organism: E. coli O55


Sequence:


SEQ ID NO: 4


mndnvlliga sgfvgtrlle taiadfnikn ldkqqshfyp eitqigdvrd


 


qqaldqalag fdtvvllaae hrddvsptsl yydvnvqgtr nvlaamekng


 


vkniiftssv avyglnkhnp denhphdpfn hygkskwqae evirewynka


 


ptersltiir ptvifgernr gnvynllkqi aggkfmmvga gtnyksmayv


 


gnivefikyk lknvaagyev ynyvdkpdln mnqlvaeveq sinkkipsmh


 


lpyplgmlgg ycfdilskit gkkyayssvr vkkfcattqf datkvhssgf


 


vapytlsqgl drtlqyefvh akkdditfvs e


 


Nucleotide Sequence for E. coli O86 gne1


Locus AAO37706 BCT 6 Dec. 2005


Definition UDP-GlcNAc C4-epimerase [Escherichia coli O86].


Accession AAO37706


Length: 993


Type: DNA


Organism: E. coli O86


Sequence:


SEQ ID NO. 5


ATGAACGATA ACGTTTTGCT CATAGGAGCT TCCGGATTCG TAGGAACCCG


 


ACTACTTGAA ACGGCAATTG CTGACTTTAA TATCAAGAAC CTGGACAAAC


 


AGCAGAGCCA CTTTTATCCA GAAATCACAC AGATTGGTGA TGTTCGTGAT


 


CAACAGGCAC TCGACCAGGC GTTAGCCGGT TTTGACACTG TTGTACTACT


 


GGCAGCGGAA CACCGCGATG ACGTCAGCCC TACTTCTCTC TATTATGATG


 


TCAACGTTCA GGGTACCCGC AATGTGCTGG CGGCCATGGA AAAAAATGGC


 


GTTAAAAATA TCATCTTTAC CAGTTCCGTT GCTGTTTATG GTTTGAACAA


 


ACACAACCCT GACGAAAACC ATCCACACGA CCCTTTCAAC CACTACGGCA


 


AAAGCAAGTG GCAGGCGGAG GAAGTGCTGC GTGAATGGTA TAACAAAGCA


 


CCAACAGAAC GTTCATTAAC TATCATCCGT CCTACCGTTA TCTTCGGTGA


 


ACGCAACCGC GGTAACGTCT ATAACTTGCT GAAACAGATC GCTGGCGGCA


 


AGTTTATGAT GGTGGGCGCA GGGACTAACT ATAAGTCCAT GGCTTATGTT


 


GGAAACATTG TTGAGTTTAT CAAGTACAAA CTGAAGAATG TTGCCGCAGG


 


TTACGAGGTT TATAACTACG TTGATAAGCC AGACCTGAAC ATGAACCAGT


 


TGGTTGCTGA AGTTGAACAA AGCCTGAACA AAAAGATCCC TTCTATGCAC


 


TTGCCTTACC CACTAGGAAT GCTGGGTGGA TATTGCTTTG ATATCCTGAG


 


CAAAATTACG GGCAAAAAAT ACGCTGTCAG CTCTGTGCGC GTGAAAAAAT


 


TCTGCGCAAC AACACAGTTT GACGCAACGA AAGTGCATTC TTCAGGTTTT


 


GTGGCACCGT ATACGCTGTC GCAAGGTCTG GATCGAACTC TGCAGTATGA


 


ATTCGTCCAT GCCAAAAAAG ACGACATAAC GTTTGTTTCT GAG


 


Amino Acid Sequence for E. coli O86 UDP-GlcNAc C4-epimerase


Locus AA037706


Definition UDP-GlcNAc C4-epimerase [Escherichia coli O86].


Accession AAO37706


Length: 331 aa linear


Type: PRT


Organism: E. coli O86


Sequence:


SEQ ID NO: 6


mndnvlliga sgfvgtrlle taiadfnikn ldkqqshfyp eitqigdvrd


 


qqaldqalag fdtvvllaae hrddvsptsl yydvnvqgtr nvlaamekng


 


vkniiftssv avyglnkhnp denhphdpfn hygkskwqae evlrewynka


 


ptersltiir ptvifgernr gnvynllkqi aggkfmmvga gtnyksmayv


 


gnivefikyk lknvaagyev ynyvdkpdln mnqlvaeveq slnkkipsmh


 


lpyplgmlgg ycfdilskit gkkyayssvr vkkfcattqf datkvhssgf


 


vapytlsqgl drtlqyefvh akkdditfvs e


 


Nucleotide Sequence for Shigella boydii O18 gne


Locus ACD09753 BCT 5 May 2008


Definition UDP-N-acetylglucosamine 4-epimerase 


[Shigella boydii CDC 3083-94].


Accession ACD09753


Length: 993


Type: DNA


Organism: Shigella boydii O18


Sequence:


SEQ ID NO: 7


ATGAACGATA ACGTTTTGCT CATAGGAGCT TCCGGATTCG TAGGAACCCG


 


ACTACTTGAA ACGGCAATTG CTGACTTTAA TATCAAGAAC CTGGACAAAC


 


AGCAGAGCCA TTTTTATCCA GCAATCACAC AGATTGGCGA TGTTCGTGAT


 


CAACAGGCAC TCGACCAGGC GTTAGCCGGT TTTGACACTG TTGTACTACT


 


GGCAGCGGAA CACCGCGATG ACGTCAGCCC TACTTCTCTC TATTATGATG


 


TCAACGTTCA GGGTACCCGC AATGTGCTGG CGGCCATGGA AAAAAATGGC


 


GTTAAAAATA TCATCTTTAC CAGTTCCGTT GCTGTTTATG GTTTGAACAA


 


ACACAACCCT GACGAAAACC ATCCACACGA CCCTTTCAAC CACTACGGCA


 


AAAGTAAGTG GCAGGCAGAG GAAGTGCTGC GTGAATGGTA TAACAAAGCA


 


CCAACAGAAC GTTCATTAAC CATCATCCGT CCTACCGTTA TCTTCGGTGA


 


ACGCAACCGC GGTAACGTCT ATAACTTGCT GAAACAGATC GCTGGCGGCA


 


AGTTTATGAT GGTGGGCGCA GGGACTAACT ATAAGTCCAT GGCTTATGTT


 


GGAAACATTG TTGAGTTTAT CAAGTACAAA CTGAAGAATG TTGCCGCAGG


 


TTATGAGGTT TATAACTATG TTGATAAGCC AGACCTGAAC ATGAACCAGT


 


TGGTTGCTGA AGTTGAACAA AGCCTGAACA AAAAGATCCC TTCTATGCAC


 


TTGCCTTACC CACTAGGAAT GCTGGGTGGA TATTGCTTTG ATATCCTGAG


 


CAAAATTACG GGCAAAAAAT ACGCTGTCAG CTCTGTGCGC GTGAAAAAAT


 


TCTGCGCAAC AACACAGTTT GACGCAACGA AAGTGCATTC TTCAGGTTTT


 


GTGGCACCGT ATACGCTGTC GCAAGGTCTG GATCGAACTC TGCAGTATGA


 


ATTCGTCCAT GCCAAAAAAG ACGACATAAC GTTTGTTTCT GAG


 


Amino Acid Sequence for Shigella boydii O18 UDP-N-


acetylglucosamine 4-epimerase


Locus ACD09753


Definition UDP-N-acetylglucosamine 4-epimerase 


[Shigella boydii CDC 3083-94].


Accession ACD09753


Length: 331 aa linear


Type: PRT


Organism: Shigella boydii O18


Sequence:


SEQ ID NO: 8


mndnvlliga sgfvgtrile taiadfnikn ldkggshfyp aitqigdvrd


 


qqaldqalag fdtvvliaae hrddvsptsi yydvnvqgtr nvlaamekng


 


vkniiftssv avyglnkhnp denhphdpfn hygkskwqae evirewynka


 


ptersltiir ptvifgernr gnvynllkqi aggkfmmvga gtnyksmayv


 


gnivefikyk lknvaagyev ynyvdkpdln mnqlvaeveq sinkkipsmh


 


lpyplgmlgg ycfdilskit gkkyayssvr vkkfcattqf datkvhssgf


 


vapytlsggl drtlqyefvh akkdditfvs


 


Nucleotide Sequence for Salmonella enterica O30 gne


Locus AAV34516 BCT 25 Oct. 2004


Definition UDP-GlcNAc 4-epimerase 


[Salmonella enterica subsp. salamae serovar Greenside].


Accession AAV34516


Length: 993


Type: DNA


Organism: Salmonella enterica O30


Sequence:


SEQ ID NO: 9


ATGAACGATA ACGTTTTGCT CATTGGTGCT TCCGGATTCG TAGGAACCCG


 


ACTCCTTGAA ACGGCAGTGG ATGATTTTAA TATCAAGAAC CTGGATAAAC


 


AGCAAAGCCA TTTCTACCCA GAGATTACAC ACATTGGCGA TGTTCGTGAC


 


CAACAAATCC TTGACCAGAC GTTGGTGGGT TTTGACACCG TAGTACTATT


 


GGCTGCGGAG CATCGTGATG ATGTTAGTCC TACCTCGCTT TATTATGATG


 


TCAACGTCCA GGGAACGCGT AATGTACTGG CGGCGATGGA AAAAAATGGT


 


GTAAAAAATA TCATTTTTAC CAGTTCCGTT GCAGTTTATG GACTCAACAA


 


GAAAAATCCT GACGAAACGC ACCCTCACGA TCCCTTTAAT CATTACGGAA


 


AAAGTAAATG GCAAGCAGAA GAAGTTCTGC GTGAGTGGCA TGCTAAAGCG


 


CCGAATGAGC GTTCTTTGAC CATAATTCGT CCTACCGTTA TTTTCGGGGA


 


GCGTAACCGC GGTAATGTAT ACAATCTCTT GAAACAGATC GCTGGTGGTA


 


AATTTGCGAT GGTTGGTCCG GGAACTAACT ATAAATCAAT GGCTTATGTT


 


GGTAATATCG TTGAGTTTAT CAAATTCAAA CTCAAGAATG TTACGGCGGG


 


CTATGAAGTT TATAATTATG TTGATAAACC TGATCTGAAT ATGAATCAAT


 


TGGTTGCTGA AGTAGAGCAG AGCCTGGGCA AAAAAATACC ATCGATGCAC


 


CTTCCATATC CATTAGGTAT GCTGGGGGGT TACTGTTTCG ATATCCTGAG


 


CAAAGTAACG GGCAAGAAGT ACGCTGTAAG TTCGGTTCGT GTTAAAAAAT


 


TCTGTGCGAC AACGCAGTTT GATGCAACAA AAGTGCATTC TTCTGGTTTT


 


GTTGCGCCAT ACACCTTATC TCAGGGGTTG GATCGTACAC TGCAATATGA


 


ATTTGTTCAT GCAAAGAAAG ATGACATTAC ATTCGTTTCA GAG


 


Amino Acid Sequence for Salmonella enterica O30 UDP-


GlcNAc 4-epimerase


Locus AAV34516


Definition UDP-GlcNAc 4-epimerase


[Salmonella enterica subsp. salamae serovar Greenside].


Accession AAV34516


Length: 331 aa linear


Type: PRT


Organism: Salmonella enterica O30


Sequence:


SEQ ID NO: 10


mndnviliga sgfvgtrlle tavddfnikn ldkggshfyp eithigdvrd


 


ggildgtivg fdtvvilaae hrddvsptsl yydvnvqgtr nvlaamekng


 


vkniiftssv avyglnkknp dethphdpfn hygkskwgae evlrewhaka


 


pnersltiir ptvifgernr gnvyralkgi aggkfamvgp gtnyksmayv


 


gnivefikfk lknvtagyev ynywdkpdln mnglvaeveg slgkkipsmh


 


lpyplgmlgg ycfdilskvt gkkyayssvr vkkfcattqf datkvhssgf


 


vapytlsggl drtlgyefvh akkdditfvs e


 


Nucleotide Sequence for C. jejuni gne


Locus YP_002344524 BCT 14 Sep. 2010


Definition UDP-GlcNAc/Glc 4-epimerase 


[Campylobacter jejuni subsp. jejuni


Accession YP_002344524


Length: 987


Type: DNA


Organism: C. jejuni


Sequence:


SEQ ID NO: 11


ATGAAAATTCTTATTAGCGGTGGTGCAGGTTATATAGGTTCTCATACTTTAAGACAATT


 


TTTAAAAACAGATCATGAAATTTGTGTTTTAGATAATCTTTCTAAGGGTTCTAAAATCG


 


CAATAGAAGATTTGCAAAAAACAAGAGCTTTTAAATTTTTCGAACAAGATTTAAGTGAT


 


TTTCAAGGCGTAAAAGCATTGTTTGAGAGAGAAAAATTTGACGCTATTGTGCATTTTGC


 


AGCAAGCATTGAAGTTTTTGAAAGTATGCAAAATCCTTTAAAATATTATATGAACAACA


 


CTGTTAATACGACAAATCTCATCGAAACTTGTTTGCAAACTGGAGTGAATAAATTTATA


 


TTTTCTTCAACGGCGGCCACTTATGGCGAACCACAAACTCCCGTTGTGAGCGAAACAAG


 


TCCTTTAGCACCTATTAATCCTTATGGGCGTAGTAAGCTTATGAGTGAAGAAGTTTTGC


 


GTGATGCAAGTATGGCAAATCCTGAATTTAAGCATTGTATTTTAAGATATTTTAATGTT


 


GCAGGTGCTTGTATGGATTATACTTTAGGACAACGCTATCCAAAAGCGACTTTGCTTAT


 


AAAAGTTGCAGCTGAATGTGCCGCAGGAAAACGTGATAAACTTTTCATATTTGGCGATG


 


ATTATGATACAAAAGATGGTACTTGCATAAGAGATTTTATCCATGTAGATGATATTTCA


 


AGTGCACATTTAGCGGCTTTGGATTATTTAAAAGAGAATGAAAGCAATGTTTTTAATGT


 


AGGTTATGGACATGGTTTTAGCGTAAAAGAAGTGATTGAAGCGATGAAAAAAGTTAGCG


 


GAGTGGATTTTAAAGTAGAACTTGCCCCACGCCGTGCGGGTGATCCTAGTGTATTGATT


 


TCTGATGCAAGTAAAATCAGAAATCTTACTTCTTGGCAGCCTAAATATGATGATTTAGA


 


GCTTATTTGTAAATCTGCTTTTGATTGGGAAAAACAGTGTTAA


 


Amino Acid Sequence for C. jejuni UDP-GlcNAc/Glc 4-epimerase


Locus YP_002344524


Definition UDP-GlcNAc/Glc 4-epimerase 


[Campylobacter jejuni subsp. jejuni


Accession YP_002344524


Length: 328 aa linear


Type: PRT


Organism: C. jejuni


Sequence:


SEQ ID NO: 12


mkilisggag yigshtlrqf lktdheicvl dnlskgskia iedlqktraf


 


kffeqdlsdf qgvkalfere kfdaivhfaa sievfesmqn plkyymnntv


 


nttnlietcl gtgvnkfifs staatygepq tpvvsetspl apinpygrsk


 


imseevirda smanpefkhc ilryfnvaga cmdytlaqry pkatllikva


 


aecaagkrdk ififgddydt kdgtcirdfi hvddissahi aaldylkene


 


snvfnvgygh gfsvkeviea mkkvsgvdfk velaprragd psvlisdask


 


irnltswqpk yddlelicks afdwekqc


 


Nucleotide Sequence for E. coli K12 galE


Locus AP_001390 BCT 30 Apr. 2010


Definition UDP-galactose-4-epimerase 


[Escherichia coli str. K-12 substr. W3110].


Accession AP_001390


Length: 1,017


Type: DNA


Organism: E. coli K12


Sequence:


SEQ ID NO: 13


ATGAGAGTTCTGGTTACCGGTGGTAGCGGTTACATTGGAAGTCATACCTGTGTGCAA


 


TTACTGCAAAACGGTCATGATGTCATCATTCTTGATAACCTCTGTAACAGTAAGCGC


 


AGCGTACTGCCTGTTATCGAGCCTTTTAGGCGGCAAACATCCAACGTTTGTTGAAGG


 


CGATATTCGTAACGAAGCGTTGATGACCGAGATCCTGCACGATCACGCTATCGACAC


 


CGTGATCCACTTCGCCGGGCTGAAAGCCGTGGGCGAATCGGTACAAAAACCGCTGGA


 


ATATTACGACAACAATGTCAACGGCACTCTGCGCCTGATTAGCGCCATGCGCGCCGC


 


TAACGTCAAAAACTTTATTTTTAGCTCCTCCGCCACCGTTTATGGCGATCAGCCCAA


 


AATTCCATACGTTGAAAGCTTCCCGACCGGCACACCGCAAAGCCCTTACGGCAAAAG


 


CAAGCTGATGGTGGAACAGATCCTCACCGATCTGCAAAAAGCCCAGCCGGACTGGAG


 


CATTGCCCTGCTGCGCTACTTCAACCCGGTTGGCGCGCATCCGTCGGGCGATATGGG


 


CGAAGATCCGCAAGGCATTCCGAATAACCTGATGCCATACATCGCCCAGGTTGCTGT


 


AGGCCGTCGCGACTCGCTGGCGATTTTTGGTAACGATTATCCGACCGAAGATGGTAC


 


TGGCGTACGCGATTACATCCACGTAATGGATCTGGCGGACGGTCACGTCGTGGCGAT


 


GGAAAAACTGGCGAACAAGCCAGGCGTACACATCTACAACCTCGGCGCTGGCGTAGG


 


CAACAGCGTGCTGGACGTGGTTAATGCCTTCAGCAAAGCCTGCGGCAAACCGGTTAA


 


TTATCATTTTGCACCGCGTCGCGAGGGCGACCTTCCGGCCTACTGGGCGGACGCCAG


 


CAAAGCCGACCGTGAACTGAACTGGCGCGTAACGCGCACACTCGATGAAATGGCGCA


 


GGACACCTGGCACTGGCAGTCACGCCATCCACAGGGATATCCCGATTAA


 


Amino Acid Sequence for E. coli K12 UDP-galactose-4-epimerase


Locus AP_001390


Definition UDP-galactose-4-epimerase


[Escherichia coli str. K-12 substr. W3110].


Accession AP_001390


Length: 338 aa linear


Type: PRT


Organism: E. coli K12


Sequence:


SEQ ID NO: 14


mrvlvtqgsgyigshtcvqllqnghdviildnlcnskrsvlpvierlggkhptfvegdi


 


rnealmteilhdhaidtvihfaglkavgesvqkpleyydnnvngtlrlisamraanvkn


 


fifsssatvygdqpkipyvesfptgtpqspygksklmveqi1tdlqkaqpdwsiallry


 


fnpvgahpsgdmgedpqgipnnlmpyiaqvavgrrdslaifgndyptedgtgvrdyihv


 


mdladghvvameklankpgvhiynigagvgnsvldvvnafskacgkpvnyhfaprregd


 


lpaywadaskadrelnwrvtrtldemaqdtwhwqsrhpqgypd


 


Nucleotide Sequence for E. coli O86 gne2


Locus AAV85952 BCT 27 Mar. 2005


Definition Gne [Escherichia coli O86[.


Accession AAV85952


Length: 1,020


Type: DNA


Organism: E. coli O86


Sequence:


SEQ ID NO: 15


ATGGTGATTT TCGTAACAGG CGGTGCAGGA TATATTGGAT CCCATACCAT


 


ACTTGAGTTA CTTAATAATC GTCATGATGT CGTTTCGATA GATAATTTTG


 


TCAATTCCTC TATAGAATCA TTAAAAAGAC TAGAGCAAAT AACTAATAAG


 


AAAATTATTT CTTATCAAGG TGATATCCGT GATAAAAATC TACTTGATGA


 


GATTTTTTCA AGACACCATA TCCATGCTGT AATTCACTTT GCATCGTTAA


 


AATCTGTAGG TGAGTCTAAG TTAAAGCCCT TAGAGTATTA TTCTAATAAT


 


GTTGGTGGAA CTTTAGTATT ACTTCAATGC ATGAAGAGAT ATAACATTAA


 


TAAAATGATA TTTAGCTCTT CTGCTACTGT TTATGGGAGT AACAGTATCC


 


CTCCCCATAC GGAAGATAGA CGAATTGGTG AAACTACAAA CCCATATGGG


 


ACATCGAAAT TTATAATAGA AATAATTTTG AGTGATTATT GTGATAGTGA


 


TAATAATAAA TCAGTAATTG CACTGCGTTA CTTTAATCCA ATCGGAGCAC


 


ATAAGTCCGG GATGATTGGT GAAAATCCTA ACGGGATCCC TAATAATCTG


 


GTTCCTTATA TATCTAAAGT TGCACAAAAT CAACTTCCTG TATTAAATAT


 


TTATGGCAAC GATTATCCAA CTAAAGATGG TACAGGAGTA AGAGACTATA


 


TACATGTCTG TGATTTGGCT AAAGGGCATG TTAAAGCATT AGAATATATG


 


TTTTTAAATG ATGTCAATTA TGAAGCTTTT AATTTAGGTA CTGGTCAAGG


 


TTATTCTGTT TTAGAGATTG TAAAAATGTT TGAGATAGTC ACTAAAAAGA


 


GTATACCTGT TGCTATTTGT AATAGACGTG AGGGGGATGT TGCGGAGTCA


 


TGGGCGTCTG CTGATTTGGC ACATAAAAAG CTTTCCTGGA AAGCGCAAAA


 


AAATTTGAAA GAAATGATCG AAGATGTATG GCGTTGGCAA ACAAACAATC


 


CAAATGGATA TAAAAAATAA


 


Amino Acid Sequence for E. coli O86 Gne


Locus AAV85952


Definition Gne [Escherichia coli O86].


Accession AAV85952


Length: 339 aa (gne2) linear


Type: PRT


Organism: E. coli O86


Sequence:


SEQ ID NO: 16


mvifvtggag yigshtilel innghdvvsi dnfvnssies lkrvegitnk


 


kiisyggdir dknlldeifs rhhidavihf aslksvgesk lkpleyysnn


 


vgctivllec mkryninkmi fsssatvygs nsipphtedr rigettnpyg


 


tskfiieiil sdycdsdnnk svialryfnp igahksgmig enpngipnnl


 


vpyiskvaqn qlpviniygn dyptkdgtgv rdyihvcdla kghvkaleym


 


findvnyeaf nlgtgqgysv leivkmfeiv tkksipvaic nrregdvaes


 


wasadlahkk lswkaeknlk emiedvwrwq tnnpngykk


 


Nucleotide Sequence for synthetic oligonucleotide Z3206-


Fw (primer) encoding an end of Z3206; restriction sites underlined


Length: 30


Type: DNA


Sequence:


SEQ ID NO: 17


AAACCCGGGATGAACGATAACGTTTTGCTC


 


Nucleotide Sequence for synthetic oligonucleotide Z3206-


RvHA (primer) encoding an end of Z3206 with a hemoaglutinin 


tag (HA tag); restriction sites underlined


Length: 60


Type: DNA


Organism:


Sequence: 


SEQ ID NO: 18


AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTACTCAGAAACAAACGTTATGTC


 


Nucleotide Sequence for synthetic oligonucleotide gne-Fw


(primer) with restriction sites underlined


Length: 29


Type: DNA


Organism:


Sequence:


SEQ ID NO: 19


AAACCATGGATGAAAATTCTTATTAGCGG


 


Nucleotide Sequence for synthetic oligonucleotide gne-RV


(primer) with restriction sites underlined


Length: 57


Type: DNA


Organism:


Sequence: 


SEQ ID NO: 20


AAATCTAGATTAAGCGTAATCTGGAACATCGTATGGGTAGCACTGTTTTTCCCAATC


 


Nucleotide Sequence for oligonucleotide containing


restriction sites for NheI restriction enzyme


Length: 11


Type: DNA


Organism:


Sequence:


SEQ ID NO: 21


AAAAAGCTAGC


 


Nucleotide Sequence for oligonucleotide containing


restriction sites for AscI restriction enzyme


Length: 8


Type: DNA


Organism:


Sequence:


SEQ ID NO: 22


CCGCGCGG


 


Nucleotide Sequence for plasmid pMLBAD: Z3206 (E. coli O157 


insert in plasmid) encoding Z3206 with a C-terminal hemagglutinin tag


Definition Ligation of product into Z3206-pMLBAD*








Features
    Location/Qualifiers


CDS
    2105..3098



/label=Z3206


CDS
    3098..3127



/label=HA







Length: 7794 bp


Type: DNA circular UNA


Sequence:


SEQ ID NO: 23








    1
TCTACGGGGT CTGACGCTCA GTGGAACGAA ATCGATGAGC TCGCACGAAC CCAGTTGACA


 


   61
TAAGCCTGTT CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC


 


  121
AGAACCTTGA CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT


 


  181
GACTGTTTTT TTGTACAGTC TAGCCTCGGG CATCCAAGCT AGCTAAGCGC GTTACGCCGT


 


  241
GGGTCGATGT TTGATGTTAT GGAACAGCAA CGATGTTACG CAGCAGGGTA GTCGCCCTAA


 


  301
AACAAAGTTA GGCAGCCGTT GTGCTGGTGC TTTCTAGTAG TTGTTGTGGG GTAGGCAGTC


 


  361
AGAGCTCGAT TTGCTTGTCG CCATAATAGA TTCACAAGAA GGATTCGACA TGGGTCAAAG


 


  421
TAGCGATGAA GCCAACGCTC CCGTTGCAGG GCAGTTTGCG CTTCCCCTGA GTGCCACCTT


 


  481
TGGCTTAGGG GATCGCGTAC GCAAGAAATC TGGTGCCGCT TGGCAGGGTC AAGTCGTCGG


 


