P-1, 3-GALACTOSYL TRANSFERASES FOR USE IN THE BIOSYNTHESIS OF OLIGOSACCHARIDES

RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63/373,468 filed on August 25, 2022. The entire teachings of the above application(s) are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: a) File name: 62271009002_Sequence_Listing.xml; created August 25, 2023, 54,093 Bytes in size.

BACKGROUND

[0003] Human milk contains a diverse set of neutral and acidic sugar oligomers collectively known as the “human milk oligosaccharides” (HMOs) (Bode and Jantscher- Krenn, 2012; Chaturvedi et al., 1997; Cheng et al., 2020; Kunz et al., 2000). More than 200 distinct oligosaccharide species have been identified in human milk, and both their particular complement of structural features and their high overall abundance are unique to humans. Although these HMO sugars are not utilized per se by infants for nutrition, they nevertheless serve critical roles in the establishment of a healthy infant gut microbiome, in the prevention of disease, and in immune function (Bode and Jantscher-Krenn, 2012; Cheng et al., 2020; Gnoth et al., 2000; Newburg and Walker, 2007; Ray et al., 2019; Rudloff and Kunz, 2012). [0004] Lacto-N-tetraose (LNT) is one of the major individual human milk oligosaccharide species and contains within its structure the most abundant HMO foundational motif (i.e. Gal(pi-3)GlcNAc), a motif called the “Type 1” glycan core. The related, but distinct, “Type 2” glycan core structure (i.e. Gal(pi-4)GlcNAc) is rarer, and is found in a smaller subset of HMOs. Most of the higher molecular weight oligosaccharides in human milk, i.e., those larger in size than three combined hexose units, are based on LNT, and therefore include the Type 1 core structure. Thus, the ability to synthesize the (Gal(pi- 3)GlcNAc) motif is critically important for the production of the broadest selection of HMOs. SUMMARY

[0005] Prior to the disclosure described herein, the ability to produce certain “Type 1” human milk oligosaccharides inexpensively was problematic. Indeed, their production through chemical synthesis remains limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost. As such, there exists a continuing need for new tools and strategies to inexpensively manufacture large quantities of LNT and its derived Type 1 HMOs.

[0006] Accordingly, the disclosure features newly discovered LNT2-accepting [3-1,3- galactosyltransferase enzymes, GatA (SEQ ID NO: 1), GatB (SEQ ID NO: 17), GatC (SEQ ID NO: 10), and GatD (SEQ ID NO: 18). These enzymes are useful for cost-effective and efficient biosynthesis of oligosaccharides.

[0007] In addition to the amino acid sequences described above, the disclosure also encompasses enzymes that are less than 100% identical to the reference sequence of SEQ ID NO: 1, 17, 10, or 18. For example, such an amino acid sequence comprises at least 50% sequence identity to the reference sequence and retain P-l,3-galactosyltransferase activity. In some examples, the sequence is at least 60%, 75%, 80%, 85%, 90%, 95%, and 99% identical to the reference sequence, e.g., SEQ ID NO: 1, 17, 10, or 18 and retain [3-1,3- galactosyltransferase activity.

[0008] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using search algorithms such as BLAST and PSLBLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25: 17, 3389- 402). For the PSLBLAST search, the following exemplary parameters are employed: (1) Expect threshold was 10; (2) Gap cost was Existence: 11 and Extension: 1; (3) The Matrix employed was BLOSUM62; (4) The filter for low complexity regions was “on”.

[0009] The P-l,3-galactosyltransferases of the disclosure include the amino acid sequences of SEQ ID NOs: 1, 17, 10, or 18 as well as fragments and variants thereof that exhibit P-1, 3 -galactosyltransferase activity.

[0010] The disclosure provides methods for producing oligosaccharides that comprise a Type 1 glycan core, i.e. Gal(pi-3)GlcNAc, (e.g., LNT or its derived Type 1 HMOs) or a Type 2 glycan core, i.e. Gal(pi-4)GlcNAc. The methods comprise providing a bacterium that expresses at least one exogenous LNT-accepting P-l,3-galactosyltransferase and culturing the bacterium to inexpensively and efficiently produce oligosaccharides. The methods may further comprise retrieving or purifying the oligosaccharide from the bacterium or from a culture supernatant of the bacterium.

[0011] For example, the disclosure includes methods for producing an oligosaccharide in a bacterium comprising expressing an enzyme in a host bacterium, wherein the amino acid sequence of said enzyme comprises at least 85% identity to GatB (SEQ ID NO: 17), thereby producing an oligosaccharide comprising a Gal(pi-3)GlcNAc motif. The disclosure also encompasses compositions for use in the production of an oligosaccharide, the composition comprising a bacterium expressing at least one P-1, 3 -galactosyltransferase enzyme, wherein the amino acid sequence of said at least one enzyme comprises at least 80% identity, at least 85%, at least 90%, at least 95%, at least 99%, and up to 100% identity to full length amino acid sequence of SEQ ID NO: 1, 17, 10, or 18. Biosynthetic oligosaccharides produced according to the disclosure are useful as ingredients in nutritional supplements and/or therapeutics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0013] FIG. 1 is a diagram of synthetic routes for neutral hMOS.