  541
TTGGTATTGC ACAAAACTCA CTCCTGAAGG CTATGCGGTC GAGTCCGAAT CCCACCCAGG


 


  601
CTCAGTGCAA ATTTATCCTG TGGCTGCACT TGAACGTGTG GCCTAAGCGA TATCTTAGGA


 


  661
TCTCCCATCG GTGATGTCGG CGATATAGGC GCCAGCAACC GCACCTGTGG CGCCGGTGAT


 


  721
GCCGGCCACG ATGCGTCCGG CGTAGAGGAT CTGCTCATGT TTGACAGCTT ATCATCGATG


 


  781
CATAATGTGC CTGTCAAATG GACGAAGCAG GGATTCTGCA AACCCTATGC TACTCCGTCA


 


  841
AGCCGTCAAT TGTCTGAATC GTTACCAATT ATGACAACTT GACGGCTACA TCATTCACTT


 


  901
TTTCTTCACA ACCGGCACGG AACTCGCTCG GGCTGGCCCC GGTGCATTTT TTAAATACCC


 


  961
GCGAGAAATA GAGTTGATCG TCAAAACCAA CATTGCGACC GACGGTGGCG ATAGGCATCC


 


 1021
GGGTGGTGCT CAAAAGCAGC TTCGCCTGGC TGATACGTTG GTCCTCGCGC CAGCTTAAGA


 


 1081
CGCTAATCCC TAACTGCTGG CGGAAAAGAT GTGACAGACG CGACGGCGAC AAGCAAACAT


 


 1141
GCTGTGCGAC GCTGGCGATA TCAAAATTGC TGTCTGCCAG GTGATCGCTG ATGTACTGAC


 


 1201
AAGCCTCGCG TACCCGATTA TCCATCGGTG GATGGAGCGA CTCGTTAATC GCTTCCATGC


 


 1261
GCCGCAGTAA CAATTGCTCA AGCAGATTTA TCGCCAGCAG CTCCGAATAG CGCCCTTCCC


 


 1321
CTTGCCCGGC GTTAATGATT TGCCCAAACA GGTCGCTGAA ATGCGGCTGG TGCGCTTCAT


 


 1381
CCGGGCGAAA GAACCCCGTA TTGGCAAATA TTGACGGCCA GTTAAGCCAT TCATGCCAGT


 


 1441
AGGCGCGCGG ACGAAAGTAA ACCCACTGGT GATACCATTC GCGAGCCTCC GGATGACGAC


 


 1501
CGTAGTGATG AATCTCTCCT GGCGGGAACA GCAAAATATC ACCCGGTCGG CAAACAAATT


 


 1561
CTCGTCCCTG ATTTTTCACC ACCCCCTGAC CGCGAATGGT GAGATTGAGA ATATAACCTT


 


 1621
TCATTCCCAG CGGTCGGTCG ATAAAAAAAT CGAGATAACC GTTGGCCTCA ATCGGCGTTA


 


 1681
AACCCGCCAC CAGATGGGCA TTAAACGAGT ATCCCGGCAG CAGGGGATCA TTTTGCGCTT


 


 1741
CAGCCATACT TTTCATACTC CCGCCATTCA GAGAAGAAAC CAATTGTCCA TATTGCATCA


 


 1301
GACATTGCCG TCACTGCGTC TTTTACTGGC TCTTCTCGCT AACCAAACCG GTAACCCCGC


 


 1861
TTATTAAAAG CATTCTGTAA CAAAGCGGGA CCAAAGCCAT GACAAAAACG CGTAACAAAA


 


 1921
GTGTCTATAA TCACGGCAGA AAAGTCCACA TTGATTATTT GCACGGCGTC ACACTTTGCT


 


 1981
ATGCCATAGC ATTTTTATCC ATAAGATTAG CGGATCCTAC CTGACGCTTT TTATCGCAAC


 


 2041
TCTCTACTGT TTCTCCATAC CCGTTTTTTT GGGCTAGCAG GAGGAATTCA CCATGGTACC


 


 2101
CGGGATGAAC GATAACGTTT TGCTCATAGG AGCTTCCGGA TTCGTAGGAA CCCGACTACT


 


 2161
TGAAACGGCA ATTGCTGACT TTAATATCAA GAACCTGGAC AAACAGCAGA GCCACTTTTA


 


 2221
TCCAGAAATC ACACAGATTG GCGATGTTCG CGATCAACAG GCACTCGACC AGGCGTTAGT


 


 2281
CGGTTTTGAC ACTGTTGTAC TACTGGCAGC GGAACACCGC GATGACGTCA GCCCTACTTC


 


 2341
TCTCTATTAT GATGTCAACG TTCAGGGTAC CCGCAATGTG CTGGCGGCCA TGGAAAAAAA


 


 2401
TGGCGTTAAA AATATCATCT TTACCAGTTC CGTTGCTGTT TATGGTTTGA ACAAACACAA


 


 2461
CCCTGACGAA AACCATCCAC ACGACCCTTT CAACCACTAC GGCAAAAGTA AGTGGCAGGC


 


 2521
AGAGGAAGTG CTGCGTGAAT GGTATAACAA AGCACCAACA GAACGTTCAT TAACCATCAT


 


 2581
CCGTCCTACC GTTATCTTCG GTGAACGCAA CCGCGGTAAC GTCTATAACT TGCTGAAACA


 


 2641
GATCGCTGGC GGCAAGTTTA TGATGGTGGG CGCAGGGACT AACTATAAGT CCATGGCTTA


 


 2701
TGTTGGAAAC ATTGTTGAGT TTATCAAGTA CAAACTGAAG AATGTTGCCG CAGGTTATGA


 


 2761
GGTTTATAAC TACGTTGATA AGCCAGACCT GAACATGAAC CAGTTGGTTG CTGAAGTTGA


 


 2821
ACAAAGCCTG AACAAAAAGA TCCCTTCTAT GCACTTGCCT TACCCACTAG GAATGCTGGG


 


 2881
TGGATATTGC TTTGATATCC TGAGCAAAAT TACGGGCAAA AAATACGCTG TCAGCTCAGT


 


 2941
GCGCGTGAAA AAATTCTGCG CAACAACACA GTTTGACGCA ACGAAAGTGC ATTCTTCAGG


 


 3001
TTTTGTGGCA CCGTATACGC TGTCGCAAGG TCTGGATCGA ACACTGCAGT ATGAATTCGT


 


 3061
TCATGCCAAA AAAGACGACA TAACGTTTGT TTCTGAGTAC CCATACGATG TTCCAGATTA


 


 3121
CGCTTAATCT AGAGTCGACC TGCAGGCATG CAAGCTTGGC TGTTTTGGCG GATGAGAGAA


 


 3181
GATTTTCAGC CTGATACAGA TTAAATCAGA ACGCAGAAGC GGTCTGATAA AACAGAATTT


 


 3241
GCCTGGCGGC AGTAGCGCGG TGGTCCCACC TGACCCCATG CCGAACTCAG AAGTGAAACG


 


 3301
CCGTAGCGCC GATGGTAGTG TGGGGTCTCC CCATGCGAGA GTAGGGAACT GCCAGGCATC


 


 3361
AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT TGTTTGTCGG


 


 3421
TGAACGCTCT CCTGAGTAGG ACAAATCCGC CGGGAGCGGA TTTGAACGTT GCGAAGCAAC


 


 3481
GGCCCGGAGG GTGGCGGGCA GGACGCCCGC CATAAACTGC CAGGCATCAA ATTAAGCAGA


 


 3541
AGGCCATCCT GACGGATGGC CTTTTTGCGT TTCTACAAAC TCTTCCACTC ACTACAGCAG


 


 3601
AGCCATTTAA ACAACATCCC CTCCCCCTTT CCACCGCGTC AGACGCCCGT AGCAGCCCGC


 


 3661
TACGGGCTTT TTCATGCCCT GCCCTAGCGT CCAAGCCTCA CGGCCGCGCT CGGCCTCTCT


 


 3721
GGCGGCCTTC TGGCGCTGAG GTCTGCCTCG TGAAGAAGGT GTTGCTGACT CATACCAGGC


 


 3781
CTGAATCGCC CCATCATCCA GCCAGAAAGT GAGGGAGCCA CGGTTGATGA GAGCTTTGTT


 


 3841
GTAGGTGGAC CAGTTGGTGA TTTTGAACTT TTGCTTTGCC ACGGAACGGT CTGCGTTGTC


 


 3901
GGGAAGATGC GTGATCTGAT CCTTCAACTC AGCAAAAGTT CGATTTATTC AACAAAGCCG


 


 3961
CCGTCCCGTC AAGTCAGCGT AATGCTCTGC CAGTGTTACA ACCAATTAAC CAATTCTGAT


 


 4021
TAGAAAAACT CATCGAGCAT CAAATGAAAC TGCAATTTAT TCATATCAGG ATTATCAATA


 


 4081
CCATATTTTT GAAAAAGCCG TTTCTGTAAT GAAGGAGAAA ACTCACCGAG GCAGTTCCAT


 


 4141
AGGATGGCAA GATCCTGGTA TCGGTCTGCG ATTCCGACTC GTCCAACATC AATACAACCT


 


 4201
ATTAATTTCC CCTCGTCAAA AATAAGGTTA TCAAGCGAGA AATCACCATG AGTGACGACT


 


 4261
GAATCCGGTG AGAATGGCAA AAGCTAAAAA GGCCGTAATA TCCAGCTGAA CGGTCTGGTT


 


 4321
ATAGGTACAT TGAGCAACTG ACTGAAATGC CTCAAAATGT TCTTTACGAT GCCATTGGGA


 


 4381
TATATCAACG GTGGTATATC CAGTGATTTT TTTCTCCATT TTAGCTTCCT TAGCTCCTGA


 


 4441
AAATCTCGAT AACTCAAAAA ATACGCCCGG TAGTGATCTT ATTTCATTAT GGTGAAAGTT


 


 4501
GGAACCTCTT ACGTGCCGAT CAACGTCTCA TTTTCGCCAA AAGTTGGCCC AGGGCTTCCC


 


 4561
GGTATCAACA GGGACACCAG GATTTATTTA TTCTGCGAAG TGATCTTCCG TCACAGGTAT


 


 4621
TTATTCGAAG ACGAAAGGGC CTCGTGATAC GCCTATTTTT ATAGGTTAAT GTCATGATAA


 


 4681
TAATGGTTTC TTAGACGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGCC CGCGTTCCTG


 


 4741
CTGGCGCTGG GCCTGTTTCT GGCGCTGGAC TTCCCGCTGT TCCGTCAGCA GCTTTTCGCC


 


 4801
CACGGCCTTG ATGATCGCGG CGGCCTTGGC CTGCATATCC CGATTCAACG GCCCCAGGGC


 


 4861
GTCCAGAACG GGCTTCAGGC GCTCCCGAAG GTCTCGGGCC GTCTCTTGGG CTTGATCGGC


 


 4921
CTTCTTGCGC ATCTCACGCG CTCCTGCGGC GGCCTGTAGG GCAGGCTCAT ACCCCTGCCG


 


 4981
AACCGCTTTT GTCAGCCGGT CGGCCACGGC TTCCGGCGTC TCAACGCGCT TTGAGATTCC


 


 5041
CAGCTTTTCG GCCAATCCCT GCGGTGCATA GGCGCGTGGC TCGACCGCTT GCGGGCTGAT


 


 5101
GGTGACGTGG CCCACTGGTG GCCGCTCCAG GGCCTCGTAG AACGCCTGAA TGCGCGTGTG


 


 5161
ACGTGCCTTG CTGCCCTCGA TGCCCCGTTG CAGCCCTAGA TCGGCCACAG CGGCCGCAAA


 


 5221
CGTGGTCTGG TCGCGGGTCA TCTGCGCTTT GTTGCCGATG AACTCCTTGG CCGACAGCCT


 


 5281
GCCGTCCTGC GTCAGCGGCA CCACGAACGC GGTCATGTGC GGGCTGGTTT CGTCACGGTG


 


 5341
GATGCTGGCC GTCACGATGC GATCCGCCCC GTACTTGTCC GCCAGCCACT TGTGCGCCTT


 


 5401
CTCGAAGAAC GCCGCCTGCT GTTCTTGGCT GGCCGACTTC CACCATTCCG GGCTGGCCGT


 


 5461
CATGACGTAC TCGACCGCCA ACACAGCGTC CTTGCGCCGC TTCTCTGGCA GCAACTCGCG


 


 5521
CAGTCGGCCC ATCGCTTCAT CGGTGCTGCT GGCCGCCCAG TGCTCGTTCT CTGGCGTCCT


 


 5581
GCTGGCGTCA GCGTTGGGCG TCTCGCGCTC GCGGTAGGCG TGCTTGAGAC TGGCCGCCAC


 


 5641
GTTGCCCATT TTCGCCAGCT TCTTGCATCG CATGATCGCG TATGCCGCCA TGCCTGCCCC


 


 5701
TCCCTTTTGG TGTCCAACCG GCTCGACGGG GGCAGCGCAA GGCGGTGCCT CCGGCGGGCC


 


 5761
ACTCAATGCT TGAGTATACT CACTAGACTT TGCTTCGCAA AGTCGTGACC GCCTACGGCG


 


 5821
GCTGCGGCGC CCTACGGGCT TGCTCTCCGG GCTTCGCCCT GCGCGGTCGC TGCGCTCCCT


 


 5881
TGCCAGCCCG TGGATATGTG GACGATGGCC GCGAGCGGCC ACCGGCTGGC TCGCTTCGCT


 


 5941
CGGCCCGTGG ACAACCCTGC TGGACAAGCT GATGGACAGG CTGCGCCTGC CCACGAGCTT


 


 6001
GACCACAGGG ATTGCCCACC GGCTACCCAG CCTTCGACCA CATACCCACC GGCTCCAACT


 


 6061
GCGCGGCCTG CGGCCTTGCC CCATCAATTT TTTTAATTTT CTCTGGGGAA AAGCCTCCGG


 


 6121
CCTGCGGCCT GCGCGCTTCG CTTGCCGGTT GGACACCAAG TGGAAGGCGG GTCAAGGCTC


 


 6181
GCGCAGCGAC CGCGCAGCGG CTTGGCCTTG ACGCGCCTGG AACGACCCAA GCCTATGCGA


 


 6241
GTGGGGGCAG TCGAAGGCGA AGCCCGCCCG CCTGCCCCCC GAGCCTCACG GCGGCGAGTG


 


 6301
CGGGGGTTCC AAGGGGGCAG CGCCACCTTG GGCAAGGCCG AAGGCCGCGC AGTCGATCAA


 


 6361
CAAGCCCCGG AGGGGCCACT TTTTGCCGGA GGGGGAGCCG CGCCGAAGGC GTGGGGGAAC


 


 6421
CCCGCAGGGG TGCCCTTCTT TGGGCACCAA AGAACTAGAT ATAGGGCGAA ATGCGAAAGA


 


 6481
CTTAAAAATC AACAACTTAA AAAAGGGGGG TACGCAACAG CTCATTGCGG CACCCCCCGC


 


 6541
AATAGCTCAT TGCGTAGGTT AAAGAAAATC TGTAATTGAC TGCCACTTTT ACGCAACGCA


 


 6601
TAATTGTTGT CGCGCTGCCG AAAAGTTGCA GCTGATTGCG CATGGTGCCG CAACCGTGCG


 


 6661
GCACCCTACC GCATGGAGAT AAGCATGGCC ACGCAGTCCA GAGAAATCGG CATTCAAGCC


 


 6721
AAGAACAAGC CCGGTCACTG GGTGCAAACG GAACGCAAAG CGCATGAGGC GTGGGCCGGG


 


 6781
CTTATTGCGA GGAAACCCAC GGCGGCAATG CTGCTGCATC ACCTCGTGGC GCAGATGGGC


 


 6841
CACCAGAACG CCGTGGTGGT CAGCCAGAAG ACACTTTCCA AGCTCATCGG ACGTTCTTTG


 


 6901
CGGACGGTCC AATACGCAGT CAAGGACTTG GTGGCCGAGC GCTGGATCTC CGTCGTGAAG


 


 6961
CTCAACGGCC CCGGCACCGT GTCGGCCTAC GTGGTCAATG ACCGCGTGGC GTGGGGCCAG


 


 7021
CCCCGCGACC AGTTGCGCCT GTCGGTGTTC AGTGCCGCCG TGGTGGTTGA TCACGACGAC


 


 7081
CAGGACGAAT CGCTGTTGGG GCATGGCGAC CTGCGCCGCA TCCCGACCCT GTATCCGGGC


 


 7141
GAGCAGCAAC TACCGACCGG CCCCGGCGAG GAGCCGCCCA GCCAGCCCGG CATTCCGGGC


 


 7201
ATGGAACCAG ACCTGCCAGC CTTGACCGAA ACGGAGGAAT GGGAACGGCG CGGGCAGCAG


 


 7261
CGCCTGCCGA TGCCCGATGA GCCGTGTTTT CTGGACGATG GCGAGCCGTT GGAGCCGCCG


 


 7321
ACACGGGTCA CGCTGCCGCG CCGGTAGCAC TTGGGTTGCG CAGCAACCCG TAAGTGCGCT


 


 7381
GTTCCAGACT ATCGGCTGTA GCCGCCTCGC CGCCCTATAC CTTGTCTGCC TCCCCGCGTT


 


 7441
GCGTCGCGGT GCATGGAGCC GGGCCACCTC GACCTGAATG GAAGCCGGCG GCACCTCGCT


 


 7501
AACGGATTCA CCGTTTTTAT CAGGCTCTGG GAGGCAGAAT AAATGATCAT ATCGTCAATT


 


 7561
ATTACCTCCA CGGGGAGAGC CTGAGCAAAC TGGCCTCAGG CATTTGAGAA GCACACGGTC


 


 7621
ACACTGCTTC CGGTAGTCAA TAAACCGGTA AACCAGCAAT AGACATAAGC GGCTATTTAA


 


 7681
CGACCCTGCC CTGAACCGAC GACCGGGTCG AATTTGCTTT CGAATTTCTG CCATTCATCC


 


 7741
GCTTATTATC ACTTATTCAG GCGTAGCACC AGGCGTTTAA GTCGACCAAT AACC


 







Nucleotide Sequence for pMLBAD: gne (E. coli O157 insert


in plasmid) which encodes Gne with a C-terminal hemagglutinin tag


Locus gne-pMLBAD


Definition Ligation of dig galE into pmlbad did (NcoI-XbaI)








Features
    Location/Qualifiers


CDS
    2097..3080



/label=galE


CDS
    3081..3107



/label=HA


Region
    3108..3110



/label=stop







Length: 7776 bp


Type: DNA circular UNA


Sequence:


SEQ ID NO: 24








    1
TCTACGGGGT CTGACGCTCA GTGGAACGAA ATCGATGAGC TCGCACGAAC CCAGTTGACA


 


   61
TAAGCCTGTT CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC


 


  121
AGAACCTTGA CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT


 


  181
GACTGTTTTT TTGTACAGTC TAGCCTCGGG CATCCAAGCT AGCTAAGCGC GTTACGCCGT


 


  241
GGGTCGATGT TTGATGTTAT GGAACAGCAA CGATGTTACG CAGCAGGGTA GTCGCCCTAA


 


  301
AACAAAGTTA GGCAGCCGTT GTGCTGGTGC TTTCTAGTAG TTGTTGTGGG GTAGGCAGTC


 


  361
AGAGCTCGAT TTGCTTGTCG CCATAATAGA TTCACAAGAA GGATTCGACA TGGGTCAAAG


 


  421
TAGCGATGAA GCCAACGCTC CCGTTGCAGG GCAGTTTGCG CTTCCCCTGA GTGCCACCTT


 


  481
TGGCTTAGGG GATCGCGTAC GCAAGAAATC TGGTGCCGCT TGGCAGGGTC AAGTCGTCGG


 


  541
TTGGTATTGC ACAAAACTCA CTCCTGAAGG CTATGCGGTC GAGTCCGAAT CCCACCCAGG


 


  601
CTCAGTGCAA ATTTATCCTG TGGCTGCACT TGAACGTGTG GCCTAAGCGA TATCTTAGGA


 


  661
TCTCCCATCG GTGATGTCGG CGATATAGGC GCCAGCAACC GCACCTGTGG CGCCGGTGAT


 


  721
GCCGGCCACG ATGCGTCCGG CGTAGAGGAT CTGCTCATGT TTGACAGCTT ATCATCGATG


 


  781
CATAATGTGC CTGTCAAATG GACGAAGCAG GGATTCTGCA AACCCTATGC TACTCCGTCA


 


  841
AGCCGTCAAT TGTCTGATTC GTTACCAATT ATGACAACTT GACGGCTACA TCATTCACTT


 


  901
TTTCTTCACA ACCGGCACGG AACTCGCTCG GGCTGGCCCC GGTGCATTTT TTAAATACCC


 


  961
GCGAGAAATA GAGTTGATCG TCAAAACCAA CATTGCGACC GACGGTGGCG ATAGGCATCC


 


 1021
GGGTGGTGCT CAAAAGCAGC TTCGCCTGGC TGATACGTTG GTCCTCGCGC CAGCTTAAGA


 


 1081
CGCTAATCCC TAACTGCTGG CGGAAAAGAT GTGACAGACG CGACGGCGAC AAGCAAACAT


 


 1141
GCTGTGCGAC GCTGGCGATA TCAAAATTGC TGTCTGCCAG GTGATCGCTG ATGTACTGAC


 


 1201
AAGCCTCGCG TACCCGATTA TCCATCGGTG GATGGAGCGA CTCGTTAATC GCTTCCATGC


 


 1261
GCCGCAGTAA CAATTGCTCA AGCAGATTTA TCGCCAGCAG CTCCGAATAG CGCCCTTCCC


 


 1321
CTTGCCCGGC GTTAATGATT TGCCCAAACA GGTCGCTGAA ATGCGGCTGG TGCGCTTCAT


 


 1381
CCGGGCGAAA GAACCCCGTA TTGGCAAATA TTGACGGCCA GTTAAGCCAT TCATGCCAGT


 


 1441
AGGCGCGCGG ACGAAAGTAA ACCCACTGGT GATACCATTC GCGAGCCTCC GGATGACGAC


 


 1501
CGTAGTGATG AATCTCTCCT GGCGGGAACA GCAAAATATC ACCCGGTCGG CAAACAAATT


 


 1561
CTCGTCCCTG ATTTTTCACC ACCCCCTGAC CGCGAATGGT GAGATTGAGA ATATAACCTT


 


 1621
TCATTCCCAG CGGTCGGTCG ATAAAAAAAT CGAGATAACC CTTGGCCTCA ATCGGCGTTA


 


 1681
AACCCGCCAC CAGATGGGCA TTAAACGAGT ATCCCGGCAG CAGGGGATCA TTTTGCGCTT


 


 1741
CAGCCATACT TTTCATACTC CCGCCATTCA GAGAAGAAAC CAATTGTCCA TATTGCATCA


 


 1801
GACATTGCCG TCACTGCGTC TTTTACTGGC TCTTCTCGCT AACCAAACCG GTAACCCCGC


 


 1861
TTATTAAAAG CATTCTGTAA CAAAGCGGGA CCAAAGCCAT GACAAAAACG CGTAACAAAA


 


 1921
GTGTCTATAA TCACGGCAGA AAAGTCCACA TTGATTATTT GCACGGCGTC ACACTTTGCT


 


 1981
ATGCCATAGC ATTTTTATCC ATAAGATTAG CGGATCCTAC CTGACGCTTT TTATCGCAAC


 


 2041
TCTCTACTGT TTCTCCATAC CCGTTTTTTT GGGCTAGCAG GAGGAATTCA CCATGGATGA


 


 2101
AAATTCTTAT TAGCGGTGGT GCAGGTTATA TAGGTTCTCA TACTTTAAGA CAATTTTTAA


 


 2161
AAACAGATCA TGAAATTTGT GTTTTAGATA ATCTTTCTAA GGGTTCTAAA ATCGCAATAG


 


 2221
AAGATTTGCA AAAAATAAGA ACTTTTAAAT TTTTTGAACA AGATTTAAGT GATTTTCAAG


 


 2281
GCGTAAAAGC ATTGTTTGAG AGAGAAAAAT TTGACGCTAT TGTGCATTTT GCAGCGAGCA


 


 2341
TTGAAGTTTT TGAAAGTATG CAAAACCCTT TAAAGTATTA TATGAATAAC ACTGTTAATA


 


 2401
CGACAAATCT CATCGAAACT TGTTTGCAAA CTGGAGTGAA TAAATTTATA TTTTCTTCAA


 


 2461
CGGCAGCCAC TTATGGCGAA CCACAAACTC CCGTTGTGAG CGAAACAAGT CCTTTAGCAC


 


 2521
CTATTAATCC TTATGGGCGT AGTAAGCTTA TGAGCGAAGA GGTTTTGCGT GATGCAAGTA


 


 2581
TGGCAAATCC TGAATTTAAG CATTGTATTT TAAGATATTT TAATGTTGCA GGTGCTTGCA


 


 2641
TGGATTATAC TTTAGGACAA CGCTATCCAA AAGCGACTTT GCTTATAAAA GTTGCAGCTG


 


 2701
AATGTGCCGC AGAAAAACGT AATAAACTTT TCATATTTGG CGATGATTAT GATACAAAAG


 


 2761
ATGGCACTTG CATAAGAGAT TTTATCCATG TGGATGATAT TTCAAGTGCG CATTTATCGG


 


 2821
CTTTGGATTA TTTAAAAGAG AATGAAAGCA ATGTTTTTAA TGTAGGTTAT GGACATGGTT


 


 2881
TTAGCGTAAA AGAAGTGATT GAAGCGATGA AAAAAGTTAG CGGAGTGGAT TTTAAAGTAG


 


 2941
AACTTGCCCC ACGCCGTGCG GGTGATCCTA GTGTATTGAT TTCTGATGCA AGTAAAATCA


 


 3001
GAAATCTTAC TTCTTGGCAG CCTAAATATG ATGATTTAGG GCTTATTTGT AAATCTGCTT


 


 3061
TTGATTGGGA AAAACAGTGC TACCCATACG ATGTTCCAGA TTACGCTTAA TCTAGAGTCG


 


 3121
ACCTGCAGGC ATGCAAGCTT GGCTGTTTTG GCGGATGAGA GAAGATTTTC AGCCTGATAC


 


 3181
AGATTAAATC AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG


 


 3241
CGGTGGTCCC ACCTGACCCC ATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA


 


 3301
GTGTGGGGTC TCCCCATGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT


 


 3361
CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGTGAACGC TCTCCTGAGT


 


 3421
AGGACAAATC CGCCGGGAGC GGATTTGAAC GTTGCGAAGC AACGGCCCGG AGGGTGGCGG


 


 3481
GCAGGACGCC CGCCATAAAC TGCCAGGCAT CAAATTAAGC AGAAGGCCAT CCTGACGGAT


 


 3541
GGCCTTTTTG CGTTTCTACA AACTCTTCCA CTCACTACAG CAGAGCCATT TAAACAACAT


 


 3601
CCCCTCCCCC TTTCCACCGC GTCAGACGCC CGTAGCAGCC CGCTACGGGC TTTTTCATGC


 


 3661
CCTGCCCTAG CGTCCAAGCC TCACGGCCGC GCTCGGCCTC TCTGGCGGCC TTCTGGCGCT


 


 3721
GAGGTCTGCC TCGTGAAGAA GGTGTTGCTG ACTCATACCA GGCCTGAATC GCCCCATCAT


 


 3781
CCAGCCAGAA AGTGAGGGAG CCACGGTTGA TGAGAGCTTT GTTGTAGGTG GACCAGTTGG


 


 3841
TGATTTTGAA CTTTTGCTTT GCCACGGAAC GGTCTGCGTT GTCGGGAAGA TGCGTGATCT


 


 3901
GATCCTTCAA CTCAGCAAAA GTTCGATTTA TTCAACAAAG CCGCCGTCCC GTCAAGTCAG


 


 3961
CGTAATGCTC TGCCAGTGTT ACAACCAATT AACCAATTCT GATTAGAAAA ACTCATCGAG


 


 4021
CATCAAATGA AACTGCAATT TATTCATATC AGGATTATCA ATACCATATT TTTGAAAAAG


 


 4081
CCGTTTCTGT AATGAAGGAG AAAACTCACC GAGGCAGTTC CATAGGATGG CAAGATCCTG


 


 4141
GTATCGGTCT GCGATTCCGA CTCGTCCAAC ATCAATACAA CCTATTAATT TCCCCTCGTC


 


 4201
AAAAATAAGG TTATCAAGCG AGAAATCACC ATGAGTGACG ACTGAATCCG GTGAGAATGG


 


 4261
CAAAAGCTAA AAAGGCCGTA ATATCCAGCT GAACGGTCTG GTTATAGGTA CATTGAGCAA


 


 4321
CTGACTGAAA TGCCTCAAAA TGTTCTTTAC GATGCCATTG GGATATATCA ACGGTGGTAT


 


 4381
ATCCAGTGAT TTTTTTCTCC ATTTTAGCTT CCTTAGCTCC TGAAAATCTC GATAACTCAA


 


 4441
AAAATACGCC CGGTAGTGAT CTTATTTCAT TATGGTGAAA GTTGGAACCT CTTACGTGCC


 


 4501
GATCAACGTC TCATTTTCGC CAAAAGTTGG CCCAGGGCTT CCCGGTATCA ACAGGGACAC


 


 4561
CAGGATTTAT TTATTCTGCG AAGTGATCTT CCGTCACAGG TATTTATTCG AAGACGAAAG


 


 4621
GGCCTCGTGA TACGCCTATT TTTATAGGTT AATGTCATGA TAATAATGGT TTCTTAGACG


 


 4681
TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GCCCGCGTTC CTGCTGGCGC TGGGCCTGTT


 


 4741
TCTGGCGCTG GACTTCCCGC TGTTCCGTCA GCAGCTTTTC GCCCACGGCC TTGATGATCG


 


 4801
CGGCGGCCTT GGCCTGCATA TCCCGATTCA ACGGCCCCAG GGCGTCCAGA ACGGGCTTCA


 


 4861
GGCGCTCCCG AAGGTCTCGG GCCGTCTCTT GGGCTTGATC GGCCTTCTTG CGCATCTCAC


 


 4921
GCGCTCCTGC GGCGGCCTGT AGGGCAGGCT CATACCCCTG CCGAACCGCT TTTGTCAGCC


 


 4981
GGTCGGCCAC GGCTTCCGGC GTCTCAACGC GCTTTGAGAT TCCCAGCTTT TCGGCCAATC


 


 5041
CCTGCGGTGC ATAGGCGCGT GGCTCGACCG CTTGCGGGCT GATGGTGACG TGGCCCACTG


 


 5101
GTGGCCGCTC CAGGGCCTCG TAGAACGCCT GAATGCGCGT GTGACGTGCC TTGCTGCCCT


 


 5161
CGATGCCCCG TTGCAGCCCT AGATCGGCCA CAGCGGCCGC AAACGTGGTC TGGTCGCGGG


 


 5221
TCATCTGCGC TTTGTTGCCG ATGAACTCCT TGGCCGACAG CCTGCCGTCC TGCGTCAGCG


 


 5281
GCACCACGAA CGCGGTCATG TGCGGGCTGG TTTCGTCACG GTGGATGCTG GCCGTCACGA


 


 5341
TGCGATCCGC CCCGTACTTG TCCGCCAGCC ACTTGTGCGC CTTCTCGAAG AACGCCGCCT


 


 5401
GCTGTTCTTG GCTGGCCGAC TTCCACCATT CCGGGCTGGC CGTCATGACG TACTCGACCG


 


 5461
CCAACACAGC GTCCTTGCGC CGCTTCTCTG GCAGCAACTC GCGCAGTCGG CCCATCGCTT


 


 5521
CATCGGTGCT GCTGGCCGCC CAGTGCTCGT TCTCTGGCGT CCTGCTGGCG TCAGCGTTGG


 


 5581
GCGTCTCGCG CTCGCGGTAG GCGTGCTTGA GACTGGCCGC CACGTTGCCC ATTTTCGCCA


 


 5641
GCTTCTTGCA TCGCATGATC GCGTATGCCG CCATGCCTGC CCCTCCCTTT TGGTGTCCAA


 


 5701
CCGGCTCGAC GGGGGCAGCG CAAGGCGGTG CCTCCGGCGG GCCACTCAAT GCTTGAGTAT


 


 5761
ACTCACTAGA CTTTGCTTCG CAAAGTCGTG ACCGCCTACG GCGGCTGCGG CGCCCTACGG


 


 5821
GCTTGCTCTC CGGGCTTCGC CCTGCGCGGT CGCTGCGCTC CCTTGCCAGC CCGTGGATAT


 


 5881
GTGGACGATG GCCGCGAGCG GCCACCGGCT GGCTCGCTTC GCTCGGCCCG TGGACAACCC


 


 5941
TGCTGGACAA GCTGATGGAC AGGCTGCGCC TGCCCACGAG CTTGACCACA GGGATTGCCC


 


 6001
ACCGGCTACC CAGCCTTCGA CCACATACCC ACCGGCTCCA ACTGCGCGGC CTGCGGCCTT


 


 6061
GCCCCATCAA TTTTTTTAAT TTTCTCTGGG GAAAAGCCTC CGGCCTGCGG CCTGCGCGCT


 


 6121
TCGCTTGCCG GTTGGACACC AAGTGGAAGG CGGGTCAAGG CTCGCGCAGC GACCGCGCAG


 


 6181
CGGCTTGGCC TTGACGCGCC TGGAACGACC CAAGCCTATG CGAGTGGGGG CAGTCGAAGG


 


 6241
CGAAGCCCGC CCGCCTGCCC CCCGAGCCTC ACGGCGGCGA GTGCGGGGGT TCCAAGGGGG


 


 6301
CAGCGCCACC TTGGGCAAGG CCGAAGGCCG CGCAGTCGAT CAACAAGCCC CGGAGGGGCC


 


 6361
ACTTTTTGCC GGAGGGGGAG CCGCGCCGAA GGCGTGGGGG AACCCCGCAG GGGTGCCCTT


 


 6421
CTTTGGGCAC CAAAGAACTA GATATAGGGC GAAATGCGAA AGACTTAAAA ATCAACAACT


 


 6481
TAAAAAAGGG GGGTACGCAA CAGCTCATTG CGGCACCCCC CGCAATAGCT CATTGCGTAG


 


 6541
GTTAAAGAAA ATCTGTAATT GACTGCCACT TTTACGCAAC GCATAATTGT TGTCGCGCTG


 


 6601
CCGAAAAGTT GCAGCTGATT GCGCATGGTG CCGCAACCGT GCGGCACCCT ACCGCATGGA


 


 6661
GATAAGCATG GCCACGCAGT CCAGAGAAAT CGGCATTCAA GCCAAGAACA AGCCCGGTCA


 


 6721
CTGGGTGCAA ACGGAACGCA AAGCGCATGA GGCGTGGGCC GGGCTTATTG CGAGGAAACC


 


 6781
CACGGCGGCA ATGCTGCTGC ATCACCTCGT GGCGCAGATG GGCCACCAGA ACGCCGTGGT


 


 6841
GGTCAGCCAG AAGACACTTT CCAAGCTCAT CGGACGTTCT TTGCGGACGG TCCAATACGC


 


 6901
AGTCAAGGAC TTGGTGGCCG AGCGCTGGAT CTCCGTCGTG AAGCTCAACG GCCCCGGCAC


 


 6961
CGTGTCGGCC TACGTGGTCA ATGACCGCGT GGCGTGGGGC CAGCCCCGCG ACCAGTTGCG


 


 7021
CCTGTCGGTG TTCAGTGCCG CCGTGGTGGT TGATCACGAC GACCAGGACG AATCGCTGTT


 


 7081
GGGGCATGGC GACCTGCGCC GCATCCCGAC CCTGTATCCG GGCGAGCAGC AACTACCGAC


 


 7141
CGGCCCCGGC GAGGAGCCGC CCAGCCAGCC CGGCATTCCG GGCATGGAAC CAGACCTGCC


 


 7201
AGCCTTGACC GAAACGGAGG AATGGGAACG GCGCGGGCAG CAGCGCCTGC CGATGCCCGA


 


 7261
TGAGCCGTGT TTTCTGGACG ATGGCGAGCC GTTGGAGCCG CCGACACGGG TCACGCTGCC


 


 7321
GCGCCGGTAG CACTTGGGTT GCGCAGCAAC CCGTAAGTGC GCTGTTCCAG ACTATCGGCT


 


 7381
GTAGCCGCCT CGCCGCCCTA TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA


 


 7441
GCCGGGCCAC CTCGACCTGA ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCGTTTT


 


 7501
TATCAGGCTC TGGGAGGCAG AATAAATGAT CATATCGTCA ATTATTACCT CCACGGGGAG


 


 7561
AGCCTGAGCA AACTGGCCTC AGGCATTTGA GAAGCACACG GTCACACTGC TTCCGGTAGT


 


 7621
CAATAAACCG GTAAACCAGC AATAGACATA AGCGGCTATT TAACGACCCT GCCCTGAACC


 


 7681
GACGACCGGG TcGAATrTGc ETTCGAATTT CTGCCATTCA TCCGCTTATT ATCACTTATT


 


 7741
CAGGCGTAGC ACCAGGCGTT TAAGTCGACC AATAAC


 







Amino Acid Sequence for modified EPA with signal sequence


Disclosed in WO 2009/104074 (as SEQ ID NO. 6)


Type: PRT


Organism: Artificial


/note=“Description of Artificial Sequence: Synthetic polypeptide”


Length: 643


Sequence:


SEQ ID NO: 25


Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser


1               5                   10                  15


 


Ala Ser Ala Ala Glu Glu Ala Phe Asp Leu Trp Asn Glu Cys Ala Lys


            20                  25                  30


 


Ala Cys Val Leu Asp Leu Lys Asp Gly Val Arg Ser Ser Arg Met Ser


        35                  40                  45


 


Val Asp Pro Ala Ile Ala Asp Thr Asn Gly Gin Gly Val Leu His Tyr


    50                  55                  60


 


Ser Met Val Leu Glu Gly Gly Asn Asp Ala Leu Lys Leu Ala Ile Asp


65                  70                  75                  80


 


Asn Ala Leu Ser Ile Thr Ser Asp Gly Leu Thr Ile Arg Leu Glu Gly


                85                  90                  95


 


Gly Val Glu Pro Asn Lys Pro Val Arg Tyr Ser Tyr Thr Arg Gin Ala


            100                 105                 110


 


Arg Gly Ser Trp Ser Leu Asn Trp Leu Val Pro Ile Gly His Glu Lys


        115                 120                 125


 


Pro Ser Asn Ile Lys Val Phe Ile His Glu Leu Asn Ala Gly Asn Gin


    130                 135                 140


 


Leu Ser His Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Leu


145                 150                 155                 160


 


Leu Ala Lys Leu Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu


                165                 170                 175


 


Ser Asn Glu Met Gln Pro Thr Leu Ala Ile Ser His Ala Gly Val Ser


            180                 185                 190


 


Val Val Met Ala Gln Ala Gln Pro Arg Arg Glu Lys Arg Trp Ser Glu


        195                 200                 205


 


Trp Ala Ser Gly Lys Val Leu Cys Leu Leu Asp Pro Leu Asp Gly Val


    210                 215                 220


 


Tyr Asn Tyr Leu Ala Gln Gln Arg Cys Asn Leu Asp Asp Thr Trp Glu


225                 230                 235                 240


 


Gly Lys Ile Tyr Arg Val Leu Ala Gly Asn Pro Ala Lys His Asp Leu


                245                 250                 255


 


Asp Ile Lys Asp Asn Asn Asn Ser Thr Pro Thr Val Ile Ser His Arg


            260                 265                 270


 


Leu His Phe Pro Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln


        275                 280                 285


 


Ala Cys His Leu Pro Leu Glu Ala Phe Thr Arg His Arg Gln Pro Arg


    290                 295                 300


 


Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val


305                 310                 315                 320


 


Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gin Val Asp Gln Val


                325                 330                 335


 


Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu


            340                 345                 350


 


Ala Ile Arg Glu Gln Pre Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala


        355                 360                 365


 


Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu


    370                 375                 380


 


Ala Gly Ala Ala Ser Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala


385                 390                 395                 400


 


Lys Asp Gln Asn Arg Thr Lys Gly Glu Cys Ala Gly Pro Ala Asp Ser


                405                 410                 415


 


Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu


            420                 425                 430


 


Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp


        435                 440                 445


 


Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly


    450                 455                 460


 


Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser


465                 470                 475                 480


 


Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile


                485                 490                 495


 


Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr


            500                 505                 510


 


Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala


        515                 520                 525


 


Leu Leu Arg Val Tyr Val Pro Arg Trp Ser Leu Pro Gly Phe Tyr Arg


    530                 535                 540


 


Thr Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg


545                 550                 555                 560


 


Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro


                565                 570                 575


 


Glu Glu Glu Gly Gly Arg Val Thr Ile Leu Gly Trp Pro Leu Ala Glu


            580                 585                 590


 


Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val


        595                 600                 605


 


Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile


    610                 615                 620


 


Ser Ala Leu Pro Asp Tyr Ala Ser Gin Pro Gly Lys Pro Pro Arg Glu


625                 630                 635                 640


 


Asp Leu Lys


 


Amino Acid Sequence for PglB


Disclosed in WO 2009/104074 (as SEQ ID NO. 2)


Length: 722


Type: PRT


Organism: Campylobacter jejuni


Sequence:


SEQ ID NO: 26


Met Leu Lys Lys Glu Tyr Leu Lys Asn Pro Tyr Leu Val Leu Phe Ala


1               5                   10                  15


 


Met Ile TIe Leu Ala Tyr Val Phe Ser Val Phe Cys Arg Phe Tyr Trp


            20                  25                  30


 


Val Trp Trp Ala Ser Glu Phe Asn Glu Tyr Phe Phe Asn Asn Gln Leu


        35                  40                  45


 


Met Ile Ile Ser Asn Asp Gly Tyr Ala Phe Ala Glu Gly Ala Arg Asp


    50                  55                  60


 


Met Ile Ala Gly Phe His Gln Pro Asn Asp Leu Ser Tyr Tyr Gly Ser


65                  70                  75                  80


 


Ser Leu Ser Ala Leu Thr Tyr Trp Leu Tyr Lys Ile Thr Pro Phe Ser


                85                  90                  95


 


Phe Glu Ser Ile Ile Leu Tyr Met Ser Thr Phe Leu Ser Ser Leu Val


            100                 105                 110


 


Val Ile Pro Thr Ile Leu Leu Ala Asn Glu Tyr Lys Arg Pro Leu Met


        115                 120                 125


 


Gly Phe Val Ala Ala Leu Leu Ala Ser Ile Ala Asn Ser Tyr Tyr Asn


    130                 135                 140


 


Arg Thr Met Ser Gly Tyr Tyr Asp Thr Asp Met Leu Val Ile Val Leu


145                 150                 155                 160


 


Pro Met Phe Ile Leu Phe Phe Met Val Arg Met Ile Leu Lys Lys Asp


                165                 170                 175


 


Phe Phe Ser Leu Ile Ala Leu Pro Leu Phe Ile Gly Ile Tyr Leu Trp


            180                 185                 190


 


Trp Tyr Pro Ser Ser Tyr Thr Leu Asn Val Ala Leu Ile Gly Leu Phe


        195                 200                 205


 


Leu Ile Tyr Thr Leu Ile Phe His Arg Lys Glu Lys Ile Phe Tyr Ile


    210                 215                 220


 


Ala Val Ile Leu Ser Ser Leu Thr Leu Ser Asn Ile Ala Trp Phe Tyr


225                 230                 235                 240


 


Gln Ser Ala Ile Ile Val Ile Leu Phe Ala Leu Phe Ala Leu Glu Gln


                245                 250                 255


 


Lys Arg Leu Asn Phe Met Ile Ile Gly Ile Leu Gly Ser Ala Thr Leu


            260                 265                 270


 


Ile Phe Leu Ile Leu Ser Gly Gly Val Asp Pro Ile Leu Tyr Gln Leu


        275                 280                 285


 


Lys Phe Tyr Ile Phe Arg Ser Asp Glu Ser Ala Asn Leu Thr Gln Gly


    290                 295                 300


 


Phe Met Tyr Phe Asn Val Asn Gln Thr Ile Gln Glu Val Glu Asn Val


305                 310                 315                 320


 


Asp Leu Ser Glu Phe Met Arg Arg Ile Ser Gly Ser Glu Ile Val Phe


                325                 330                 335


 


Leu Phe Ser Leu Phe Gly Phe Val Trp Leu Leu Arg Lys His Lys Ser


            340                 345                 350


 


Met Ile Met Ala Leu Pro Ile Leu Val Leu Gly Phe Leu Ala Leu Lys


        355                 360                 365


 


Gly Gly Leu Arg Phe Thr Ile Tyr Ser Val Pro Val Met Ala Leu Gly


    370                 375                 380


 


Phe Gly Phe Leu Leu Ser Glu Phe Lys Ala Ile Met Val Lys Lys Tyr


385                 390                 395                 400


 


Ser Gln Leu Thr Ser Asn Val Cys Ile Val Phe Ala Thr Ile Leu Thr


                405                 410                 415


 


Leu Ala Pro Val Phe Ile His Ile Tyr Asn Tyr Lys Ala Pro Thr Val


            420                 425                 430


 


Phe Ser Gln Asn Glu Ala Ser Leu Leu Asn Gln Leu Lys Asn Ile Ala


        435                 440                 445


 


Asn Arg Glu Asp Tyr Val Val Thr Trp Ala Ala Tyr Gly Tyr Pro Val


    450                 455                 460


 


Arg Tyr Tyr Ser Asp Val Lys Thr Leu Val Asp Gly Gly Lys His Leu


465                 470                 475                 480


 


Gly Lys Asp Asn Phe Phe Pro Ser Phe Ala Leu Ser Lys Asp Glu Gln


                485                 490                 495


 


Ala Ala Ala Asn Met Ala Arg Leu Ser Val Glu Tyr Thr Glu Lys Ser


            500                 505                 510


 


Phe Tyr Ala Pro Gln Asn Asp Ile Leu Lys Thr Asp Ile Leu Gln Ala


        515                 520                 525


 


Met Met Lys Asp Tyr Asn Gln Ser Asn Val Asp Leu Phe Leu Ala Ser


    530                 535                 540


 


Leu Ser Lys Pro Asp Phe Lys Ile Asp Thr Pro Lys Thr Arg Asp Ile


545                 550                 555                 560


 


Tyr Leu Tyr Met Pro Ala Arg Met Ser Leu Ile Phe Ser Thr Val Ala


                565                 570                 575


 


Ser Phe Ser Phe Ile Asn Leu Asp Thr Gly Val Leu Asp Lys Pro Phe


            580                 585                 590


 


Thr Phe Ser Thr Ala Tyr Pro Leu Asp Val Lys Asn Gly Glu Ile Tyr


        595                 600                 605


 


Leu Ser Asn Gly Val Val Leu Ser Asp Asp Phe Arg Ser Phe Lys Ile


    610                 615                 620


 


Gly Asp Asn Val Val Ser Val Asn Ser Ile Val Glu Ile Asn Ser Ile


625                 630                 635                 640


 


Lys Gln Gly Glu Tyr Lys Ile Thr Pro Ile Asp Asp Lys Ala Gln Phe


                645                 650                 555


 


Tyr Ile Phe Tyr Leu Lys Asp Ser Ala Ile Pro Tyr Ala Gln Phe Ile


            660                 665                 670


 


Leu Met Asp Lys Thr Met Phe Asn Ser Ala Tyr Val Gln Met Phe Phe


        675                 680                 685


 


Leu Gly Asn Tyr Asp Lys Asn Leu Phe Asp Leu Val Ile Asn Ser Arg


    690                 695                 700


 


Asp Ala Lys Val Phe Lys Leu Lys Ile Tyr Pro Tyr Asp Val Pro Asp


705                 710                 715                 720


 


Tyr Ala


 


Nucleotide Sequence for pCC1FOS Empty plasmid


Locus pCC1FOS with MCS cassette








Features
    Location/Qualifiers


Region
    230..256



/label=“pCC1/pEpiFOS fwd”


Region
    311..330



/label=“T7 promoter”


Region
    complement(504..529)



/label=“pCC1pEpiFOS rv”


CDS
    complement(805..1464)



/label=cat


CDS
    1683..2030



/label=redF


CDS
    3425..4180



/label=repE


CDS
    4759..5934



/label=parA


CDS
    5934..6905



/label=parB







ORIGIN


Length: 8171 bp


Type: DNA circular TNA


Organism: Artificial


Sequence:


SEQ ID NO: 27








    1
GCGGCCGCAA GGGGTTCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG


 


   61
CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT


 


  121
GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT CAGCTGCGCA ACTGTTGGGA


 


  181
AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC


 


  241
AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC


 


  301
CAGTGAATTG TAATACGACT CACTATAGGG CGAATTCGAG CTCGGTACCC GGGGATCCCA


 


  361
CGTGGCGCGC CACTAGTGCT AGCGACGTCG TGGGATCCTC TAGAGTCGAC CTGCAGGCAT


 


  421
GCAAGCTTGA GTATTCTATA GTCTCACCTA AATAGCTTGG CGTAATCATG GTCATAGCTG


 


  481
TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA


 


  541
AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA


 


  601
CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC


 


  661
GAACCCCTTG CGGCCGCCCG GGCCGTCGAC CAATTCTCAT GTTTGACAGC TTATCATCGA


 


  721
ATTTCTGCCA TTCATCCGCT TATTATCACT TATTCAGGCG TAGCAACCAG GCGTTTAAGG


 


  781
GCACCAATAA CTGCCTTAAA AAAATTACGC CCCGCCCTGC CACTCATCGC AGTACTGTTG


 


  841
TAATTCATTA AGCATTCTGC CGACATGGAA GCCATCACAA ACGGCATGAT GAACCTGAAT


 


  901
CGCCAGCGGC ATCAGCACCT TGTCGCCTTG CGTATAATAT TTGCCCATGG TGAAAACGGG


 


  961
GGCGAAGAAG TTOTCCATAT TGGCCACGTT TAAATCAAAA CTGGTGAAAC TCACCCAGGG


 


 1021
ATTGGCTGAG ACGAAAAACA TATTCTCAAT AAACCCTTTA GGGAAATAGG CCAGGTTTTC


 


 1081
ACCGTAACAC GCCACATCTT GCGAATATAT GTGTAGAAAC TGCCGGAAAT CGTCGTGGTA


 


 1141
TTCACTCCAG AGCGATGAAA ACGTTTCAGT TTGCTCATGG AAAACGGTGT AACAAGGGTG


 


 1201
AACACTATCC CATATCACCA GCTCACCGTC TTTCATTGCC ATACGAAATT CCGGATGAGC


 


 1261
ATTCATCAGG CGGGCAAGAA TGTGAATAAA GGCCGGATAA AACTTGTGCT TATTTTTCTT


 


 1321
TACGGTCTTT AAAAAGGCCG TAATATCCAG CTGAACGGTC TGGTTATAGG TACATTGAGC


 


 1381
AACTGACTGA AATGCCTCAA AATGTTCTTT ACGATGCCAT TGGGATATAT CAACGGTGGT


 


 1441
ATATCCAGTG ATTTTTTTCT CCATTTTAGC TTCCTTAGCT CCTGAAAATC TCGATAACTC


 


 1501
AAAAAATACG CCCGGTAGTG ATCTTATTTC ATTATGGTGA AAGTTGGAAC CTCTTACGTG


 


 1561
CCGATCAACG TCTCATTTTC GCCAAAAGTT GGCCCAGGGC TTCCCGGTAT CAACAGGGAC


 


 1621
ACCAGGATTT ATTTATTCTG CGAAGTGATC TTCCGTCACA GGTATTTATT CGCGATAAGC


 


 1681
TCATGGAGCG GCGTAACCGT CGCACAGGAA GGACAGAGAA AGCGCGGATC TGGGAAGTGA


 


 1741
CGGACAGAAC GGTCAGGACC TGGATTGGGG AGGCGGTTGC CGCCGCTGCT GCTGACGGTG


 


 1801
TGACGTTCTC TGTTCCGGTC ACACCACATA CGTTCCGCCA TTCCTATGCG ATGCACATGC


 


 1861
TGTATGCCGG TATACCGCTG AAAGTTCTGC AAAGCCTGAT GGGACATAAG TCCATCAGTT


 


 1921
CAACGGAAGT CTACACGAAG GTTTTTGCGC TGGATGTGGC TGCCCGGCAC CGGGTGCAGT


 


 1981
TTGCGATGCC GGAGTCTGAT GCGGTTGCGA TGCTGAAACA ATTATCCTGA GAATAAATGC


 


 2041
CTTGGCCTTT ATATGGAAAT GTGGAACTGA GTGGATATGC TGTTTTTGTC TGTTAAACAG


 


 2101
AGAAGCTGGC TGTTATCCAC TGAGAAGCGA ACGAAACAGT CGGGAAAATC TCCCATTATC


 


 2161
GTAGAGATCC GCATTATTAA TCTCAGGAGC CTGTGTAGCG TTTATAGGAA GTAGTGTTCT


 


 2221
GTCATGATGC CTGCAAGCGG TAACGAAAAC GATTTGAATA TGCCTTCAGG AACAATAGAA


 


 2281
ATCTTCGTGC CGTGTTACGT TGAAGTGGAG CGGATTATGT CAGCAATGGA CAGAACAACC


 


 2341
TAATGAACAC AGAACCATGA TGTGGTCTGT CCTTTTACAG CCAGTAGTGC TCGCCGCAGT


 


 2401
CGAGCGACAG GGCGAAGCCC TCGGCTGGTT GCCCTCGCCG CTGGGCTGGC GGCCGTCTAT


 


 2461
GGCCCTGCAA ACGCGCCAGA AACGCCGTCG AAGCCGTGTG CGAGACACCG CGGCCGGCCG


 


 2521
CCGGCGTTGT GGATACCTCG CGGAAAACTT GGCCCTCACT GACAGATGAG GGGCGGACGT


 


 2581
TGACACTTGA GGGGCCGACT CACCCGGCGC GGCGTTGACA GATGAGGGGC AGGCTCGATT


 


 2641
TCGGCCGGCG ACGTGGAGCT GGCCAGCCTC GCAAATCGGC GAAAACGCCT GATTTTACGC


 


 2701
GAGTTTCCCA CAGATGATGT GGACAAGCCT GGGGATAAGT GCCCTGCGGT ATTGACACTT


 


 2761
GAGGGGCGCG ACTACTGACA GATGAGGGGC GCGATCCTTG ACACTTGAGG GGCAGAGTGC


 


 2821
TGACAGATGA GGGGCGCACC TATTGACATT TGAGGGGCTG TCCACAGGCA GAAAATCCAG


 


 2881
CATTTGCAAG GGTTTCCGCC CGTTTTTCGG CCACCGCTAA CCTGTCTTTT AACCTGCTTT


 


 2941
TAAACCAATA TTTATAAACC TTGTTTTTAA CCAGGGCTGC GCCCTGTGCG CGTGACCGCG


 


 3001
CACGCCGAAG GGGGGTGCCC CCCCTTCTCG AACCCTCCCG GTCGAGTGAG CGAGGAAGCA


 


 3061
CCAGGGAACA GCACTTATAT ATTCTGCTTA CACACGATGC CTGAAAAAAC TTCCCTTGGG


 


 3121
GTTATCCACT TATCCACGGG GATATTTTTA TAATTATTTT TTTTATAGTT TTTAGATCTT


 


 3181
CTTTTTTAGA GCGCCTTGTA GGCCTTTATC CATGCTGGTT CTAGAGAAGG TGTTGTGACA


 


 3241
AATTGCCCTT TCAGTGTGAC AAATCACCCT CAAATGACAG TCCTGTCTGT GACAAATTGC


 


 3301
CCTTAACCCT GTGACAAATT GCCCTCAGAA GAAGCTGTTT TTTCACAAAG TTATCCCTGC


 


 3361
TTATTGACTC TTTTTTATTT AGTGTGACAA TCTAAAAACT TGTCACACTT CACATGGATC


 


 3421
TGTCATGGCG GAAACAGCGG TTATCAATCA CAAGAAACGT AAAAATAGCC CGCGAATCGT


 


 3481
CCAGTCAAAC GACCTCACTG AGGCGGCATA TAGTCTCTCC CGGGATCAAA AACGTATGCT


 


 3541
GTATCTGTTC GTTGACCAGA TCAGAAAATC TGATGGCACC CTACAGGAAC ATGACGGTAT


 


 3601
CTGCGAGATC CATGTTGCTA AATATGCTGA AATATTCGGA TTGACCTCTG COGAAGCCAG


 


 3661
TAAGGATATA CGGCAGGCAT TGAAGAGTTT CGCGGGGAAG GAAGTGGTTT TTTATCGCCC


 


 3721
TGAACAGGAT GCCGGCGATG AAAAAGGCTA TGAATCTTTT CCTTGGTTTA TCAAACGTGC


 


 3781
GCACAGTCCA TCCAGAGGGC TTTACAGTGT ACATATCAAC CCATATCTCA TTCCCTTCTT


 


 3841
TATCGGGTTA CAGAACCGGT TTACGCAGTT CGGCTTAGTG GAAACAAAAG AAATCACCAA


 


 3901
TCCGTATCCC ATGCGTTTAT ACGAATCCCT GTGTCAGTAT CGTAAGCCGG ATGGCTCAGG


 


 3961
CATCGTCTCT CTGAAAATCG ACTGGATCAT AGAGCGTTAC CAGCTGCCTC AAAGTTACCA


 


 4021
GCGTATGCCT GACTTCCGCC GCCGCTTCCT GCAGGTCTGT GTTAATGAGA TCAACAGCAG


 


 4081
AACTCCAATG CGCCTCTCAT ACATTGAGAA AAAGAAAGGC CGCCAGACGA CTCATATCGT


 


 4141
ATTTTCCTTC CGCGATATCA CTTCCATGAC GACAGGATAG TCTGAGGGTT ATCTGTCACA


 


 4201
GATTTGAGGG TGGTTCGTCA CATTTGTTCT GACCTACTGA GGGTAATTTG TCACAGTTTT


 


 4261
GCTGTTTCCT TCAGCCTGCA TGGATTTTCT CATACTTTTT GAACTGTAAT TTTTAAGGAA


 


 4321
GCCAAATTTG AGGGCAGTTT GTCACAGTTG ATTTCCTTCT CTTTCCCTTC GTCATGTGAC


 


 4381
CTGATATCGG GGGTTAGTTC GTCATCATTG ATGAGGGTTG ATTATCACAG TTTATTACTC


 


 4441
TGAATTGGCT ATCCGCGTGT GTACCTCTAC CTGGAGTTTT TCCCACGGTG GATATTTCTT


 


 4501
CTTGCGCTGA GCGTAAGAGC TATCTGACAG AACAGTTCTT CTTTGCTTCC TCGCCAGTTC


 


 4561
GCTCGCTATG CTCGGTTACA CGGCTGCGGC GAGCGCTAGT GATAATAAGT GACTGAGGTA


 


 4621
TGTGCTCTTC TTATCTCCTT TTGTAGTGTT GCTCTTATTT TAAACAACTT TGCGGTTTTT


 


 4681
TGATGACTTT GCGATTTTGT TGTTGCTTTG CAGTAAATTG CAAGATTTAA TAAAAAAACG


 


 4741
CAAAGCAATG ATTAAAGGAT GTTCAGAATG AAACTCATGG AAACACTTAA CCAGTGCATA


 


 4801
AACGCTGGTC ATGAAATGAC GAAGGCTATC GCCATTGCAC AGTTTAATGA TGACAGCCCG


 


 4861
GAAGCGAGGA AAATAACCCG GCGCTGGAGA ATAGGTGAAG CAGCGGATTT AGTTGGGGTT


 


 4921
TCTTCTCAGG CTATCAGAGA TGCCGAGAAA GCAGGGCGAC TACCGCACCC GGATATGGAA


 


 4981
ATTCGAGGAC GGGTTGAGCA ACGTGTTGGT TATACAATTG AACAAATTAA TCATATGCGT


 


 5041
GATGTGTTTG GTACGCGATT GCGACGTGCT GAAGACGTAT TTCCACCGGT GATCGGGGTT


 


 5101
GCTGCCCATA AAGGTGGCGT TTACAAAACC TCAGTTTCTG TTCATCTTGC TCAGGATCTG


 


 5161
GCTCTGAAGG GGCTACGTGT TTTGCTCGTG GAAGGTAACG ACCCCCAGGG AACAGCCTCA


 


 5221
ATGTATCACG GATGGGTACC AGATCTTCAT ATTCATGCAG AAGACACTCT CCTGCCTTTC


 


 5281
TATCTTGGGG AAAAGGACGA TGTCACTTAT GCAATAAAGC CCACTTGCTG GCCGGGGCTT


 


 5341
GACATTATTC CTTCCTGTCT GGCTCTGCAC CGTATTGAAA CTGAGTTAAT GGGCAAATTT


 


 5401
GATGAAGGTA AACTGCCCAC CGATCCACAC CTGATGCTCC GACTGGCCAT TGAAACTCTT


 


 5461
GCTCATGACT ATGATGTCAT AGTTATTGAC AGCGCGCCTA ACCTGGGTAT CGGCACGATT


 


 5521
AATGTCGTAT GTGCTGCTGA TGTGCTGATT GTTCCCACGC CTGCTGAGTT GTTTGACTAC


 


 5581
ACCTCCGCAC TGCAGTTTTT CGATATGCTT CGTGATCTGC TCAAGAACGT TGATCTTAAA


 


 5641
GGGTTCGAGC CTGATGTACG TATTTTGCTT ACCAAATACA GCAATAGTAA TGGCTCTCAG


 


 5701
TCCCCGTGGA TGGAGGAGCA AATTCGGGAT GCCTGGGGAA GCATGGTTCT AAAAAATGTT


 


 5761
GTACGTGAAA CGGATGAAGT TGGTAAAGGT CAGATCCGGA TGAGAACTGT TTTTGAACAG


 


 5821
GCCATTGATC AACGCTCTTC AACTGGTGCC TGGAGAAATG CTCTTTCTAT TTGGGAACCT


 


 5881
GTCTGCAATG AAATTTTCGA TCGTCTGATT AAACCACGCT GGGAGATTAG ATAATGAAGC


 


 5941
GTGCGCCTGT TATTCCAAAA CATACGCTCA ATACTCAACC GGTTGAAGAT ACTTCGTTAT


 


 6001
CGACACCAGC TGCCCCGATG GTGGATTCGT TAATTGCGCG CGTAGGAGTA ATGGCTCGCG


 


 6061
GTAATGCCAT TACTTTGCCT GTATGTGGTC GGGATGTGAA GTTTACTCTT GAAGTGCTCC


 


 6121
GGGGTGATAG TGTTGAGAAG ACCTCTCGGG TATGGTCAGG TAATGAACGT GACCAGGAGC


 


 6181
TGCTTACTGA GGACGCACTG GATGATCTCA TCCCTTCTTT TCTACTGACT GGTCAACAGA


 


 6241
CACCGGCGTT CGGTCGAAGA GTATCTGGTG TCATAGAAAT TGCCGATGGG AGTCGCCGTC


 


 6301
GTAAAGCTGC TGCACTTACC GAAAGTGATT ATCGTGTTCT GGTTGGCGAG CTGGATGATG


 


 6361
AGCAGATGGC TGCATTATCC AGATTGGGTA ACGATTATCG CCCAACAAGT GCTTATGAAC


 


 6421
GTGGTCAGCG TTATGCAAGC CGATTGCAGA ATGAATTTGC TGGAAATATT TCTGCGCTGG


 


 6481
CTGATGCGGA AAATATTTCA CGTAAGATTA TTACCCGCTG TATCAACACC GCCAAATTGC


 


 6541
CTAAATCAGT TGTTGCTCTT TTTTCTCACC CCGGTGAACT ATCTGCCCGG TCAGGTGATG


 


 6601
CACTTCAAAA AGCCTTTACA GATAAAGAGG AATTACTTAA GCAGCAGGCA TCTAACCTTC


 


 6661
ATGAGCAGAA AAAAGCTGGG GTGATATTTG AAGCTGAAGA AGTTATCACT CTTTTAACTT


 


 6721
CTGTGCTTAA AACGTCATCT GCATCAAGAA CTAGTTTAAG CTCACGACAT CACTTTGCTC


 


 6781
CTGGAGCGAC AGTATTGTAT AAGGGCCATA AAATGGTGCT TAACCTGGAC AGGTCTCGTG


 


 6841
TTCCAACTGA GTGTATAGAG AAAATTGAGG CCATTCTTAA GGAACTTGAA AAGCCAGCAC


 


 6901
CCTGATGCGA CCACGTTTTA GTTTACTTTT ATCTGTCTTT ACTTAATGTC CTTTGTTACA


 


 6961
GGCCAGAAAG CATAACTGGC CTGAATATTC TCTCTGGGCC CACTGTTCCA CTTGTATCGT


 


 7021
CGGTCTGATA ATCAGACTGG GACCACGGTC CCACTCGTAT CGTCGGTCTG ATTATTAGTC


 


 7081
TGGGACCACG GTCCCACTCG TATCGTCGGT CTGATTATTA GTCTGGGACC ACGGTCCCAC


 


 7141
TCGTATCGTC GGTCTGATAA TCAGACTGGG ACCACGGTCC CACTCGTATC GTCGGTCTGA


 


 7201
TTATTAGTCT GGGACCATGG TCCCACTCGT ATCGTCGGTC TGATTATTAG TCTGGGACCA


 


 7261
CGGTCCCACT CGTATCGTCG GTCTGATTAT TAGTCTGGAA CCACGGTCCC ACTCGTATCG


 


 7321
TCGGTCTGAT TATTAGTCTG GGACCACGGT CCCACTCGTA TCGTCGGTCT GATTATTAGT


 


 7381
CTGGGACCAC GATCCCACTC GTGTTGTCGG TCTGATTATC GGTCTGGGAC CACGGTCCCA


 


 7441
CTTGTATTGT CGATCAGACT ATCAGCGTGA GACTACGATT CCATCAATGC CTGTCAAGGG


 


 7501
CAAGTATTGA CATGTCGTCG TAACCTGTAG AACGGAGTAA CCTCGGTGTG CGGTTGTATG


 


 7561
CCTGCTGTGG ATTGCTGCTG TGTCCTGCTT ATCCACAACA TTTTGCGCAC GGTTATGTGG


 


 7621
ACAAAATACC TGGTTACCCA GGCCGTGCCG CCACGTTAAC CGGGCTGCAT CCGATGCAAG


 


 7681
TGTGTCGCTG TCGACGAGCT CGCGAGCTCG GACATGAGGT TGCCCCGTAT TCAGTGTCGC


 


 7741
TGATTTGTAT TGTCTGAAGT TGTTTTTACG TTAAGTTGAT GCAGATCAAT TAATACGATA


 


 7801
CCTGCGTCAT AATTGATTAT TTGACGTGGT TTGATGGCCT CCACGCACGT TGTGATATGT


 


 7861
AGATGATAAT CATTATCACT TTACGGGTCC TTTCCGGTGA TCCGACAGGT TACGGGGCGG


 


 7921
CGACCTCGCG GGTTTTCGCT ATTTATGAAA ATTTTCCGGT TTAAGGCGTT TCCGTTCTTC


 


 7981
TTCGTCATAA CTTAATGTTT TTATTTAAAA TACCCTCTGA AAAGAAAGGA AACGACAGGT


 


 8041
GCTGAAAGCG AGCTTTTTGG CCTCTGTCGT TTCCTTTCTC TGTTTTTGTC CGTGGAATGA


 


 8101
ACAATGGAAG TCCGAGCTCA TCGCTAATAA CTTCGTATAG CATACATTAT ACGAAGTTAT


 


 8161
ATTCGATCCA C


 







Nucleotide Sequence for pCC1FOS cut (pFOS) and


S. flexneri 6 O-antigen without Z3206


Locus pFOS cut and O-antige cut (-Z3206)


Definition Ligation of inverted pCC1FOS with MCS cassette cut with


NheI and into S. flexneri 6 O antigen cluster amplified with 


galFNheI and wzzAscI cut with NheI and AscI








FEATURES
    Location/Qualifiers


CDS
    3..411



/label=′galF


CDS
    784..1869



/label=rmlB


CDS
    1869..2768



/label=rmlD


CDS
    2826..3704



/label=rmlA


CDS
    3709..4266



/label=rmlC


CDS
    4263..5495



/label=wzx


CDS
    5551..6738



/label=wzy


CDS
    6755..7624



/label=wfbY


CDS
    7621..8454



/label=wfbZ


CDS
    8559..9965



/label=gnd


CDS
    10187..11380



/label=ugd


CDS
    complement(11416..12450)



/label=uge


CDS
    12802..12828



/label=wzz′


Region
    complement(12868..12887)



/label=“T7 promoter”


Region
    complement(12942..12968)



/label=“pCC1/pEpiRDS fwd”


CDS
    complement(14460..15431)



/label=parB


CDS
    complement(15431..16606)



/label=parA


CDS
    complement(7185..17940)



/label=repE


CDS
    complement(19335..19682)



/label=redF


CDS
    19901..20560



/label=cat


Region
    20836..20861



/label=“pCC1pEpiFOS rv”







Length: 20982 bp


Type: DNA circular UNA


Sequence:


SEQ ID NO: 28








    1
CTAGCGGCAA AACGTATGCC GGGTGACCTC TCTGAATACT CCGTCATCCA GACCAAAGAA


 


   61
CCGCTGGATC GCGAAGGTAA AGTCAGCCGC ATTGTTGAAT TTATCGAAAA ACCGGATCAG


 


  121
CCGCAGACGC TGGACTCAGA CATCATGGCC GTTGGTCGCT ATGTGCTTTC TGCCGATATT


 


  181
TGGCCGGAAC TTGAACGTAC TCAGCCTGGT GCATGGGGAC GTATTCAGCT GACTGATGCC


 


  241
ATTGCCGAGC TGGCGAAAAA ACAGTCCGTT GATGCAATGC TGATGACCGG CGACAGCTAC


 


  301
GACTGCGGTA AAAAAATGGG CTATATGCAG GCGTTTGTGA AGTATGGGCT GCGCAACCTG


 


  361
AAAGAAGGGG CGAAGTTCCG TAAAGGTATT GAGAAGCTGT TAAGCGAATA ATGAAAATCT


 


  421
GACCGGATGT AACGGTTGAT AAGAAAATTA TAACGGCAGT GAAGATTCGT GGTGAAAGTA


 


  481
ATTTGTTGCG AATATTCCTG CCGTTGTTTT ATATAAACAA TCAGAATAAC AACGAGTTAG


 


  541
CAATAGGATT TTAGTCAAAG TTTTCCAGGA TTTTCCTTGT TTCCAGAGCG GATTGGTAAG


 


  601
ACAATTAGCT TTTGAATTTT TCGGGTTTAG CGCGAGTGGG TAACGCTCGT CACATCGTAG


 


  661
GCATGCATGC AGTGCTCTGG TAGCTGTAAA GCCAGGGGCG GTAGCGTGCA TTAATACTTC


 


  721
TATTAATCAA ACTGAGAGCC GCTTATTTCA CAGCATGCTC TGAAGCAATA TGGAATAAAT


 


  781
TAGGTGAAAA TACTTGTTAC TGGTGGCGCA GGATTTATTG GTTTTGCTGT AGTTCGTCAC


 


  841
ATTATAAATA ATACGCAGGA TAGTGTTGTT AATGTCGATA AATTAACGTA CGCCGGAAAC


 


  901
CTGGAATCAC TTGCTGATGT TTCTGATTCT GAACGCTATG TTTTTGAACA TGCGGATATT


 


  961
TGCGATGCAG CTGCAATGGC ACGGATTTTT GCTCAGCATC AGCCAGATGC AGTGATGCAC


 


 1021
CTGGCTGCTG AAAGCCATGT TGACCGTTCA ATTACAGGTC CTGCGGCATT TATTGAAACC


 


 1081
AATATTGTTG GTACATATGT CCTTTTGGAA GCCGCTCGCA ATTATTGGTC TGCTCTTGAT


 


 1141
AGCGACAAGA AAACTAGATT CCGTTTTCAT CATATTTCTA CTGACGAAGT CTATGGTGAT


 


 1201
TTGCCTCATC CTGACGAGGT AAATAATACA GAAGAATTAC CCTTATTTAC AGAGACAACA


 


 1261
GCTTACGCGC CAAGCAGCCC TTATTCCGCT TCAAAAGCAT CCAGCGATCA TTTAGTCCGC


 


 1321
GCGTGGAAAC GTACCTATGG TTTACCAACC ATTGTGACTA ATTGCTCTAA TAATTATGGT


 


 1381
CCTTATCATT TCCCGGAAAA ATTGATTCCA TTGGTTATTC TGAATGCTCT GGAAGGTAAG


 


 1441
GCATTACCTA TTTATGGCAA AGGGGATCAA ATTCGTGACT GGCTGTATGT TGAAGATCAT


 


 1501
GCGCGTGCGT TATATACCGT CGTAACCGAA GGTAAAGCGG GTGAAACTTA TAACATTGGT


 


 1561
GGACACAACG AAAAGAAAAA CATCGATGTA GTGCTCACTA TTTGTGATTT GCTGGATGAG


 


 1621
ATTGTACCGA AAGAGAAATC TTACCGCGAG CAAATTACTT ATGTTGCCGA TCGCCCGGGA


 


 1681
CACGATCGCC GTTATGCGAT TGATGCAGAG AAGATTAGCC GCGAATTGGG CTGGAAACCG


 


 1741
CAGGAAACGT TTGAGAGCGG GATTCGGAAG ACATTGGAAT GGTACCTGTC CAATACAAAA


 


 1801
TGGGTTGATA ATGTGAAAAG TGGTGCTTAT CAATCGTGGA TTGAACAGAA CTATGAGGGC


 


 1861
CGCCAGTAAT GAATATCCTC CTTTTCGGCA AAACAGGGCA GGTAGGTTGG GAACTACAGC


 


 1921
GTGCTCTGGC ACCTTTGGGT AATTTGATTG CTCTTGATGT TCACTCCACT GATTATTGTG


 


 1981
GTGATTTTAG TAATCCTGAA GGTGTAGCTG AAACAGTCAA AAGAATTCGA CCTGATGTTA


 


 2041
TTGTTAATGC TGCGGCTCAC ACCGCAGTAG ATAAGGCTGA GTCAGAACCC GAATTTGCAC


 


 2101
AATTACTCAA TGCGACTAGT GTTGAATCAA TTGCAAAAGA GGCTAATGAA GTTGGGGCTT


 


 2161
GGGTAATTCA TTACTCAACT GACTACGTAT TCCCTGGAAA TGGCGACACG CCATGGCTGG


 


 2221
AGACGGATGC AACCGCACCG CTAAATGTTT ACGGTGAAAC CAAGTTAGCC GGAGAAAAAG


 


 2281
CGTTACAGGA ACATTGCGCG AAGCATCTTA TTTTCCGTAC CAGCTGGGTA TACGCAGCTA


 


 2341
AAGGAAATAA CTTCGCCAAA ACGATGTTGC GTCTGGCAAA AGAGCGCGAA GAACTGGCTG


 


 2401
TGATAAATGA TCAATTTGGT GCGCCAACAG GTGCTGAGCT GCTGGCTGAT TGTACGGCAC


 


 2461
ATGCTATTCG TGTGGCACTG AATAAACCGG AAGTCGCAGG TTTGTACCAT CTGGTAGCCA


 


 2521
GTGGTACCAC AACCTGGCAC GATTATGCTG CGCTGGTTTT TGAAGAGGCG CGCAAAGCAG


 


 2581
GTATTCCCCT TGCACTCAAC AAGCTCAACG CAGTACCAAC AACAGCCTAT CCTACACCAG


 


 2641
CTCGTCGTCC ACATAACTCT CGCCTTAATA CAGAAAAATT TCAGCAGAAC TTTGCGCTTG


 


 2701
TCTTGCCTGA CTGGCAGGTT GGTGTGAAAC GAATGCTCAA CGAATTAATT ACGACTACAG


 


 2761
CAATTTAATA GTTTTTGCAT CTTGTTCGTG ATGGTGGAGC AAGATGAATT AAAAGGAATG


 


 2821
ATGAAATGAA AACGCGTAAA GGTATTATTT TAGCGGGTGG TTCTGGTACA CGTCTTTATC


 


 2881
CTGTGACTAT GGCTGTCAGT AAACAGCTAT TACCTATTTA TGATAAGCCG ATGATCTATT


 


 2941
ACCCGCTCTC TACACTGATG TTGGCGGGTA TTCGCGATAT TCTGATTATT AGTACGCCAC


 


 3001
AGGATACTCC TCGTTTTCAA CAACTGCTAG GTGACGGTAG CCAGTGGGGG CTAAATCTTC


 


 3061
AGTACAAAGT GCAACCGACT CCAGATGGGC TTGCGCAGGC GTTTATTATC GGTGAAGAGT


 


 3121
TTATCGGTGG TGATGATTGT GCTTTGGTTC TTGGTGATAA TATCTTCTAC GGTCATGATC


 


 3181
TGCCGAAGTT AATGGATGTC GCTGTTAACA AAGAAAGTGG TGCAACGGTA TTTGCCTATC


 


 3241
ACGTTAATGA TCCTGAACGC TACGGCGTCG TTGAGTTTGA TAAAAACGGT ACGGCAATAA


 


 3301
GCCTGGAAGA AAAACCGCTA CAACCAAAAA GTAATTATGC GGTAACCGGG CTTTATTTCT


 


 3361
ATGATAACGA CGTTGTCGAA ATGGCGAAAA ACCTTAAGCC TTCTGCCCGT GGTGAACTGG


 


 3421
AAATTACCGA TATTAACCGT ATTTATATGG AACAGGGGCG TTTATCCGTT GCCATGATGG


 


 3481
GGCGTGGTTA TGCATGGCTG GATACGGGGA CACATCAGAG TCTTATTGAA GCAAGCAACT


 


 3541
TCATTGCCAC CATTGAAGAG CGCCAGGGAC TAAAGGTTTC CTGCCCAGAA GAAATTGCTT


 


 3601
ACCGTAAAGG GTTTATTGAT GCTGAACAGG TGAAAGCATT AGCGGAGCCG CTGAAAAAAA


 


 3661
ATGCTTATGG ACAGTATCTG CTGAAAATGA TTAAAGGTTA TTAATAAAAT GAACGTAATT


 


 3721
AAAACAGAAA TTCCTGATGT GTTAATTTTC GAGCCGAAAG TTTTTGGTGA TGAGCGTGGT


 


 3781
TTCTTTATGG AAAGCTTTAA TCAGAAAGTT TTCGAAGAAG CTGTAGGACG TAAGGTTGAA


 


 3841
TTTGTTCAGG ATAACCATTC GAAGTCTAGT AAAGGTGTTT TACGCGGGCT GCATTATCAG


 


 3901
TTAGAACCTT ATGCGCAAGG GAAACTGGTA CGTTGCGTTG TTGGTGAGGT TTTTGATGTA


 


 3961
GCTGTTGATA TTCGTAAATC GTCGCCTACC TTTGGTAAAT GGGTTGGGGT GAATTTATCT


 


 4021
GCTGAGAATA AGCGGCAATT GTGGATCCCT GAGGGATTTG CACATGGTTT TTTGGTGCTG


 


 4081
AGCGAGACTG CGGAATTTTT ATATAAAACG ACGAACTATT ATCATCCTGA TAGTGATAGA


 


 4141
GGGATTGTAT GGAATGATCC TATTCTGAGC ATAAAATGGC CGACGATAGA ACATAATAAT


 


 4201
TATATTTTAT CGATTAAAGA TGCAAGGGCT AAAGAATTGC ATAACATGAA GGAATTATTT


 


 4261
TTGTGAGTAT TGTAAAGAAT ACTTTATGGA ATATAAGTGG GTATATTATA CCATCATTAA


 


 4321
TAGCAATTCC TGCGTTAGGT ATACTGTCTA GAATTCTAGG GACCGAGCAA TTTGGCCTTT


 


 4381
TTACGTTAGC TATTGCCTTA GTTGGATATG CAAGTATTTT TGATGCTGGA TTGACCAGAG


 


 4441
CTGTTATAAG AGAAGTATCA ATATATAAAA ATGTTCATAA AGAATTAAGA GCGATCATTT


 


 4501
CAACTTCAAC GGTAATTCTA ACTATATTGG GCTTGATTGG CGGTAGTGTA CTATTTTTGA


 


 4561
GTAGCAATGT AATTGTTAAA TTATTAAACA TTAACGCGAA TCATGTTGTA GAATCTGTCA


 


 4621
AAGCAATATA TATTATTTCA GCTACCATAC CCTTATACTT GTTAAACCAA GTCTGGTTGG


 


 4681
GGATTTTTGA GGGGATGGAA AAGTTCAGAA AAGTAAATTT AATAAAATCA ATTAACAACT


 


 4741
CTTTTGTGGC TGGATTACCA GTGATTTTCT GTTTTTTTCA TGGAGGATTA CTAAGTGCTA


 


 4801
TATATGGTTT AGTTATGGCA AGAGTCTTAT CACTTATAGT GACCTTTATA TTTAGTCGAA


 


 4861
AACTAATAAT ATCATCTGGG CTGTCTGTAA AAATTGTAAC AGTTAAAAGA TTAATCGGCT


 


 4921
TTGGAAGCTG GATAACAGTT AGCAATATTA TTAGCCCTAT TATGACATAT ATGGATCGTT


 


 4981
TTATTCTTTC ACACATTGTG GGGGCTGATA AAGTTTCTTT TTATACTGCT CCGTCTGAAG


 


 5041
GTATACAACG CTTAACGATA TTACCAAGTG CGTTGTCCAG AGCTATTTTT CCAAGATTAA


 


 5101
GTTCAGAATT GCAATCGGTA AAGCAAACTA AAATATTATC ATATTTTATA ATGGTTATTG


 


 5161
GTATACTTCC AATTGTAATG TTGATAATTA TTTTATCAGA TTTTATAATG TCCGCTTGGA


 


 5221
TGGGACCTAC ATATCATGGG ACGCCAGGTA TAGTATTAAA AATTCTTGCA ATAGGTTTCT


 


 5281
TTTTTAATTG CATTGCACAA ATCCCATTTG TTTCAGTTCA GGCTAGTGGA AGATCAAAAA


 


 5341
TTACAGCTAT TATTCATTTG CTCGAAGTTA TCCCATATTT ATGCATATTA TATATTTTTA


 


 5401
TTTATCATTG GGGAATTGTT GGAGCCGCAA TAGCATGGTC TGTAAGAACA TCGTTAGATT


 


 5461
TTTTGATATT ATTATTAATT GATACGAAAT ATTAATAGCG AATTGATTTT AGGGATTACT


 


 5521
TCCTCAAGCC CATCTAATTA GAGTGCAAAC ATGACTTCTG ATTTTTATAA CTCAAAAGAC


 


 5581
AAAAGTTTAA GTGTTCTTTT GTTTTTTGGG TTTATATTTT TCCTTACACG TAGCTTTCCA


 


 5641
TTTATTCAAT ATAGTTOGAT TATGGAGGGG TTTTTATGTC TTTGTATCAT GTCATTTACA


 


 5701
AAGAAAATTG CAAACGGAAT ATATCACTAT CCTGTTATTT TAATATTTCT ATTAGCTCTT


 


 5761
TTTATAAATT TTATTTATTC CTATATCAAG GGTAACGATA TAGCGATAAT AATTAGGTTT


 


 5821
TATATTATCA TATTATTTAT ATTATGTGCT TATTTCTGCT CTTATGGAAC CATCTCGATT


 


 5881
GTTAAAATAT TTTTATATTT AATGGTATTA CAGGCGGTTA TTATATCCAT CATTAGTATT


 


 5941
TATATGACAA AAACATATGG TATTGGTGAT TATTCAGCAC TAAGACATTA TTTTTTGGAG


 


 6001
AATGATTATG GTGATGTTTA TACATATGGA AGTGGTTTCT ATAGAGTTCA AATTAAAGGA


 


 6061
AATGCTCTCA TTCCATTTGC CTTTATGTTG CATATAGTCA TAAAAGATTA TTTCTATTAT


 


 6121
CGATTCAAAA ATACAATAAC CGTTATTCTG GCTATAGGTA CTATAGTGGC TGGTAATTTT


 


 6181
GCATATTTTG TTTCGATATG CTTGTTTTTT ATGTATATTA TACTATGTTC TAAATCTAAC


 


 6241
TCACGATACG CTAAATTAAG GAAAATTATT TTTGGGGTTT TTCTTACTGT GATTCTCCCT


 


 6301
TTTTTTATTA CATATTCAAT TGAGTTGATA ATCATGAAAT CAAATGGAGC TGATTCTTCT


 


 6361
TTAGGAGTTA GATGGGATCA GTTTACTGTA TTAATTAATG ATCTTACAGA GTCTGTATCA


 


 6421
AATTTTGTTA TAGGTTCTGG TTTGGGTAAT GTCATCAAAA TTCAAACTCC TATCCGTGAT


 


 6481
TATAGTGCAT ATATATATTA TGAATTGCAG TCAGTTTATT TTTTAAATCA ACTTGGCGTT


 


 6541
ATTTTATTTA CTTTGTTTTT ATTAATTAAT CTCCTTCTCA CGATTAAAAT CATAAAATAC


 


 6601
AGTGAGTTGT GTGTGCTATA TTTTCTATAT GTTTCTTATG CAATTACTAA TCCTTATATT


 


 6661
TTAGACTCTA ACCATGTTGC TGTAATAATT GTATTAGTGA CATTAAGTAA TGTTCTAAAA


 


 6721
AAGATGAAAG CTAAATGAAG GTTTTAAGGT GAAGATGGAC ACTGTATATG CCGTTTTGGT


 


 6781
TGCTTACAAC CCAGAACATA ATGATTTAAA AAATGCGGTT GAATTATTGT TGAGACAAGT


 


 6841
TACTAAAGTT GTCGTTTGCA ATAACTCTAC AAATGGTTAT AAATATGCTG AAAATTCTTC


 


 6901
AGGCGATGTA AAAATATTCA ATTTCAATGA TAATTTAGGC ATAGCAGAAG CCCAAAGTAT


 


 6961
AGGAATGAAA TGGGCTTTTG AAAATGGCGC TGATTTTATA TTGCAAATGG ATCAGGATAG


 


 7021
TATTCCTGAT CCTAAGATGG TAGAGCAGTT ACTTACTTGT TACAAAAAAT TGCTTAAACA


 


 7081
AAATGTCAAT GTTGGTTTAG TTGGTTCACA AGATTTTGAT AAAGTAACTG GTGAATTAAA


 


 7141
TAAAGCAAGG GTAAAAAAAG GGAAACCACT TACAGAAGTT TATTATGAGG TAGATAGTAC


 


 7201
AlTAAGTTCT GGCAGTCTAA TACCAAAAAA TAGTTGGTTG ATTGTTGGAG GAATGAAAGA


 


 7261
TGAGCTTTTT ATCGATGCGG TAGACCATGA ATATTGTTGG AGATTAAGAG CTGCTGGGTT


 


 7321
TAAAGTAATT AGGAATAAAA ATGCGTTACT TGCACATAGA CTTGGAGATG GGCGATTTAA


 


 7381
GATCTTAAAT ATTCTTTCTG TCGGTTTGCC AAGCCCATTT CGTCATTATT ATGCTACTCG


 


 7441
AAATATCTTT CTTTTATTAA ATAAAAATTA TGTACCCATC TACTGGAAAA TTTCTAGTCT


 


 7501
GGTTAAATTA ATTGGAAAGG TTTTTTTATA TCCTATTTTC CTTCCAAATG GTAATAAAAG


 


 7561
GTTATATTTT TTTTTAAAAG GCATTAATGA CGGTTTAATG GGTCGAAGTG GTAAAATGAA


 


 7621
ATGAATCATA GATTAGAAAA ATTCTCAGTT TTAATTAGCA TTTATAAAAA TGATCTACCG


 


 7681
CAATTTTTTG AGGTGGCTCT ACGCTCTATT TTTCACGATC AAACACTTAA GCCAGATCAA


 


 7741
ATAGTAATTG TTGCAGATGG AGAACTCCAT CAAACACACA TCGATATTAT AAATTCATTC


 


 7801
ATTGATGATG TTGGCAATAA AATAGTAACA TTTGTACCTT TACCTAGAAA TGTTGGATTG


 


 7861
GCTAATGCCT TAAATGAAGG ATTAAAGGCT TGTAGGAATG AGTTAGTGGC AAGAATGGAT


 


 7921
GCTGATGATA TTTCTTTGCC TCATCGGTTT GAGAAACAAA TTTCTTTTAT GATTAATAAT


 


 7981
TCAGAAATAG ATGTATGTGG CAGTTTTATT GATGAAATTG AAACTGTTAC TGAGGAGTTT


 


 8041
ATTTCAACAC GCAAAGTGCC TCTCGAACAT AGAGAAATAG TTAAATTCGC GAGGAAACGA


 


 8101
AGCGCAGTTA GCCATCCTTC TGTAATTTTT AGAAAGAATA CAGTATTAGC TGTTGGTGGT


 


 8161
TATCCTCCAT TCAGAAAATC TCAAGATTTT GCATTGTGGA GCCTATTAAT TGTACATAAT


 


 8221
GCAAGATTTG CAAATCTTCC AGATATTTTA TTAAAAATGC GAACTGGTCG TAATCTTATG


 


 8281
GCTCGACGTG GATTGTCATA TTTATTGTAC GAGTATAAAG TATTGTATTA TCAATATAAA


 


 8341
ATTGGTTTTA TTCGAAAAAA TGAATTAATA AGTAATGCTA TGTTGAGAAC ATTTTTTCGT


 


 8401
ATAATGCCAT CTAAATTAAA GGAGCTGATG TATTCAATCG TTAGGAATCG ATAATAATAA


 


 8461
TTTTCTGATT AAGTGTTATG GATTTATTTT TATTAGGCAT ATTCTATAAT TAAGCATAAC


 


 8521
CCGCATACCA CCCAGCGGTA TCCTGACAGG AGTAAACAAT GTCAAAGCAA CAGATCGGCG


 


 8581
TCGTCGGTAT GGCAGTGATG GGGCGCAACC TTGCGCTCAA TATCGAAAGC CGTGGTTATA


 


 8641
CCGTCTCTAT TTTCAACCGT TCCCGTGAAA AGACCGAAGA AGTGATTACC GAAAATCCAG


 


 8701
GCAAGAAACT GGTTCCTTAC TATACGGTGA AAGAATTTGT TGAATCTCTG GAAACGCCTC


 


 8761
GTCGCATCCT GTTAATGGTG AAAGCAGGTG CTGGCACGGA TGCTGCTATT GATTCCCTCA


 


 8821
AGCCATACCT CGATAAAGGT GACATCATCA TTGATGGTGG TAACACCTTC TTCCATGACA


 


 8881
CCATTCGTCG TAACCGTGAG CTTTCTGCAG AAGGCTTTAA CTTTATCGGT ACCGGTGTTT


 


 8941
CCGGTGGTGA AGAAGGTGCG CTGAAAGGTC CTTCCATTAT GCCTGGTGGG CAGAAAGAAG


 


 9001
CTTATGAACT GATTGCGCCG ATCCTGACCA AAATCGCCGC TGTGGCTGAA GACGGCGAAC


 


 9061
CGTGCGTTAC CTATATTGGT GCCGATGGTG CAGGTCATTA TGTGAAGATG GTTCACAACG


 


 9121
GTATTGAATA CGGTGATATG CAGCTGATTG CTGAAGCCTA TTCTCTGCTT AAAGGTGGCT


 


 9181
TGAACCTCAC CAACGAAGAA CTGGCGCAGA CCTTTACCGA GTGGAATAAC GGTGAACTGA


 


 9241
GCAGCTACCT GATCGACATC ACCAAAGATA TCTTCACCAA AAAAGATGAA GAGGGTAACT


 


 9301
ACCTGGTTGA TGTGATTCTG GATGAAGCAG CAAACAAAGG TACGGGCAAA TGGACCAGCC


 


 9361
AGAGCGCGCT GGATCTCGGC GAACCGCTGT CGCTGATTAC CGAGTCTGTG TTTGCACGTT


 


 9421
ATATCTCTTC TCTGAAAGAG CAGCGTGTTG CCGCATCTAA AGTTCTCTCT GGCCCGCAAG


 


 9481
CGCAGCCAGC TGGCGACAAT GCTGAGTTCA TCGAAAAAGT TCGCCGTGCG CTGTATCTGG


 


 9541
GCAAAATCGT TTCTTACGCT CAGGGCTTCT CTCAGCTACG CGCTGCGTCT GAAGAGTACA


 


 9601
ACTGGGATCT GAACTACGGT GAAATCGCGA AGATTTTCCG TGCTGGCTCC ATCATCCGTG


 


 9661
CGCAGTTCCT GCAGAAAATC ACCGATGCTT ATGCCGAAAA TCCGCAGATC GCTAACCTGT


 


 9721
TGCTGGCTCC TTACTTCAAG CAAATTGCCG ATGACTACCA GCAGGCGCTG CGCGATGTCG


 


 9781
TCGCTTACGC AGTACAGAAC GGTATCCCGG TGCCCTACCT CGCCGCTGCG GTTGCCTATT


 


 9841
ACGACAGCTA CCGCGCCGCT GTTCTGCCTG CGAACCTGAT CCAGGCACAG CGTGACTATT


 


 9901
TCGGTGCGCA TACTTATAAG CGCATTGATA AAGAAGGTGT GTTCCATACC GAATGGCTGG


 


 9961
ATTAATCTGA TTTAAATCAA TTAATCAAAG CAAGGCCCGG AGAAACCCTC CGGGCTTTTT


 


10021
TATTATACAA AGCGGCAGGT TAGGGCCTTT TTTTATAATT TATAGTTAAA AACGCGATAT


 


10081
AATACAGCGC CGCACAGCAG GATCGCTGCC TTGACAGTTC ATCTACATCA GCGTTAAAAA


 


10141
TCCCGCAGTA GATGAAGCTG TGGTGGTGGA TTAATGACCA CTCTAAATGT TTAACCGGAA


 


10201
GAAGTCAGAG CTAATGAAAA TAACAATTTC AGGAACAGGT TATGTTGGTC TTTCAAATGG


 


10261
TATTCTGATT GCGCAAAACC ACGAAGTGGT TGCACTGGAT ATCGTTCAGG CCAAAGTGGA


 


10321
CATGCTTAAC AAGAGGCAGT CACCGCTTGT TGATAAGGAG ATTGAAGAGT ATCTGGCGAC


 


10381
TAAAGATCTC AATTTCCGCG CTACGACAGA TAAGTATGAC GCGTATAAAA ATGCCGATTA


 


10441
CGTTATTATT GCCACACCTA CCGATTATGA TCCGAAAACA AATTACTTTA ATACCTCAAG


 


10501
CGTGGAAGCG GTCATTCGTG ATGTGACAGA AATTAATCCC AACGCGGTAA TGATTATAAA


 


10561
ATCAACTATC CCTGTTGGTT TTACAGAGTC CATTAAAGAA CGTTTTGGTA TTGAAAATGT


 


10621
GATCTTTTCG CCTGAGTTTT TGCGTGAAGG TAAAGCACTT TATGATAACT TACACCCATC


 


10681
ACGCATTGTG ATTGGCGAGC AGTCTGAACG CGCTAAACGT TTTGCTGCGT TATTACAGGA


 


10741
AGGCGCCATT AAGCAAGACA TACCAACATT GTTTACTGAC TCAACCGAGG CTGAGGCGAT


 


10801
TAAACTTTTT GCGAACACTT ATCTGGCGAT GCGTGTAGCG TATTTCAATG AACTTGATAG


 


10861
TTATGCTGAA AGCCTGGGAC TTAATTCACG CCAGATTATT GAGGGCGTAT GCCTTGACCC


 


10921
GCGTATCGGT AATCACTACA ACAACCCGTC ATTCGGTTAT GGTGGTTATT GTCTGCCGAA


 


10981
AGATACTAAG CAGTTACTGG CAAATTACCA GTCTGTGCCG AATAACCTGA TCTCGGCAAT


 


11041
TGTTGACGCC AACCGCACGC GCAAAGATTT TATTGCCGAT TCTATCCTTG CACGTAAACC


 


11101
GAAAGTTGTT GGCGTCTATC GTTTGATTAT GAAGAATGGT TCAGACAATT TTCGTGCTTC


 


11161
CTCGATTCAG GGTATTATGA AGCGAATCAA GGCGAAAGGT GTGCCTGTAA TCGTTTATGA


 


11221
GCCAGCTATG AAAGAGGACG ATTTTTTCCG GTCGCGCGTG GTACGTGATC TGGATGCGTT


 


11281
CAAACAAGAA GCTGATGTTA TTATTTCTAA CCGTATGTCT GCCGATCTGG CTGATGTAGC


 


11341
AGATAAAGTT TATACGCGCG ACTTGTTTGG CAATGATTAA TTATTTTGTT TCATTCTAAG


 


11401
AAAAGGCCCT AATAAATTAG GGCCTTTTCT TATGGTTTTG TAAAATCAAA CTTTATAGAA


 


11461
GTTACGATAC CATTCTACAA AGTTCTTTAC CCCTTCTTTA ACTGACGTTT CAGGTTTGAA


 


11521
TCCTATTACG TCATACAGTG CTTTTGTATC AGCACTGGTT TCCAGTACAT CACCGGGTTG


 


11581
GAGAGGCATC ATATTTTTGT TGGCTTCAAT ACCCAGAGCC TCTTCTAACG CATTGATATA


 


11641
GTCCATCAAC TCCACAGGCG AACTATTACC AATGTTATAG ACACGATATG GTGCTGAACT


 


11701
TGTTGCAGGC GAGCCTGTTT CTACAGCCCA CTGTGGGTTT TTTTCTGGAA TAACATCCTG


 


11761
TAAGCGAATA ATAGCTTCGG CAATATCATC AATGTAAGTA AAGTCACGCT TCATTTTGCC


 


11821
GAAGTTGTAA ACATCAATGC TTTTACCTTC CAGCATGGCT TTAGTGAATT TAAATAATGC


 


11881
CATATCCGGA CGTCCCCATG GACCATAAAC CGTAAAGAAA CGCAGCCCTG TGGTCGGTAA


 


11941
GCCATACAAA TGAGAATATG TATGGGCCAT GAGTTCATTC GCTTTTTTAG TTGCTGCATA


 


12001
AAGCGAAACA GGATGATCTA CAGAGTCATC TGTAGAGAAA GGCATCTTGC GGTTCATGCC


 


12061
ATAAACAGAA CTGGAGGAAG CGTAAAGTAG ATGCTGAACA TTATTATGGC GACATCCTTC


 


12121
TAGTATGTTC AGGAATCCAA TCAGGTTTGC ATCTGCATAT GCATTGGGAT TTTCAAGAGA


 


12181
GTAACGTACA CCGGCTTGCG CAGCGAGGTT TATTACGCGT TCGAACCGCT CGTCTGCAAA


 


12241
CAGTGCCGCC ATTTTCTCAC GATCGGCCAG GTCAATTTTA TAAAAACTGA AGTTGTCGTG


 


12301
CTTGAGTAAA TCAAGTCGTG CTTGTTTGAG GTTGACATCG TAATAATCAT TTAAGTTGTC


 


12361
AATGCCTACA ACCTGATGAC CAGCTGCAAG AAGCCGTTTA CTTAGATAGA AACCGATAAA


 


12421
GCCAGCAGCT CCCGTAACCA GAAATTTCAT TTATAATCCT CGCTCAGGCT AGAATATAGC


 


12481
CAATCTTCAT CTGGCATAAC TGAAAGTTAA ATTATACCGT TAGACAAGAA AAAAAGATAA


 


12541
TCGGTATCAG TTCTAAACTT GGCTGTTTTT TCTGGTAACG TGCTCATTTT ACAATCAAAG


 


12601
CTGTTCTAAG CTGACTATAC AAGCCGACGT CATTATCTCC AACCGTATGG CAGAAGAGCT


 


12661
TAAGGATGTG GCAGACAAAG TCTACACCCG CGATCTCTTT GGCAGTGACT AACATCCTGT


 


12721
TATCATGGCG ATTTTCGCCC TGATTCTCTT ATGTTCCCTT TGTAATAATT CATTATTTTT


 


12781
ATCATTTATC CTATAGCATT CATGGCGATT ATCGCTAAAC TATGGCGGCG CGCCACGTGG


 


12841
GATCCCCGGG TACCGAGCTC GAATTCGCCC TATAGTGAGT CGTATTACAA TTCACTGGCC


 


12901
GTCGTTTTAC AACGTCGTGA CTGGGAAAAC CCTGGCGTTA CCCAACTTAA TCGCCTTGCA


 


12961
GCACATCCCC CTTTCGCCAG CTGGCGTAAT AGCGAAGAGG CCCGCACCGA TCGCCCTTCC


 


13021
CAACAGTTGC GCAGCTGAAT GGCGAATGGC GCCTGATGCG GTATTTTCTC CTTACGCATC


 


13081
TGTGCGGTAT TTCACACCGC ATATGGTGCA CTCTCAGTAC AATCTGCTCT GATGCCGCAT


 


13141
AGTTAAGCCA GCCCCGACAC CCGCCAACAC CCGCTGACGC GAACCCCTTG CGGCCGCATC


 


13201
GAATATAACT TCGTATAATG TATGCTATAC GAAGTTATTA GCGATGAGCT CGGACTTCCA


 


13261
TTGTTCATTC CACGGACAAA AACAGAGAAA GGAAACGACA GAGGCCAAAA AGCTCGCTTT


 


13321
CAGCACCTGT CGTTTCCTTT CTTTTCAGAG GGTATTTTAA ATAAAAACAT TAAGTTATGA


 


13381
CGAAGAAGAA CGGAAACGCC TTAAACCGGA AAATTTTCAT AAATAGCGAA AACCCGCGAG


 


13441
GTCGCCGCCC CGTAACCTGT CGGATCACCG GAAAGGACCC GTAAAGTGAT AATGATTATC


 


13501
ATCTACATAT CACAACGTGC GTGGAGGCCA TCAAACCACG TCAAATAATC AATTATGACG


 


13561
CAGGTATCGT ATTAATTGAT CTGCATCAAC TTAACGTAAA AACAACTTCA GACAATACAA


 


13621
ATCAGCGACA CTGAATACGG GGCAACCTCA TGTCCGAGCT CGCGAGCTCG TCGACAGCGA


 


13681
CACACTTGCA TCGGATGCAG CCCGGTTAAC GTGCCGGCAC GGCCTGGGTA ACCAGGTATT


 


13741
TTGTCCACAT AACCGTGCGC AAAATGTTGT GGATAAGCAG GACACAGCAG CAATCCACAG


 


13801
CAGGCATACA ACCGCACACC GAGGTTACTC CGTTCTACAG GTTACGACGA CATGTCAATA


 


13861
CTTGCCCTTG ACAGGCATTG ATGGAATCGT AGTCTCACGC TGATAGTCTG ATCGACAATA


 


13921
CAAGTGGGAC CGTGGTCCCA GACCGATAAT CAGACCGACA ACACGAGTGG GATCGTGGTC


 


13981
CCAGACTAAT AATCAGACCG ACGATACGAG TGGGACCGTG GTCCCAGACT AATAATCAGA


 


14041
CCGACGATAC GAGTGGGACC GTGGTTCCAG ACTAATAATC AGACCGACGA TACGAGTGGG


 


14101
ACCGTGGTCC CAGACTAATA ATCAGACCGA CGATACGAGT GGGACCATGG TCCCAGACTA


 


14161
ATAATCAGAC CGACGATACG AGTGGGACCG TGGTCCCAGT CTGATTATCA GACCGACGAT


 


14221
ACGAGTGGGA CCGTGGTCCC AGACTAATAA TCAGACCGAC GATACGAGTG GGACCGTGGT


 


14281
CCCAGACTAA TAATCAGACC GACGATACGA GTGGGACCGT GGTCCCAGTC TGATTATCAG


 


14341
ACCGACGATA CAAGTGGAAC AGTGGGCCCA GAGAGAATAT TCAGGCCAGT TATGCTTTCT


 


14401
GGCCTGTAAC AAAGGACATT AAGTAAAGAC AGATAAACGT AGACTAAAAC GTGGTCGCAT


 


14461
CAGGGTGCTG CCTTTTCAAG TTCCTTAAGA ATGGCCTCAA TTTTCTCTAT ACACTCAGTT


 


14521
GGAACACGAG ACCTGTCCAG GTTAAGCACC ATTTTATCGC CCTTATACAA TACTGTCGCT


 


14581
CCAGGAGCAA ACTGATGTCG TGAGCTTAAA CTAGTTCTTG ATGCAGATGA CGTTTTAAGC


 


14641
ACAGAAGTTA AAAGAGTGAT AACTTCTTCA GCTTCAAATA TCACCCCAGC TTTTTTCTGC


 


14701
TCATGAAGGT TAGATGCCTG CTGCTTAAGT AATTCCTCTT TATCTGTAAA TTTTTTTTGA


 


14761
AGTGCATCAC CTGACCGGGC AGATAGTTCA CCGGGGTGAG AAAAAAGAGC AACAACTGAT


 


14821
TTAGGCAATT TGGCGGTGTT GATACAGCGG GTAATAATCT TACGTGAAAT ATTTTCCGCA


 


14881
TCAGCCAGCG CAGAAATATT TCCAGCAAAT TCATTCTGCA ATCGGCTTGC ATAACGCTGA


 


14941
CCACGTTCAT AAGCACTTGT TGGGCGATAA TCGTTACCCA ATCTGGATAA TGCAGCCATC


 


15001
TGCTCATCAT CCAGCTCGCC AACCAGAACA CGATAATCAC TTTCGGTAAG TGCAGCAGCT


 


15061
TTACGACGGC GACTCCCATC GGCAATTTCT ATGACACCAG ATACTCTTCG ACCGAACGCC


 


15121
GGTGTCTGTT GACCAGTCAG TAGAAAAGAA GGGATGAGAT CATCCAGTGC GTCCTCAGTA


 


15181
AGCAGCTCCT GGTCACGTTC ATTACCTGAC CATACCCGAG AGGTCTTCTC AACACTATCA


 


15241
CCCCGGAGCA CTTCAAGAGT AAACTTCACA TCCCGACCAC ATACAGGCAA AGTAATGGCA


 


15301
TTACCGCGAG CCATTACTCC TACGCGCGCA ATTAACGAAT CCACCATCGG GGCAGCTGGT


 


15361
GTCGATAACG AAGTATCTTC AACCGGTTGA GTATTGAGCG TATGTTTTGG AATAACAGGC


 


15421
GCACGCTTCA TTATCTAATC TCCCAGCGTG GTTTAATCAG ACGATCGAAA ATTTCATTGC


 


15481
AGACAGGTTC CCAAATAGAA AGAGCATTTC TCCAGGCACC AGTTGAAGAG CGTTGATCAA


 


15541
TGGCCTGTTC AAAAACAGTT CTCATCCGGA TCTGACCTTT ACCAACTTCA TCCGTTTCAC


 


15601
GTACAACATT TTTTAGAACC ATGCTTCCCC AGGCATCCCG AATTTGCTCC TCCATCCACG


 


15661
GGGACTGAGA GCCATTACTA TTGCTGTATT TGGTAAGCAA AATACGTACA TCAGGCTCGA


 


15721
ACCCTTTAAG ATCAACGTTC TTGAGCAGAT CACGAAGCAT ATCGAAAAAC TGCAGTGCGG


 


15781
AGGTGTAGTC AAACAACTCA GCAGGCGTGG GAACAATCAG CACATCAGCA GCACATACGA


 


15841
CATTAATCGT GCCGATACCC AGGTTAGGCG CGCTGTCAAT AACTATGACA TCATAGTCAT


 


15901
GAGCAACAGT TTCAATGGCC AGTCGGAGCA TCAGGTGTGG ATCGGTGGGC AGTTTACCTT


 


15961
CATCAAATTT GCCCATTAAC TCAGTTTCAA TACGGTGCAG AGCCAGACAG GAAGGAATAA


 


16021
TGTCAAGCCC CGGCCAGCAA GTGGGCTTTA TTGCATAAGT GACATCGTCC TTTTCCCCAA


 


16081
GATAGAAAGG CAGGAGAGTG TCTTCTGCAT GAATATGAAG ATCTGGTACC CATCCGTGAT


 


16141
ACATTGAGGC TGTTCCCTGG GGGTCGTTAC CTTCCACGAG CAAAACACGT AGCCCCTTCA


 


16201
GAGCCAGATC CTGAGCAAGA TGAACAGAAA CTGAGGTTTT GTAAACGCCA CCTTTATGGG


 


16261
CAGCAACCCC GATCACCGGT GGAAATACGT CTTCAGCACG TCGCAATCGC GTACCAAACA


 


16321
CATCACGCAT ATGATTAATT TGTTCAATTG TATAACCAAC ACGTTGCTCA ACCCGTCCTC


 


16381
GAATTTCCAT ATCCGGGTGC GGTAGTCGCC CTGCTTTCTC GGCATCTCTG ATAGCCTGAG


 


16441
AAGAAACCCC AACTAAATCC GCTGCTTCAC CTATTCTCCA GCGCCGGGTT ATTTTCCTCG


 


16501
CTTCCGGGCT GTCATCATTA AACTGTGCAA TGGCGATAGC CTTCGTCATT TCATGACCAG


 


16561
CGTTTATGCA CTGGTTAAGT GTTTCCATGA GTTTCATTCT GAACATCCTT TAATCATTGC


 


16621
TTTGCGTTTT TTTATTAAAT CTTGCAATTT ACTGCAAAGC AACAACAAAA TCGCAAAGTC


 


16681
ATCAAAAAAC CGCAAAGTTG TTTAAAATAA GAGCAACACT ACAAAAGGAG ATAAGAAGAG


 


16741
CACATACCTC AGTCACTTAT TATCACTAGC GCTCGCCGCA GCCGTGTAAC CGAGCATAGC


 


16801
GAGCGAACTG GCGAGGAAGC AAAGAAGAAC TGTTCTGTCA GATAGCTCTT ACGCTCAGCG


 


16861
CAAGAAGAAA TATCCACCGT GGGAAAAACT CCAGGTAGAG GTACACACGC GGATAGCCAA


 


16921
TTCAGAGTAA TAAACTGTGA TAATCAACCC TCATCAATGA TGACGAACTA ACCCCCGATA


 


16981
TCAGGTCACA TGACGAAGGG AAAGAGAAGG AAATCAACTG TGACAAACTG CCCTCAAATT


 


17041
TGGCTTCCTT AAAAATTACA GTTCAAAAAG TATGAGAAAA TCCATGCAGG CTGAAGGAAA


 


17101
CAGCAAAACT GTGACAAATT ACCCTCAGTA GGTCAGAACA AATGTGACGA ACCACCCTCA


 


17161
AATCTGTGAC AGATAACCCT CAGACTATCC TGTCGTCATG GAAGTGATAT CGCGGAAGGA


 


17221
AAATACGATA TGAGTCGTCT GGCGGCCTTT CTTTTTCTCA ATGTATGAGA GGCGCATTGG


 


17281
AGTTCTGCTG TTGATCTCAT TAACACAGAC CTGCAGGAAG CGGCGGCGGA AGTCAGGCAT


 


17341
ACGCTGGTAA CTTTGAGGCA GCTGGTAACG CTCTATGATC CAGTCGATTT TCAGAGAGAC


 


17401
GATGCCTGAG CCATCCGGCT TACGATACTG ACACAGGGAT TCGTATAAAC GCATGGCATA


 


17461
CGGATTGGTG ATTTCTTTTG TTTCACTAAG CCGAAACTGC GTAAACCGGT TCTGTAACCC


 


17521
GATAAAGAAG GGAATGAGAT ATGGGTTGAT ATGTACACTG TAAAGCCCTC TGGATGGACT


 


17581
GTGCGCACGT TTGATAAACC AAGGAAAAGA TTCATAGCCT TTTTCATCGC CGGCATCCTC


 


17641
TTCAGGGCGA TAAAAAACCA CTTCCTTCCC CGCGAAACTC TTCAATGCCT GCCGTATATC


 


17701
CTTACTGGCT TCCGCAGAGG TCAATCCGAA TATTTCAGCA TATTTAGCAA CATGGATCTC


 


17761
GCAGATACCG TCATGTTCCT GTAGGGTGCC ATCAGATTTT CTGATCTGGT CAACGAACAG


 


17821
ATACAGCATA CGTTTTTGAT CCCGGGAGAG ACTATATGCC GCCTCAGTGA GGTCGTTTGA


 


17881
CTGGACGATT CGCGGGCTAT TTTTACGTTT CTTGTGATTG ATAACCGCTG TTTCCGCCAT


 


17941
GACAGATCCA TGTGAAGTGT GACAAGTTTT TAGATTGTCA CACTAAATAA AAAAGAGTCA


 


18001
ATAAGCAGGG ATAACTTTGT GAAAAAACAG CTTCTTCTGA GGGCAATTTG TCACAGGGTT


 


18061
AAGGGCAATT TGTCACAGAC AGGACTGTCA TTTGAGGGTG ATTTGTCACA CTGAAAGGGC


 


18121
AATTTGTCAC AACACCTTCT CTAGAACCAG CATGGATAAA GGCCTACAAG GCGCTCTAAA


 


18181
AAAGAAGATC TAAAAACTAT AAAAAAAATA ATTATAAAAA TATCCCCGTG GATAAGTGGA


 


18241
TAACCCCAAG GGAAGTTTTT TCAGGCATCG TGTGTAAGCA GAATATATAA GTGCTGTTCC


 


18301
CTGGTGCTTC CTCGCTCACT CGACCGGGAG GGTTCGAGAA GGGGGGGCAC CCCCCTTCGG


 


18361
CGTGCGCGGT CACGCGCACA GGGCGCAGCC CTGGTTAAAA ACAAGGTTTA TAAATATTGG


 


18421
TTTAAAAGCA GGTTAAAAGA CAGGTTAGCG GTGGCCGAAA AACGGGCGGA AACCCTTGCA


 


18481
AATGCTGGAT TTTCTGCCTG TGGACAGCCC CTCAAATGTC AATAGGTGCG CCCCTCATCT


 


18541
GTCAGCACTC TGCCCCTCAA GTGTCAAGGA TCGCGCCCCT CATCTGTCAG TAGTCGCGCC


 


13601
CCTCAAGTGT CAATACCGCA GGGCACTTAT CCCCAGGCTT GTCCACATCA TCTGTGGGAA


 


18661
ACTCGCGTAA AATCAGGCGT TTTCGCCGAT TTGCGAGGCT GGCCAGCTCC ACGTCGCCGG


 


18721
CCGAAATCGA GCCTGCCCCT CATCTGTCAA CGCCGCGCCG GGTGAGTCGG CCCCTCAAGT


 


18781
GTCAACGTCC GCCCCTCATC TGTCAGTGAG GGCCAAGTTT TCCGCGAGGT ATCCACAACG


 


18841
CCGGCGGCCG GCCGCGGTGT CTCGCACACG GCTTCGACGG CGTTTCTGGC GCGTTTGCAG


 


18901
GGCCATAGAC GGCCGCCAGC CCAGCGGCGA GGGCAACCAG CCGAGGGCTT CGCCCTGTCG


 


18961
CTCGACTGCG GCGAGCACTA CTGGCTGTAA AAGGACAGAC CACATCATGG TTCTGTGTTC


 


19021
ATTAGGTTGT TCTGTCCATT GCTGACATAA TCCGCTCCAC TTCAACGTAA CACCGCACGA


 


19081
AGATTTCTAT TGTTCCTGAA GGCATATTCA AATCGTTTTC GTTACCGCTT GCAGGCATCA


 


19141
TGACAGAACA CTACTTCCTA TAAACGCTAC ACAGGCTCCT GAGATTAATA ATGCGGATCT


 


19201
CTACGATAAT GGGAGATTTT CCCGACTGTT TCGTTCGCTT CTCAGTGGAT AACAGCCAGC


 


19261
TTCTCTGTTT AACAGACAAA AACAGCATAT CCACTCAGTT CCACATTTCC ATATAAAGGC


 


19321
CAAGGCATTT ATTCTCAGGA TAATTGTTTC AGCATCGCAA CCGCATCAGA CTCCGGCATC


 


19381
GCAAACTGCA CCCGGTGCCG GGCAGCCACA TCCAGCGCAA AAACCTTCGT GTAGACTTCC


 


19441
GTTGAACTGA TGGACTTATG TCCCATCAGG CTTTGCAGAA CTATCAGCGG TATACCGGCA


 


19501
TACAGCATGT GCATCGCATA GGAATGGCGG AACGTATGTG GTGTGACCGG AACAGAGAAC


 


19561
GTCACACCGT CAGCAGCAGC GGCGGCAACC GCCTCCCCAA TCCAGGTCCT GACCGTTCTG


 


19621
TCCGTCACTT CCCAGATCCG CGCTTTCTCT GTCCTTCCTG TGCGACGGTT ACGCCGCTCC


 


19681
ATGAGCTTAT CGCGAATAAA TACCTGTGAC GGAAGATCAC TTCGCAGAAT AAATAAATCC


 


19741
TGGTGTCCCT GTTGATACCG GGAAGCCCTG GGCCAACTTT TGGCGAAAAT GAGACGTTGA


 


19801
TCGGCACGTA AGAGGTTCCA ACTTTCACCA TAATGAAATA AGATCACTAC CGGGCGTATT


 


19861
TTTTGAGTTA TCGAGATTTT CAGGAGCTAA GGAAGCTAAA ATGGAGAAAA AAATCACTGG


 


19921
ATATACCACC GTTGATATAT CCCAATGGCA TCGTAACTAA CATTTTGAGG CATTTCAGTC


 


19981
AGTTGCTCAA TGTACCTATA ACCAGACCGT TCAGCTGGAT ATTACGGCCT TTTTAAAGAC


 


20041
CGTAAAGAAA AATAAGCACA AGTTTTATCC GGCCTTTATT CACATTCTTG CCCGCCTGAT


 


20101
GAATGCTCAT CCGGAATTTC GTATGGCAAT GAAAGACGGT GAGCTGGTGA TATGGGATAG


 


20161
TGTTCACCCT TGTTACACCG TTTTCCATGA GCAAACTGAA ACGTTTTCAT CGCTCTGGAG


 


20221
TGAATACCAC GACGATTTCC GGCAGTTTCT ACACATATAT TCGCAAGATG TGGCGTGTTA


 


20281
CGGTGAAAAC CTGGCCTATT TCCCTAAAGG GTTTATTGAG AATATGTTTT TCGTCTCAGC


 


20341
CAATCCCTGG GTGAGTTTCA CCAGTTTTGA TTTAAACGTG GCCAATATGG ACAACTTCTT


 


20401
CGCCCCCGTT TTCACCATGG GCAAATATTA TACGCAAGGC GACAAGGTGC TGATGCCGCT


 


20461
GGCGATTCAG GTTCATCATG CCCTTTGTGA TGGCTTCCAT GTCGGCAGAA TGCTTAATGA


 


20521
ATTACAACAG TACTGCGATG AGTGGCAGGG CGGGGCGTAA TTTTTTTAAG GCAGTTATTG


 


20581
GTGCCCTTAA ACGCCTGGTT GCTACGCCTG AATAAGTGAT AATAAGCGGA TGAATGGCAG


 


20641
AAATTCGATG ATAAGCTGTC AAACATGAGA ATTGGTCGAC GGCCCGGGCG GCCGCAAGGG


 


20701
GTTCGCGTTG GCCGATTCAT TAATGCAGCT GGCACGACAG GTTTCCCGAC TGGAAAGCGG


 


20761
GCAGTGAGCG CAACGCAATT AATGTGAGTT AGCTCACTCA TTAGGCACCC CAGGCTTTAC


 


20821
ACTTTATGCT TCCGGCTCGT ATGTTGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG


 


20881
GAAACAGCTA TGACCATGAT TACGCCAAGC TATTTAGGTG AGACTATAGA ATACTCAAGC


 


20941
TTGCATGCCT GCAGGTCGAC TCTAGAGGAT CCCACGACGT CG


 







Nucleotide Sequence for pCC1FOS cut (pFOS)


and S. flexneri 6 O-antigen with Z3206


Locus pFOS cut and O-antigen cut (Z3206+)


Definition Ligation of inverted S. flexneri 6 O antigen cluster


amplified with Z3206Nhe and wzzAscI cut with NheI and AscI into


pCC1FOS with MCS cassette cut with NheI and AscI








Features
    Location/Qualifiers


CDS
    complement(370..396)



/label=wzz′


CDS
    748..1752



/label=uge


CDS
    complement(1818..3011)



/label=ugd


CDS
    complement(3233..4639)



/label=gnd


CDS
    complement(4744..5577)



/label=wfbZ


CDS
    complement(5574..6443)



/label=wfbY


CDS
    complement(6460..7647)



/label=wzy


CDS
    complement(7703..8935)



/label=wzx


CDS
    complement(8932..9489)



/label=rmlC


CDS
    complement(9494..10372)



/label=rmlA


CDS
    complement(10430..11329)



/label=rmlD


CDS
    complement(11329..12414)



/label=rmlB


CDS
    complement(12787..13680)



/label=galF


CDS
    complement(13912..14907)



/label=Z3206


CDS
    complement(15065..15097)



/label=′weaM


CDS
    complement(15525..16184)



/label=cat


CDS
    16403..16750



/label=redF


CDS
    18145..18900



/label=repE


CDS
    19479..20654



/label=parA


CDS
    20654..21625



/label=parB







Length: 22887 bp


Type: DNA circular UNA


Sequence:


SEQ ID NO: 29








    1
GCGGCCGCAA GGGGTTCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG


 


   61
CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA CCGCACAGAT


 


  121
GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT CAGCTGCGCA ACTGTTGGGA


 


  181
AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC


 


  241
AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CGACGTTGTA AAACGACGGC


 


  301
CAGTGAATTG TAATACGACT CACTATAGGG CGAATTCGAG CTCGGTACCC GGGGATCCCA


 


  361
CGTGGCGCGC CGCCATAGTT TAGCGATAAT CGCCATGAAT GCTATAGGAT AAATGATAAA


 


  421
AATAATGAAT TATTACAAAG GGAACATAAG AGAATCAGGG CGAAAATCGC CATGATAACA


 


  481
GGATGTTAGT CACTGCCAAA GAGATCGCGG GTGTAGACTT TGTCTGCCAC ATCCTTAAGC


 


  541
TCTTCTGCCA TACGGTTGGA GATAATGACG TCGGCTTGTA TAGTCAGCTT AGAACAGCTT


 


  601
TGATTGTAAA ATGAGCACGT TACCAGAAAA AACAGCCAAG TTTAGAACTG ATACCGATTA


 


  661
TCTTTTTTTC TTGTCTAACG GTATAATTTA ACTTTCAGTT ATGCCAGATG AAGATTGGCT


 


  721
ATATTCTAGC CTGAGCGAGG ATTATAAATG AAATTTCTGG TTACGGGAGC TGCTGGCTTT


 


  781
ATCGGTTTCT ATCTAAGTAA ACGGCTTCTT GCAGCTGGTC ATCAGGTTGT AGGCATTGAC


 


  841
AACTTAAATG ATTATTACGA TGTCAACCTC AAACAAGCAC GACTTGATTT ACTCAAGCAC


 


  901
GACAACTTCA GTTTTTATAA AATTGACCTG GCCGATCGTG AGAAAATGGC GGCACTGTTT


 


  961
GCAGACGAGC GGTTCGAACG CGTAATAAAC CTCGCTGCGC AAGCCGGTGT ACGTTACTCT


 


 1021
CTTGAAAATC CCAATGCATA TGCAGATGCA AACCTGATTG GATTCCTGAA CATACTAGAA


 


 1081
GGATGTCGCC ATAATAATGT TCAGCATCTA CTTTACGCTT CCTCCAGTTC TGTTTATGGC


 


 1141
ATGAACCGCA AGATGCCTTT CTCTACAGAT GACTCTGTAG ATCATCCTGT TTCGCTTTAT


 


 1201
GCAGCAACTA AAAAAGCGAA TGAACTCATG GCCCATACAT ATTCTCATTT GTATGGCTTA


 


 1261
CCGACCACAG GGCTGCGTTT CTTTACGGTT TATGGTCCAT GGGGACGTCC GGATATGGCA


 


 1321
TTATTTAAAT TCACTAAAGC CATGCTGGAA GGTAAAAGCA TTGATGTTTA CAACTTCGGC


 


 1381
AAAATGAAGC GTGACTTTAC TTACATTGAT GATATTGCCG AAGCTATTAT TCGCTTACAG


 


 1441
GATGTTATTC CAGAAAAAAA CCCACAGTGG GCTGTAGAAA CAGGCTCGCC TGCAACAAGT


 


 1501
TCAGCACCAT ATCGTGTCTA TAACATTGGT AATAGTTCGC CTGTGGAGTT GATGGACTAT


 


 1561
ATCAATGCGT TAGAAGAGGC TCTGGGTATT GAAGCCAACA AAAATATGAT GCCTCTCCAA


 


 1621
CCCGGTGATG TACTGGAAAC CAGTGCTGAT ACAAAAGCAC TGTATGACGT AATAGGATTC


 


 1681
AAACCTGAAA CGTCAGTTAA AGAAGGGGTA AAGAACTTTG TAGAATGGTA TCGTAACTTC


 


 1741
TATAAAGTTT GATTTTACAA AACCATAAGA AAAGGCCCTA ATTTATTAGG GCCTTTTCTT


 


 1801
AGAATGAAAC AAAATAATTA ATCATTGCCA AACAAGTCGC GCGTATAAAC TTTATCTGCT


 


 1861
ACATCAGCCA GATCGGCAGA CATACGGTTA GAAATAATAA CATCAGCTTC TTGTTTGAAC


 


 1921
GCATCCAGAT CACGTACCAC GCGCGACCGG AAAAAATCGT CCTCTTTCAT AGCTGGCTCA


 


 1981
TAAACGATTA CAGGCACACC TTTCGCCTTG ATTCGCTTCA TAATACCCTG AATCGAGGAA


 


 2041
GCACGAAAAT TGTCTGAACC ATTCTTCATA ATCAAACGAT AGACGCCAAC AACTTTCGGT


 


 2101
TTACGTGCAA GGATAGAATC GGCAATAAAA TCTTTGCGCG TGCGGTTGGC GTCAACAATT


 


 2161
GCCGAGATCA GGTTATTCGG CACAGACTGG TAATTTGCCA GTAACTGCTT AGTATCTTTC


 


 2221
GGCAGACAAT AACCACCATA ACCGAATGAC GGGTTGTTGT AGTGATTACC GATACGCGGG


 


 2281
TCAAGGCATA CGCCCTCAAT AATCTGGCGT GAATTAAGTC CCAGGCTTTC AGCATAACTA


 


 2341
TCAAGTTCAT TGAAATACGC TACACGCATC GCCAGATAAG TGTTCGCAAA AAGTTTAATC


 


 2401
GCCTCAGCCT CGGTTGAGTC AGTAAACAAT GTTGGTATGT CTTGCTTAAT GGCGCCTTCC


 


 2461
TGTAATAACG CAGCAAAACG TTTAGCGCGT TCAGACTGCT CGCCAATCAC AATGCGTGAT


 


 2521
GGGTGTAAGT TATCATAAAG TGCTTTACCT TCACGCAAAA ACTCAGGCGA AAAGATCACA


 


 2581
TTTTCAATAC CAAAACGTTC TTTAATGGAC TCTGTAAAAC CAACAGGGAT AGTTGATTTT


 


 2641
ATAATCATTA CCGCGTTGGG ATTAATTTCT GTCACATCAC GAATGACCGC TTCCACGCTT


 


 2701
GAGGTATTAA AATAATTTGT TTTCGGATCA TAATCGGTAG GTGTGGCAAT AATAACGTAA


 


 2761
TCGGCATTTT TATACGCGTC ATACTTATCT GTCGTAGCGC GGAAATTGAG ATCTTTAGTC


 


 2821
GCCAGATACT CTTCAATCTC CTTATCAACA AGCGGTGACT GCCTCTTGTT AAGCATGTCC


 


 2881
ACTTTGGCCT GAACGATATC CAGTGCAACC ACTTCGTGGT TTTGCGCAAT CAGAATACCA


 


 2941
TTTGAAAGAC CAACATAACC TGTTCCTGAA ATTGTTATTT TCATTAGCTC TGACTTCTTC


 


 3001
CGGTTAAACA TTTAGAGTGG TCATTAATCC ACCACCACAG CTTCATCTAC TGCGGGATTT


 


 3061
TTAACGCTGA TGTAGATGAA CTGTCAAGGC AGCGATCCTG CTGTGCGGCG CTGTATTATA


 


 3121
TCGCGTTTTT AACTATAAAT TATAAAAAAA GGCCCTAACC TGCCGCTTTG TATAATAAAA


 


 3181
AAGCCCGGAG GGTTTCTCCG GGCCTTGCTT TGATTAATTG ATTTAAATCA GATTAATCCA


 


 3241
GCCATTCGGT ATGGAACACA CCTTCTTTAT CAATGCGCTT ATAAGTATGC GCACCGAAAT


 


 3301
AGTCACGCTG TGCCTGGATC AGGTTCGCAG GCAGAACAGC GGCGCGGTAG CTGTCGTAAT


 


 3361
AGGCAACCGC AGCGGCGAAG GTCGGCACCG GGATACCGTT CTGTACTGCG TAAGCGACGA


 


 3421
CATCGCGCAG CGCCTGCTGG TAGTCATCGG CAATTTGCTT GAAGTAAGGA GCCAGCAACA


 


 3481
GGTTAGCGAT CTGCGGATTT TCGGCATAAG CATCGGTGAT TTTCTGCAGG AACTGCGCAC


 


 3541
GGATGATGCA GCCAGCACGG AAAATCTTCG CGATTTCACC GTAGTTCAGA TCCCAGTTGT


 


 3601
ACTCTTCAGA CGCAGCGCGT AGCTGAGAGA AGCCCTGAGC GTAAGAAACG ATTTTGCCCA


 


 3661
GATACAGCGC ACGGCGAACT TTTTCGATGA ACTCAGCATT GTCGCCAGCT GGCTGCGCTT


 


 3721
GCGGGCCAGA GAGAACTTTA GATGCGGCAA CACGCTGCTC TTTCAGAGAA GAGATATAAC


 


 3781
GTGCAAACAC AGACTCGGTA ATCAGCGACA GCGGTTCGCC GAGATCCAGC GCGCTCTGGC


 


 3841
TGGTCCATTT GCCCGTACCT TTGTTTGCTG CTTCATCCAG AATCACATCA ACCAGGTAGT


 


 3901
TACCCTCTTC ATCTTTTTTG GTGAAGATAT CTTTGGTGAT GTCGATCAGG TAGCTGCTCA


 


 3961
GTTCACCGTT ATTCCACTCG GTAAAGGTCT GCGCCAGTTC TTCGTTGGTG AGGTTCAAGC


 


 4021
CACCTTTAAG CAGAGAATAG GCTTCAGCAA TCAGCTGCAT ATCACCGTAT TCAATACCGT


 


 4081
TGTGAACCAT CTTCACATAA TGACCTGCAC CATCGGCACC AATATAGGTA ACGCACGGTT


 


 4141
CGCCGTCTTC AGCCACAGCG GCGATTTTGG TCAGGATCGG CGCAATCAGT TCATAAGCTT


 


 4201
CTTTCTGCCC ACCAGGCATA ATGGAAGGAC CTTTCAGCGC ACCTTCTTCA CCACCGGAAA


 


 4261
CACCGGTACC GATAAAGTTA AAGCCTTCTG CAGAAAGCTC ACGGTTACGA CGAATGGTGT


 


 4321
CATGGAAGAA GGTGTTACCA CCATCAATGA TGATGTCACC TTTATCGAGG TATGGCTTGA


 


 4381
GGGAATCAAT AGCAGCATCC GTGCCAGCAC CTGCTTTCAC CATTAACAGG ATGCGACGAG


 


 4441
GCGTTTCCAG AGATTCAACA AATTCTTTCA CCGTATAGTA AGGAACCAGT TTCTTGCCTG


 


 4501
GATTTTCGGT AATCACTTCT TCGGTCTTTT CACGGGAACG GTTGAAAATA GAGACGGTAT


 


 4561
AACCACGGCT TTCGATATTG AGCGCAAGGT TGCGCCCCAT CACTGCCATA CCGACGACGC


 


 4621
CGATCTGTTG CTTTGACATT GTTTACTCCT GTCAGGATAC CGCTGGGTGG TATGCGGGTT


 


 4681
ATGCTTAATT ATAGAATATG CCTAATAAAA ATAAATCCAT AACACTTAAT CAGAAAATTA


 


 4741
TTATTATCGA TTCCTAACGA TTGAATACAT CAGCTCCTTT AATTTAGATG GCATTATACG


 


 4801
AAAAAATGTT CTCAACATAG CATTACTTAT TAATTCATTT TTTCGAATAA AACCAATTTT


 


 4861
ATATTGATAA TACAATACTT TATACTCGTA CAATAAATAT GACAATCCAC GTCGAGCCAT


 


 4921
AAGATTACGA CCAGTTCGCA TTTTTAATAA AATATCTGGA AGATTTGCAA ATCTTGCATT


 


 4981
ATGTACAATT AATAGGCTCC ACAATGCAAA ATCTTGAGAT TTTCTGAATG GAGGATAACC


 


 5041
ACCAACAGCT AATACTGTAT TCTTTCTAAA AATTACAGAA GGATGGCTAA CTGCGCTTCG


 


 5101
TTTCCTCGCG AATTTAACTA TTTCTCTATG TTCGAGAGGC ACTTTGCGTG TTGAAATAAA


 


 5161
CTCCTCAGTA ACAGTTTCAA TTTCATCAAT AAAACTGCCA CATACATCTA TTTCTGAATT


 


 5221
ATTAATCATA AAAGAAATTT GTTTCTCAAA CCGATGAGGC AAAGAAATAT CATCAGCATC


 


 5281
CATTCTTGCC ACTAACTCAT TCCTACAAGC CTTTAATCCT TCATTTAAGG CATTAGCCAA


 


 5341
TCCAACATTT CTAGGTAAAG GTACAAATGT TACTATTTTA TTGCCAACAT CATCAATGAA


 


 5401
TGAATTTATA ATATCGATGT GTGTTTGATG GAGTTCTCCA TCTGCAACAA TTACTATTTG


 


 5461
ATCTGGCTTA AGTGTTTGAT CGTGAAAAAT AGAGCGTAGA GCCACCTCAA AAAATTGCGG


 


 5521
TAGATCATTT TTATAAATGC TAATTAAAAC TGAGAATTTT TCTAATCTAT GATTCATTTC


 


 5581
ATTTTACCAC TTCGACCCAT TAAACCGTCA TTAATGCCTT TTAAAAAAAA ATATAACCTT


 


 5641
TTATTACCAT TTGGAAGGAA AATAGGATAT AAAAAAACCT TTCCAATTAA TTTAACCAGA


 


 5701
CTAGAAATTT TCCAGTAGAT GGGTACATAA TTTTTATTTA ATAAAAGAAA GATATTTCGA


 


 5761
GTAGCATAAT AATGACGAAA TGGGCTTGGC AAACCGACAG AAAGAATATT TAAGATCTTA


 


 5821
AATCGCCCAT CTCCAAGTCT ATGTGCAAGT AACGCATTTT TATTCCTAAT TACTTTAAAC


 


 5881
CCAGCAGCTC TTAATCTCCA ACAATATTCA TGGTCTACCG CATCGATAAA AAGCTCATCT


 


 5941
TTCATTCCTC CAACAATCAA CCAACTATTT TTTGGTATTA GACTGCCAGA ACTTAATGTA


 


 6001
CTATCTACCT CATAATAAAC TTCTGTAAGT GGTTTCCCTT TTTTTACCCT TGCTTTATTT


 


 6061
AATTCACCAG TTACTTTATC AAAATCTTGT GAACCAACTA AACCAACATT GACATTTTGT


 


 6121
TTAAGCAATT TTTTGTAACA AGTAAGTAAC TGCTCTACCA TCTTAGGATC AGGAATACTA


 


 6181
TCCTGATCCA TTTGCAATAT AAAATCAGCG CCATTTTCAA AAGCCCATTT CATTCCTATA


 


 6241
CTTTGGGCTT CTGCTATGCC TAAATTATCA TTGAAATTGA ATATTTTTAC ATCGCCTGAA


 


 6301
GAATTTTCAG CATATTTATA ACCATTTGTA GAGTTATTGC AAACGACAAC TTTAGTAACT


 


 6361
TGTCTCAACA ATAATTCAAC CGCATTTTTT AAATCATTAT GTTCTGGGTT GTAAGCAACC


 


 6421
AAAACGGCAT ATACAGTGTC CATCTTCACC TTAAAACCTT CATTTAGCTT TCATCTTTTT


 


 6481
TAGAACATTA CTTAATGTCA CTAATACAAT TATTACAGCA ACATGGTTAG AGTCTAAAAT


 


 6541
ATAAGGATTA GTAATTGCAT AAGAAACATA TAGAAAATAT AGCACACACA ACTCACTGTA


 


 6601
TTTTATGATT TTAATCGTGA GAAGGAGATT AATTAATAAA AACAAAGTAA ATAAAATAAC


 


 6661
GCCAAGTTGA TTTAAAAAAT AAACTGACTG CAATTCATAA TATATATATG CACTATAATC


 


 6721
ACGGATAGGA GTTTGAATTT TGATGACATT ACCCAAACCA GAACCTATAA CAAAATTTGA


 


 6781
TACAGACTCT GTAAGATCAT TAATTAATAC AGTAAACTGA TCCCATCTAA CTCCTAAAGA


 


 6841
AGAATCAGCT CCATTTGATT TCATGATTAT CAACTCAATT GAATATGTAA TAAAAAAAGG


 


 6901
GAGAATCACA GTAAGAAAAA CCCCAAAAAT AATTTTCCTT AATTTAGCGT ATCGTGAGTT


 


 6961
AGATTTAGAA CATAGTATAA TATACATAAA AAACAAGCAT ATCGAAACAA AATATGCAAA


 


 7021
ATTACCAGCC ACTATAGTAC CTATAGCCAG AATAACGGTT ATTGTATTTT TGAATCGATA


 


 7081
ATAGAAATAA TCTTTTATGA CTATATGCAA CATAAAGGCA AATGGAATGA GAGCATTTCC


 


 7141
TTTAATTTGA ACTCTATAGA AACCACTTCC ATATGTATAA ACATCACCAT AATCATTCTC


 


 7201
CAAAAAATAA TGTCTTAGTG CTGAATAATC ACCAATACCA TATGTTTTTG TCATATAAAT


 


 7261
ACTAATGATG GATATAATAA CCGCCTGTAA TACCATTAAA TATAAAAATA TTTTAACAAT


 


 7321
CGAGATGGTT CCATAAGAGC AGAAATAAGC ACATAATATA AATAATATGA TAATATAAAA


 


 7381
CCTAATTATT ATCGCTATAT CGTTACCCTT GATATAGGAA TAAATAAAAT TTATAAAAAG


 


 7441
AGCTAATAGA AATATTAAAA TAACAGGATA GTGATATATT CCGTTTGCAA TTTTCTTTGT


 


 7501
AAATGACATG ATACAAAGAC ATAAAAACCC CTCCATAATC CAACTATATT GAATAAATGG


 


 7561
AAAGCTACGT GTAAGGAAAA ATATAAACCC AAAAAACAAA AGAACACTTA AACTTTTGTC


 


 7621
TTTTGAGTTA TAAAAATCAG AAGTCATGTT TGCACTCTAA TTAGATGGGC TTGAGGAAGT


 


 7681
AATCCCTAAA ATCAATTCGC TATTAATATT TCGTATCAAT TAATAATAAT ATCAAAAAAT


 


 7741
CTAACGATGT TCTTACAGAC CATGCTATTG CGGCTCCAAC AATTCCCCAA TGATAAATAA


 


 7801
AAATATATAA TATGCATAAA TATGGGATAA CTTCGAGCAA ATGAATAATA GCTGTAATTT


 


 7861
TTGATCTTCC ACTAGCCTGA ACTGAAACAA ATGGGATTTG TGCAATGCAA TTAAAAAAGA


 


 7921
AACCTATTGC AAGAATTTTT AATACTATAC CTGGCGTCCC ATGATATGTA GGTCCCATCC


 


 7981
AAGCGGACAT TATAAAATCT GATAAAATAA TTATCAACAT TACAATTGGA AGTATACCAA


 


 8041
TAACCATTAT AAAATATGAT AATATTTTAG TTTGCTTTAC CGATTGCAAT TCTGAACTTA


 


 8101
ATCTTGGAAA AATAGCTCTG GACAACGCAC TTGGTAATAT CGTTAAGCGT TGTATACCTT


 


 8161
CAGACGGAGC AGTATAAAAA GAAACTTTAT CAGCCCCCAC AATGTGTGAA AGAATAAAAC


 


 8221
GATCCATATA TGTCATAATA GGGCTAATAA TATTGCTAAC TGTTATCCAG CTTCCAAAGC


 


 8281
CGATTAATCT TTTAACTGTT ACAATTTTTA CAGACAGCCC AGATGATATT ATTAGTTTTC


 


 8341
GACTAAATAT AAAGGTCACT ATAAGTGATA AGACTCTTGC CATAACTAAA CCATATATAG


 


 8401
CACTTAGTAA TCCTCCATGA AAAAAACAGA AAATCACTGG TAATCCAGCC ACAAAAGAGT


 


 8461
TGTTAATTGA TTTTATTAAA TTTACTTTTC TGAACTTTTC CATCCCCTCA AAAATCCCCA


 


 8521
ACCAGACTTG GTTTAACAAG TATAAGGGTA TGGTAGCTGA AATAATATAT ATTGCTTTGA


 


 8581
CAGATTCTAC AACATGATTC GCGTTAATGT TTAATAATTT AACAATTACA TTGCTACTCA


 


 8641
AAAATAGTAC ACTACCGCCA ATCAAGCCCA ATATAGTTAG AATTACCGTT GAAGTTGAAA


 


 8701
TGATCGCTCT TAATTCTTTA TGAACATTTT TATATATTGA TACTTCTCTT ATAACAGCTC


 


 8761
TGGTCAATCC AGCATCAAAA ATACTTGCAT ATCCAACTAA GGCAATAGCT AACGTAAAAA


 


 8821
GGCCAAATTG CTCGGTCCCT AGAATTCTAG ACAGTATACC TAACGCAGGA ATTGCTATTA


 


 8881
ATGATGGTAT AATATACCCA CTTATATTCC ATAAAGTATT CTTTACAATA CTCACAAAAA


 


 8941
TAATTCCTTC ATGTTATGCA ATTCTTTAGC CCTTGCATCT TTAATCGATA AAATATAATT


 


 9001
ATTATGTTCT ATCGTCGGCC ATTTTATGCT CAGAATAGGA TCATTCCATA CAATCCCTCT


 


 9061
ATCACTATCA GGATGATAAT AGTTCGTCGT TTTATATAAA AATTCCGCAG TCTCGCTCAG


 


 9121
CACCAAAAAA CCATGTGCAA ATCCCTCAGG GATCCACAAT TGCCGCTTAT TCTCAGCAGA


 


 9181
TAAATTCACC CCAACCCATT TACCAAAGGT AGGCGACGAT TTACGAATAT CAACAGCTAC


 


 9241
ATCAAAAACC TCACCAACAA CGCAACGTAC CAGTTTCCCT TGCGCATAAG GTTCTAACTG


 


 9301
ATAATGCAGC CCGCGTAAAA CACCTTTACT AGACTTCGAA TGGTTATCCT GAACAAATTC


 


 9361
AACCTTACGT CCTACAGCTT CTTCGAAAAC TTTCTGATTA AAGCTTTCCA TAAAGAAACC


 


 9421
ACGCTCATCA CCAAAAACTT TCGGCTCGAA AATTAACACA TCAGGAATTT CTGTTTTAAT


 


 9481
TACGTTCATT TTATTAATAA CCTTTAATCA TTTTCAGCAG ATACTGTCCA TAAGCATTTT


 


 9541
TTTTCAGCGC CTCCGCTAAT GCTTTCACCT GTTCAGCATC AATAAACCCT TTACGGTAAG


 


 9601
CAATTTCTTC TGGGCAGGAA ACCTTTAGTC CCTGGCGCTC TTCAATGGTG GCAATGAAGT


 


 9661
TGCTTGCTTC AATAAGACTC TGATGTGTCC CCGTATCCAG CCATGCATAA CCACGCCCCA


 


 9721
TCATGGCAAC GGATAAACGC CCCTGTTCCA TATAAATACG GTTAATATCG GTAATTTCCA


 


 9781
GTTCACCACG GGCAGAAGGC TTAAGGTTTT TCGCCATTTC GACAACGTCG TTATCATAGA


 


 9841
AATAAAGCCC GGTTACCGCA TAATTACTTT TTGGTTGTAG CGGTTTTTCT TCCAGGCTTA


 


 9901
TTGCCGTACC GTTTTTATCA AACTCAACGA CGCCGTAGCG TTCAGGATCA TTAACGTGAT


 


 9961
AGGCAAATAC CGTTGCACCA CTTTCTTTGT TAACAGCGAC ATCCATTAAC TTCGGCAGAT


 


10021
CATGACCGTA GAAGATATTA TCACCAAGAA CCAAAGCACA ATCATCACCA CCGATAAACT


 


10081
CTTCACCGAT AATAAACGCC TGCGCAAGCC CATCTGGAGT CGGTTGCACT TTGTACTGAA


 


10141
GATTTAGCCC CCACTGGCTA CCGTCACCTA GCAGTTGTTG AAAACGAGGA GTATCCTGTG


 


10201
GCGTACTAAT AATCAGAATA TCGCGAATAC CCGCCAACAT CAGTGTAGAG AGCGGGTAAT


 


10261
AGATCATCGG CTTATCATAA ATAGGTAATA GCTGTTTACT GACAGCCATA GTCACAGGAT


 


10321
AAAGACGTGT ACCAGAACCA CCCGCTAAAA TAATACCTTT ACGCGTTTTC ATTTCATCAT


 


10381
TCCTTTTAAT TCATCTTGCT CCACCATCAC GAACAAGATG CAAAAACTAT TAAATTGCTG


 


10441
TAGTCGTAAT TAATTCGTTG AGCATTCGTT TCACACCAAC CTGCCAGTCA GGCAAGACAA


 


10501
GCGCAAAGTT CTGCTGAAAT TTTTCTGTAT TAAGGCGAGA GTTATGTGGA CGACGAGCTG


 


10561
GTGTAGGATA GGCTGTTGTT GGTACTGCGT TGAGCTTGTT GAGTGCAAGG GGAATACCTG


 


10621
CTTTGCGCGC CTCTTCAAAA ACCAGCGCAG CATAATCGTG CCAGGTTGTG GTACCACTGG


 


10681
CTACCAGATG GTACAAACCT GCGACTTCCG GTTTATTCAG TGCCACACGA ATAGCATGTG


 


10741
CCGTACAATC AGCCAGCAGC TCAGCACCTG TTGGCGCACC AAATTGATCA TTTATCACAG


 


10801
CCAGTTCTTC GCGCTCTTTT GCCAGACGCA ACATCGTTTT GGCGAAGTTA TTTCCTTTAG


 


10861
CTGCGTATAC CCAGCTGGTA CGGAAAATAA GATGCTTCGC GCAATGTTCC TGTAACGCTT


 


10921
TTTCTCCGGC TAACTTGGTT TCACCGTAAA CATTTAGCGG TGCGGTTGCA TCCGTCTCCA


 


10981
GCCATGGCGT GTCGCCATTT CCAGGGAATA CGTAGTCAGT TGAGTAATGA ATTACCCAAG


 


11041
CCCCAACTTC ATTAGCCTCT TTTGCAATTG ATTCAACACT AGTCGCATTG AGTAATTGTG


 


11101
CAAATTCGGG TTCTGACTCA GCCTTATCTA CTGCGGTGTG AGCCGCAGCA TTAACAATAA


 


11161
CATCAGGTCG AATTCTTTTG ACTGTTTCAG CTACACCTTC AGGATTACTA AAATCACCAC


 


11221
AATAATCAGT GGAGTGAACA TCAAGAGCAA TCAAATTACC CAAAGGTGCC AGAGCACGCT


 


11281
GTAGTTCCCA ACCTACCTGC CCTGTTTTGC CGAAAAGGAG GATATTCATT ACTGGCGGCC


 


11341
CTCATAGTTC TGTTCAATCC ACGATTGATA AGCACCACTT TTCACATTAT CAACCCATTT


 


11401
TGTATTGGAC AGGTACCATT CCAATGTCTT CCGAATCCCG CTCTCAAACG TTTCCTGCGG


 


11461
TTTCCAGCCC AATTCGCGGC TAATCTTCTC TGCATCAATC GCATAACGGC GATCGTGTCC


 


11521
CGGGCGATCG GCAACATAAG TAATTTGCTC GCGGTAAGAT TTCTCTTTCG GTACAATCTC


 


11581
ATCCAGCAAA TCACAAATAG TGAGCACTAC ATCGATGTTT TTCTTTTCGT TGTGTCCACC


 


11641
AATGTTATAA GTTTCACCCG CTTTACCTTC GGTTACGACG GTATATAACG CACGCGCATG


 


11701
ATCTTCAACA TACAGCCAGT CACGAATTTG ATCCCCTTTG CCATAAATAG GTAATGCCTT


 


11761
ACCTTCCAGA GCATTCAGAA TAACCAATGG AATCAATTTT TCCGGGAAAT GATAAGGACC


 


11821
ATAATTATTA GAGCAATTAG TCACAATGGT TGGTAAACCA TAGGTACGTT TCCACGCGCG


 


11881
GACTAAATGA TCGCTGGATG CTTTTGAAGC GGAATAAGGG CTGCTTGGCG CGTAAGCTGT


 


11941
TGTCTCTGTA AATAAGGGTA ATTCTTCTGT ATTATTTACC TCGTCAGGAT GAGGCAAATC


 


12001
ACCATAGACT TCGTCAGTAG AAATATGATG AAAACGGAAT CTAGTTTTCT TGTCGCTATC


 


12061
AAGAGCAGAC CAATAATTGC GAGCGGCTTC CAAAAGGACA TATGTACCAA CAATATTGGT


 


12121
TTCAATAAAT GCCGCAGGAC CTGTAATTGA ACGGTCAACA TGGCTTTCAG CAGCCAGGTG


 


12181
CATCACTGCA TCTGGCTGAT GCTGAGCAAA AATCCGTGCC ATTGCAGCTG CATCGCAAAT


 


12241
ATCCGCATGT TCAAAAACAT AGCGTTCAGA ATCAGAAACA TCAGCAAGTG ATTCCAGGTT


 


12301
TCCGGCGTAC GTTAATTTAT CGACATTAAC AACACTATCC TGCGTATTAT TTATAATGTG


 


12361
ACGAACTACA GCAAAACCAA TAAATCCTGC GCCACCAGTA ACAAGTATTT TCACCTAATT


 


12421
TATTCCATAT TGCTTCAGAG CATGCTGTGA AATAAGCGGC TCTCAGTTTG ATTAATAGAA


 


12481
GTATTAATGC ACGCTACCGC CCCTGGCTTT ACAGCTACCA GAGCACTGCA TGCATGCCTA


 


12541
CGATGTGACG AGCGTTACCC ACTCGCGCTA AACCCGAAAA ATTCAAAAGC TAATTGTCTT


 


12601
ACCAATCCGC TCTGGAAACA AGGAAAATCC TGGAAAACTT TGACTAAAAT CCTATTGCTA


 


12661
ACTCGTTGTT ATTCTGATTG TTTATATAAA ACAACGGCAG GAATATTCGC AACAAATTAC


 


12721
TTTCACCACG AATCTTCACT GCCGTTATAA TTTTCTTATC AACCGTTACA TCCGGTCAGA


 


12781
TTTTCATTAT TCGCTTAACA GCTTCTCAAT ACCTTTACGG AACTTCGCCC CTTCTTTCAG


 


12841
GTTGCGCAGC CCATACTTCA CAAACGCCTG CATATAGCCC ATTTTTTTAC CGCAGTCGTA


 


12901
GCTGTCGCCG GTCATCAGCA TTGCATCAAC GGACTGTTTT TTCGCCAGCT CGGCAATGGC


 


12961
ATCAGTCAGC TGAATACGTC CCCATGCACC AGGCTGAGTA CGTTCAAGTT CCGGCCAAAT


 


13021
ATCGGCAGAA AGCACATAGC GACCAACGGC CATGATGTCT GAGTCCAGCG TCTGCGGCTG


 


13081
ATCCGGTTTT TCGATAAATT CAACAATGCG GCTGACTTTA CCTTCGCGAT CCAGCGGTTC


 


13141
TTTGGTCTGG ATGACGGAGT ATTCAGAGAG GTCACCCGGC ATACGTTTTG CCAGCACCTG


 


13201
GCTACGGCCC GTTTCATTGA AGCGCGCAAT CATGGCAGCA AGGTTGTAGC GTAGCGGGTC


 


13261
GGCGCTGGCG TCGTCGATCA CAACGTCTGG CAGCACCACG ACAAATGGAT TGTCACCAAT


 


13321
GGCGGGTCGT GCACACAAAA TGGAGTGACC TAAACCTAAA GGTTCGCCCT GACGCACGTT


 


13381
CATAATAGTC ACGCCCGGCG GGCAGATAGA TTGCACTTCC GCCAGTAGTT GACGCTTCAC


 


13441
GCGCTGCTCA AGGAGAGATT CTAATTCATA AGAGGTGTCG AAGTGGTTTT CGACCGCGTT


 


13501
CTTGGACGCA TGAGTTACCA GGAGGATTTC TTTGATCCCT GCAGCCACAA TCTCGTCAAC


 


13561
AATGTACTGA ATCATTGGCT TGTCGACGAT CGGTAGCATC TCTTTGGGTA TCGCCTTAGT


 


13621
GGCAGGCAAC ATATGCATCC CAAGACCCGC TACCGGTATA ACTGCTTTTA AATTCGTCAT


 


13681
TATTTTCCTA CCTCTAAGGG GCTGATAGTG CGTAAATTAT TGTCATAGGT TAGCCAAACG


 


13741
GTATGGCTAT ATACCAAGCA TAACTTTGAT TAAACCTTAC GATAACACTA CACACCATCA


 


13801
GCATCTGGGT TACTCGGATT ACTCGGAAAT CCACATACTG ATAATTTAAT CAGTACCTCT


 


13861
TTCCGAATAA TCGTAGTCCA ACCTGGTCCT TTTTTCTCTG ACTCGTCTGC ATTACTCAGA


 


13921
AACAAACGTT ATGTCGTCTT TTTTGGCATG GACGAATTCA TACTGCAGAG TTCGATCCAG


 


13981
ACCTTGCGAC AGCGTATACG GTGCAACAAA ACCTGAAGAA TGCACTTTCG TTGCGTCAAA


 


14041
CTGTGTTGTT GCGCAGAATT TTTTCACGCG CACAGAGCTG ACAGCGTATT TTTTGCCCGT


 


14101
AATTTTGCTC AGGATATCAA AGCAATATCC ACCCAGCATT CCTAGTGGGT AAGGCAAGTG


 


14161
CATAGAAGGG ATCTTTTTGT TCAGGCTTTG TTCAACTTCA GCAACCAACT GGTTCATGTT


 


14221
CAGGTCTGGC TTATCAACAT AGTTATAAAC CTCATAACCT GCGGCAACAT TCTTCAGTTT


 


14281
GTACTTGATA AACTCAACAA TGTTTCCAAC ATAAGCCATG GACTTATAGT TAGTCCCTGC


 


14341
GCCCACCATC ATAAACTTGC CGCCAGCGAT CTGTTTCAGC AAGTTATAGA CGTTACCGCG


 


14401
GTTGCGTTCA CCGAAGATAA CGGTAGGACG GATGATGGTT AATGAACGTT CTGTTGGTGC


 


14461
TTTGTTATAC CATTCACGCA GCACTTCCTC TGCCTGCCAC TTACTTTTGC CGTAGTGGTT


 


14521
GAAAGGGTCG TGTGGATGGT TTTCGTCAGG GTTGTGTTTG TTCAAACCAT AAACAGCAAC


 


14581
GGAACTGGTA AAGATGATAT TTTTAACGCC ATTTTTTTCC ATGGCCGCCA GCACATTGCG


 


14641
GGTACCCTGA ACGTTGACAT CATAATAGAG AGAAGTAGGG CTGACGTCAT CGCGGTGTTC


 


14701
CGCTGCCAGT AGTACAACAG TGTCAAAACC GGCTAACGCC TGGTCGAGTG CCTGTTGATC


 


14761
ACGAACATCA CCAATCTGTG TGATTTCTGG ATAAAAGTGG CTCTGCCGTT TGTCCAGGTT


 


14821
CTTGATATTA AAGTCAGCAA TTGCCGTTTC AAGTAGTCGG GTTCCTACGA ATCCGGAAGC


 


14881
TCCTATGAGC AAAACGTTAT TGTTCATAAA TCACTTTAGT CTGGTTGTTA CGTAAGAAAC


 


14941
ACAAGATAAA GATGAGTACC TTCCCTGAGT AGTCAATGCT GCCCAGCCCC AGCTTTAACA


 


15001
GTTAGTGTGA GGATTATAAT CTTTTAGAAC ATTATATCCA GTAAGTTTAT GAATGGTCGC


 


15061
AAATCTACTC TCTCCGTTCC GGCAATCTAA AGTTAATGCT AGCGACGTCG TGGGATCCTC


 


15121
TAGAGTCGAC CTGCAGGCAT GCAAGCTTGA GTATTCTATA GTCTCACCTA AATAGCTTGG


 


15181
CGTAATCATG GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA


 


15241
ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA


 


15301
CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC


 


15361
ATTAATGAAT CGGCCAACGC GAACCCCTTG CGGCCGCCCG GGCCGTCGAC CAATTCTCAT


 


15421
GTTTGACAGC TTATCATCGA ATTTCTGCCA TTCATCCGCT TATTATCACT TATTCAGGCG


 


15481
TAGCAACCAG GCGTTTAAGG GCACCAATAA CTGCCTTAAA AAAATTACGC CCCGCCCTGC


 


15541
CACTCATCGC AGTACTGTTG TAATTCATTA AGCATTCTGC CGACATGGAA GCCATCACAA


 


15601
ACGGCATGAT GAACCTGAAT CGCCAGCGGC ATCAGCACCT TGTCGCCTTG CGTATAATAT


 


15661
TTGCCCATGG TGAAAACGGG GGCGAAGAAG TTGTCCATAT TGGCCACGTT TAAATCAAAA


 


15721
CTGGTGAAAC TCACCCAGGG ATTGGCTGAG ACGAAAAACA TATTCTCAAT AAACCCTTTA


 


15781
GGGAAATAGG CCAGGTTTTC ACCGTAACAC GCCACATCTT GCGAATATAT GTGTAGAAAC


 


15841
TGCCGGAAAT CGTCGTGGTA TTCACTCCAG AGCGATGAAA ACGTTTCAGT TTGCTCATGG


 


15901
AAAACGGTGT AACAAGGGTG AACACTATCC CATATCACCA GCTCACCGTC TTTCATTGCC


 


15961
ATACGAAATT CCGGATGAGC ATTCATCAGG CGGGCAAGAA TGTGAATAAA GGCCGGATAA


 


16021
AACTTGTGCT TATTTTTCTT TACGGTCTTT AAAAAGGCCG TAATATCCAG CTGAACGGTC


 


16081
TGGTTATAGG TACATTGAGC AACTGACTGA AATGCCTCAA AATGTTCTTT ACGATGCCAT


 


16141
TGGGATATAT CAACGGTGGT ATATCCAGTG ATTTTTTTCT CCATTTTAGC TTCCTTAGCT


 


16201
CCTGAAAATC TCGATAACTC AAAAAATACG CCCGGTAGTG ATCTTATTTC ATTATGGTGA


 


16261
AAGTTGGAAC CTCTTACGTG CCGATCAACG TCTCATTTTC GCCAAAAGTT GGCCCAGGGC


 


16321
TTCCCGGTAT CAACAGGGAC ACCAGGATTT ATTTATTCTG CGAAGTGATC TTCCGTCACA


 


16381
GGTATTTATT CGCGATAAGC TCATGGAGCG GCGTAACCGT CGCACAGGAA GGACAGAGAA


 


16441
AGCGCGGATC TGGGAAGTGA CGGACAGAAC GGTCAGGACC TGGATTGGGG AGGCGGTTGC


 


16501
CGCCGCTGCT GCTGACGGTG TGACGTTCTC TGTTCCGGTC ACACCACATA CGTTCCGCCA


 


16561
TTCCTATGCG ATGCACATGC TGTATGCCGG TATACCGCTG AAAGTTCTGC AAAGCCTGAT


 


16621
GGGACATAAG TCCATCAGTT CAACGGAAGT CTACACGAAG GTTTTTGCGC TGGATGTGGC


 


16681
TGCCCGGCAC CGGGTGCAGT TTGCGATGCC GGAGTCTGAT GCGGTTGCGA TGCTGAAACA


 


16741
ATTATCCTGA GAATAAATGC CTTGGCCTTT ATATGGAAAT GTGGAACTGA GTGGATATGC


 


16801
TGTTTTTGTC TGTTAAACAG AGAAGCTGGC TGTTATCCAC TGAGAAGCGA ACGAAACAGT


 


16861
CGGGAAAATC TCCCATTATC GTAGAGATCC GCATTATTAA TCTCAGGAGC CTGTGTAGCG


 


16921
TTTATAGGAA GTAGTGTTCT GTCATGATGC CTGCAAGCGG TAACGAAAAC GATTTGAATA


 


16981
TGCCTTCAGG AACAATAGAA ATCTTCGTGC GGTGTTACGT TGAAGTGGAG CGGATTATGT


 


17041
CAGCAATGGA CAGAACAACC TAATGAACAC AGAACCATGA TGTGGTCTGT CCTTTTACAG


 


17101
CCAGTAGTGC TCGCCGCAGT CGAGCGACAG GGCGAAGCCC TCGGCTGGTT GCCCTCGCCG


 


17161
CTGGGCTGGC GGCCGTCTAT GGCCCTGCAA ACGCGCCAGA AACGCCGTCG AAGCCGTGTG


 


17221
CGAGACACCG CGGCCGGCCG CCGGCGTTGT GGATACCTCG CGGAAAACTT GGCCCTCACT


 


17281
GACAGATGAG GGGCGGACGT TGACACTTGA GGGGCCGACT CACCCGGCGC GGCGTTGACA


 


17341
GATGAGGGGC AGGCTCGATT TCGGCCGGCG ACGTGGAGCT GGCCAGCCTC GCAAATCGGC


 


17401
GAAAACGCCT GATTTTACGC GAGTTTCCCA CAGATGATGT GGACAAGCCT GGGGATAAGT


 


17461
GCCCTGCGGT ATTGACACTT GAGGGGCGCG ACTACTGACA GATGAGGGGC GCGATCCTTG


 


17521
ACACTTGAGG GGCAGAGTGC TGACAGATGA GGGGCGCACC TATTGACATT TGAGGGGCTG


 


17581
TCCACAGGCA GAAAATCCAG CATTTGCAAG GGTTTCCGCC CGTTTTTCGG CCACCGCTAA


 


17641
CCTGTCTTTT AACCTGCTTT TAAACCAATA TTTATAAACC TTGTTTTTAA CCAGGGCTGC


 


17701
GCCCTGTGCG CGTGACCGCG CACGCCGAAG GGGGGTGCCC CCCCTTCTCG AACCCTCCCG


 


17761
GTCGAGTGAG CGAGGAAGCA CCAGGGAACA GCACTTATAT ATTCTGCTTA CACACGATGC


 


17821
CTGAAAAAAC TTCCCTTOGG GTTATCCACT TATCCACGGG GATATTTTTA TAATTATTTT


 


17881
TTTTATAGTT TTTAGATCTT CTTTTTTAGA GCGCCTTGTA GGCCTTTATC CATGCTGGTT


 


17941
CTAGAGAAGG TGTTGTGACA AATTGCCCTT TCAGTGTGAC AAATCACCCT CAAATGACAG


 


18001
TCCTGTCTGT GACAAATTGC CCTTAACCCT GTGACAAATT GCCCTCAGAA GAAGCTGTTT


 


18061
TTTCACAAAG TTATCCCTGC TTATTGACTC TTTTTTATTT AGTGTGACAA TCTAAAAACT


 


18121
TGTCACACTT CACATGGATC TGTCATGGCG GAAACAGCGG TTATCAATCA CAAGAAACGT


 


18181
AAAAATAGCC CGCGAATCGT CCAGTCAAAC GACCTCACTG AGGCGGCATA TAGTCTCTCC


 


18241
CGGGATCAAA AACGTATGCT GTATCTGTTC GTTGACCAGA TCAGAAAATC TGATGGCACC


 


18301
CTACAGGAAC ATGACGGTAT CTGCGAGATC CATGTTGCTA AATATGCTGA AATATTCGGA


 


18361
TTGACCTCTG CGGAAGCCAG TAAGGATATA CGGCAGGCAT TGAAGAGTTT CGCGGGGAAG


 


18421
GAAGTGGTTT TTTATCGCCC TGAAGAGGAT GCCGGCGATG AAAAAGGCTA TGAATCTTTT


 


18481
CCTTGGTTTA TCAAACGTGC GCACAGTCCA TCCAGAGGGC TTTACAGTGT ACATATCAAC


 


18541
CCATATCTCA TTCCCTTCTT TATCGGGTTA CAGAACCGGT TTACGCAGTT TCGGCTTAGT


 


18601
GAAACAAAAG AAATCACCAA TCCGTATGCC ATGCGTTTAT ACGAATCCCT GTGTCAGTAT


 


18661
CGTAAGCCGG ATGGCTCAGG CATCGTCTCT CTGAAAATCG ACTGGATCAT AGAGCGTTAC


 


18721
CAGCTGCCTC AAAGTTACCA GCGTATGCCT GACTTCCGCC GCCGCTTCCT GCAGGTCTGT


 


18781
GTTAATGAGA TCAACAGCAG AACTCCAATG CGCCTCTCAT ACATTGAGAA AAAGAAAGGC


 


18841
CGCCAGACGA CTCATATCGT ATTTTCCTTC CGCGATATCA CTTCCATGAC GACAGGATAG


 


18901
TCTGAGGGTT ATCTGTCACA GATTTGAGGG TGGTTCGTCA CATTTGTTCT GACCTACTGA


 


18961
GGGTAATTTG TCACAGTTTT GCTGTTTCCT TCAGCCTGCA TGGATTTTCT CATACTTTTT


 


19021
GAACTGTAAT TTTTAAGGAA GCCAAATTTG AGGGCAGTTT GTCACAGTTG ATTTCCTTCT


 


19081
CTTTCCCTTC GTCATGTGAC CTGATATCGG GGGTTAGTTC GTCATCATTG ATGAGGGTTG


 


19141
ATTATCACAG TTTATTACTC TGAATTGGCT ATCCGCGTGT GTACCTCTAC CTGGAGTTTT


 


19201
TCCCACGGTG GATATTTCTT CTTGCGCTGA GCGTAAGAGC TATCTGACAG AACAGTTCTT


 


19261
CTTTGCTTCC TCGCCAGTTC GCTCGCTATG CTCGGTTACA CGGCTGCGGC GAGCGCTAGT


 


19321
GATAATAAGT GACTGAGGTA TGTGCTCTTC TTATCTCCTT TTGTAGTGTT GCTCTTATTT


 


19381
TAAACAACTT TGCGGTTTTT TGATGACTTT GCGATTTTGT TGTTGCTTTG CAGTAAATTG


 


19441
CAAGATTTAA TAAAAAAACG CAAAGCAATG ATTAAAGGAT GTTCAGAATG AAACTCATGG


 


19501
AAACACTTAA CCAGTGCATA AACGCTGGTC ATGAAATGAC GAAGGCTATC GCCATTGCAC


 


19561
AGTTTAATGA TGACAGCCCG GAAGCGAGGA AAATAACCCG GCGCTGGAGA ATAGGTGAAG


 


19621
CAGCGGATTT AGTTGGGGTT TCTTCTCAGG CTATCAGAGA TGCCGAGAAA GCAGGGCGAC


 


19681
TACCGCACCC GGATATGGAA ATTCGAGGAC GGGTTGAGCA ACGTGTTGGT TATACAATTG


 


19741
AACAAATTAA TCATATGCGT GATGTGTTTG GTACGCGATT GCGACGTGCT GAAGACGTAT


 


19801
TTCCACCGGT GATCGGGGTT GCTGCCCATA AAGGTGGCGT TTACAAAACC TCAGTTTCTG


 


19861
TTCATCTTGC TCAGGATCTG GCTCTGAAGG GGCTACGTGT TTTGCTCGTG GAAGGTAACG


 


19921
ACCCCCAGGG AACAGCCTCA ATGTATCACG GATGGGTACC AGATCTTCAT ATTCATGCAG


 


19981
AAGACACTCT CCTGCCTTTC TATCTTGGGG AAAAGGACGA TGTCACTTAT GCAATAAAGC


 


20041
CCACTTGCTG GCCGGGGCTT GACATTATTC CTTCCTGTCT GGCTCTGCAC CGTATTGAAA


 


20101
CTGAGTTAAT GGGCAAATTT GATGAAGGTA AACTGCCCAC CGATCCACAC CTGATGCTCC


 


20161
GACTGGCCAT TGAAACTGTT GCTCATGACT ATGATGTCAT AGTTATTGAC AGCGCGCCTA


 


20221
ACCTGGGTAT CGGCACGATT AATGTCGTAT GTGCTGCTGA TGTGCTGATT GTTCCCACGC


 


20281
CTGCTGAGTT GTTTGACTAC ACCTCCGCAC TGCAGTTTTT CGATATGCTT CGTGATCTGC


 


20341
TCAAGAACGT TGATCTTAAA GGGTTCGAGC CTGATGTACG TATTTTGCTT ACCAAATACA


 


20401
GCAATAGTAA TGGCTCTCAG TCCCCGTGGA TGGAGGAGCA AATTCGGGAT GCCTGGGGAA


 


20461
GCATGGTTCT AAAAAATGTT GTACGTGAAA CGGATGAAGT TGGTAAAGGT CAGATCCGGA


 


20521
TGAGAACTGT TTTTGAACAG GCCATTGATC AACGCTCTTC AACTGGTGCC TGGAGAAATG


 


20581
CTCTTTCTAT TTGGGAACCT GTCTGCAATG AAATTTTCGA TCGTCTGATT AAACCACGCT


 


20641
GGGAGATTAG ATAATGAAGC GTGCGCCTGT TATTCCAAAA CATACGCTCA ATACTCAACC


 


20701
GGTTGAAGAT ACTTCGTTAT CGACACCAGC TGCCCCGATG GTGGATTCGT TAATTGCGCG


 


20761
CGTAGGAGTA ATGGCTCGCG GTAATGCCAT TACTTTGCCT GTATGTGGTC GGGATGTGAA


 


20821
GTTTACTCTT GAAGTGCTCC GGGGTGATAG TGTTGAGAAG ACCTCTCGGG TATGGTCAGG


 


20881
TAATGAACGT GACCAGGAGC TGCTTACTGA GGACGCACTG GATGATCTCA TCCCTTCTTT


 


20941
TCTACTGACT GGTCAACAGA CACCGGCGTT CGGTCGAAGA GTATCTGGTG TCATAGAAAT


 


21001
TGCCGATGGG AGTCGCCGTC GTAAAGCTGC TGCACTTACC GAAAGTGATT ATCGTGTTCT


 


21061
GGTTGGCGAG CTGGATGATG AGCAGATGGC TGCATTATCC AGATTGGGTA ACGATTATCG


 


21121
CCCAACAAGT GCTTATGAAC GTGGTCAGCG TTATGCAAGC CGATTGCAGA ATGAATTTGC


 


21181
TGGAAATATT TCTGCGCTGG CTGATGCGGA AAATATTTCA CGTAAGATTA TTACCCGCTG


 


21241
TATCAACACC GCCAAATTGC CTAAATCAGT TGTTGCTCTT TTTTCTCACC CCGGTGAACT


 


21301
ATCTGCCCGG TCAGGTGATG CACTTCAAAA AGCCTTTACA GATAAAGAGG AATTACTTAA


 


21361
GCAGCAGGCA TCTAACCTTC ATGAGCAGAA AAAAGCTGGG GTGATATTTG AAGCTGAAGA


 


21421
AGTTATCACT CTTTTAACTT CTGTGCTTAA AACGTCATCT GCATCAAGAA CTAGTTTAAG


 


21481
CTCACGACAT CAGTTTGCTC CTGGAGCGAC AGTATTGTAT AAGGGCGATA AAATGGTGCT


 


21541
TAACCTGGAC AGGTCTCGTG TTCCAACTGA GTGTATAGAG AAAATTGAGG CCATTCTTAA


 


21601
GGAACTTGAA AAGCCAGCAC CCTGATGCGA CCACGTTTTA GTCTACGTTT ATCTGTCTTT


 


21661
ACTTAATGTC CTTTGTTACA GGCCAGAAAG CATAACTGGC CTGAATATTC TCTCTGGGCC


 


21721
CACTGTTCCA CTTGTATCGT CGGTCTGATA ATCAGACTGG GACCACGGTC CCACTCGTAT


 


21781
CGTCGGTCTG ATTATTAGTC TGGGACCACG GTCCCACTCG TATCGTCGGT CTGATTATTA


 


21841
GTCTGGGACC ACGGTCCCAC TCGTATCGTC GGTCTGATAA TCAGACTGGG ACCACGGTCC


 


21901
CACTCGTATC GTCGGTCTGA TTATTAGTCT GGGACCATGG TCCCACTCGT ATCGTCGGTC


 


21961
TGATTATTAG TCTGGGACCA CGGTCCCACT CGTATCGTCG GTCTGATTAT TAGTCTGGAA


 


22021
CCACGGTCCC ACTCGTATCG TCGGTCTGAT TATTAGTCTG GGACCACGGT CCCACTCGTA


 


22081
TCGTCGGTCT GATTATTAGT CTGGGACCAC GATCCCACTC GTGTTGTCGG TCTGATTATC


 


22141
GGTCTGGGAC CACGGTCCCA CTTGTATTGT CGATCAGACT ATCAGCGTGA GACTACGATT


 


22201
CCATCAATGC CTGTCAAGGG CAAGTATTGA CATGTCGTCG TAACCTGTAG AACGGAGTAA


 


22261
CCTCGGTGTG CGGTTGTATG CCTGCTGTGG ATTGCTGCTG TGTCCTGCTT ATCCACAACA


 


22321
TTTTGCGCAC GGTTATGTGG ACAAAATACC TGGTTACCCA GGCCGTGCCG GCACGTTAAC


 


22381
CGGGCTGCAT CCGATGCAAG TGTGTCGCTG TCGACGAGCT CGCGAGCTCG GACATGAGGT


 


22441
TGCCCCGTAT TCAGTGTCGC TGATTTGTAT TGTCTGAAGT TGTTTTTACG TTAAGTTGAT


 


22501
GCAGATCAAT TAATACGATA CCTGCGTCAT AATTGATTAT TTGACGTGGT TTGATGGCCT


 


22561
CCACGCACGT TGTGATATGT AGATGATAAT CATTATCACT TTACGGGTCC TTTCCGGTGA


 


22621
TCCGACAGGT TACGGGGCGG CGACCTCGCG GGTTTTCGCT ATTTATGAAA ATTTTCCGGT


 


22681
TTAAGGCGTT TCCGTTCTTC TTCGTCATAA CTTAATGTTT TTATTTAAAA TACCCTCTGA


 


22741
AAAGAAAGGA AACGACAGGT GCTGAAAGCG AGCTTTTTGG CCTCTGTCGT TTCCTTTCTC


 


22801
TGTTTTTGTC CGTGGAATGA ACAATGGAAG TCCGAGCTCA TCGCTAATAA CTTCGTATAG


 


22861
CATACATTAT ACGAAGTTAT ATTCGAT

## Claims

1. A composition comprising a bioconjugate, said bioconjugate comprising a carrier protein linked to an oligosaccharide or polysaccharide, wherein said oligosaccharide or polysaccharide comprises N-acetylgalactosamine at the reducing terminus, and wherein said carrier protein comprises the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline; and
a recombinant prokaryotic host cell that comprises
(a) a heterologous nucleic acid encoding an epimerase that synthesizes N-acetylgalactosamine on undecaprenyl pyrophosphate, wherein said epimerase comprises the amino acid sequence of SEQ ID NO. 2;
(b) a heterologous nucleic acid encoding an oligosaccharyl transferase; and
(c) a heterologous nucleic acid encoding said carrier protein.

2. The composition of claim 1, wherein said carrier protein is linked to an oligosaccharide.

3. The composition of claim 1, wherein said carrier protein is linked to a polysaccharide.

4. The composition of claim 1, wherein said oligosaccharide or polysaccharide is from a Gram-negative bacterium.

5. The composition of claim 1, wherein said oligosaccharide or polysaccharide is from E. coli.

6. The composition of claim 5, wherein said oligosaccharide or polysaccharide is from E. coli 0157.

7. The composition of claim 1, wherein said oligosaccharide or polysaccharide is from Shigella flexneri.

8. The composition of claim 7, wherein said oligosaccharide or polysaccharide is from Shigella flexneri 6.

9. The composition of claim 1, wherein said oligosaccharide or polysaccharide comprises a structure:

10. The composition of claim 1, wherein said oligosaccharide or polysaccharide comprises a structure, α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc.

11. The composition of claim 1, wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.

12. The composition of claim 4, wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.

13. The composition of claim 5, wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.

14. The composition of claim 6, wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.

15. The composition of claim 7, wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.

16. The composition of claim 8, wherein said carrier protein has been modified to comprise the amino acid sequence D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.

17. The composition of claim 1, wherein said carrier protein is P. aeruginosa exoprotein that has been modified to comprise the amino acid sequence D/E-X-N-ZSIT, wherein X and Z can be any natural amino acid except proline.

18. The composition of claim 1, wherein said carrier protein is the Campylobacter AcrA protein.

19. The composition of claim 1, wherein said nucleic acid encoding an oligosaccharyl transferase encodes the oligosaccharyl transferase from Campylobacter jejuni.

20. The composition of claim 1, wherein said nucleic acid encoding an oligosaccharyl transferase is heterologous to said host cell.