[0014] FIG. 2 is a diagram of synthetic routes for acidic hMOS.

[0015] FIG. 3 is a diagram of Type 1 and Type 2 glycan motifs.

[0016] FIG. 4 is a diagram of a configuration of genes engineered at the thyA gene locus.

[0017] FIG. 5 is a diagram of the first step in the production of lacto-N-tetraose (LNT) in

E. coli.

[0018] FIG. 6 is a schematic diagram of an exemplary plasmid, pG292, used for production of LNT2.

[0019] FIG. 7 is a diagram showing the conversion of LNT2 to LNT by a P(l,3) galactosyltransferase.

[0020] FIG. 8 is a schematic diagram of an exemplary plasmid, pG221, for production of LNT. [0021] FIG. 9 is a photograph of a thin layer chromatogram showing production of LNT2 and LNT.

[0022] FIG. 10 is a photograph of a thin layer chromatogram showing LNT production: comparison of P-1,3 galactosyltransferases WbgO and WbbD with newly discovered P-1,3 galactosyltransferase GatA

[0023] FIG. 11 is a table of pairwise amino acid sequence identity comparisons to GatA. [0024] FIG. 12 is a photograph of a thin layer chromatogram showing results from PSL BLAST search 1 candidate P-1,3 galactosyltransferases.

[0025] FIG. 13 is a table of pairwise amino acid sequence identity comparisons to GatA. [0026] FIG. 14 is a photograph of a thin layer chromatogram showing LNT2 utilizing P-1,3 galactosyltransferases (comparison).

[0027] FIG. 15 is a table showing pairwise amino acid identity comparisons of newly discovered P-1,3 galactosyltransferase enzymes described herein with previously identified P- 1,3 galactosyltransferases.

DETAILED DESCRIPTION

[0028] The preferred route for efficient, industrial-scale synthesis of HMOs is through metabolic engineering of fermentable microbes, especially bacteria. This approach typically involves the construction of microbial strains expressing heterologous glycosyltransferases with desired specificities. In these strains, new metabolic pathways are often introduced, or existing pathways enhanced, to enable and increase production of regenerating nucleotide sugar pools for use as biosynthetic precursors in glycosyltransferase reactions (Bych et al., 2018; Dumon et al., 2004; Faijes et al., 2019; Mao et al., 2006; Petschacher and Nidetzky, 2016; Ruffing and Chen, 2006). These strains also need to express appropriate membrane transporters for both import of precursor sugars into the cell cytosol, and for export of products to the culture medium. A key aspect of the approach is selection of the particular heterologous glycosyltransferase, or combination of glycosyltransferases, to produce the desired HMO product. This choice, given that such enzymes can vary greatly in terms of kinetics, substrate specificity, affinity for donor and acceptor molecules, stability, solubility, and toxicity to the microbial host strain, can significantly affect final product yield and quality. Several glycosyltransferases derived from different bacterial species have previously been identified and characterized in terms of their ability to catalyze the biosynthesis of certain HMOs in E. coli host strains (Blixt et al., 1999; Drouillard et al., 2010; Dumon et al., 2006; Dumon et al., 2004; Li et al., 2008a; Li et al., 2008b; Zhu et al., 2021). However, there exists a continuing need to identify and characterize additional glycosyltransferases useful for biosynthesis or improved biosynthesis of particular HMOs in metabolically engineered microbes. The identification of additional glycosyltransferases with faster kinetics, greater affinity for nucleotide sugar donors and/or particular acceptor structures, greater stability within the heterologous microbial host, or higher specificity in producing desired molecules, has the potential to further improve HMO product yield and purity, and to make these molecules more broadly available for use as nutritional supplements and as therapeutics. p-l,3-galactosyltransferases (p(l,3)GTs) for the biosynthesis of p(l,3)-galactosyl-linked oligosaccharides in metabolically engineered microbes

[0029] To this end, we have undertaken a candidate gene screening approach to identify new p-l,3-galactosyltransferases (p(l,3)GTs) for the synthesis of p(l,3)-galactosyl-linked oligosaccharides in metabolically engineered microbes. Of particular interest are new (P(l,3)GTs that are capable of forming the (Gal(pi-3)GlcNAc) “Type 1” motif as found in the human milk tetrasaccharide, lacto-N-tetraose (LNT). LNT is one of the most abundant oligosaccharides of human milk (Austin et al., 2016), and is thought to function with other HMOs as an important natural prebiotic, promoting the growth of beneficial commensal bacteria such as Bifidobacterium spp. in the infant gut, (James et al., 2016; Sakurama et al., 2013; Wada et al., 2008). LNT is not only itself a major individual component of the HMO mixture, but it forms the foundation of many higher molecular weight human milk oligosaccharides comprising the “Type 1” core, including but not limited to; lacto-N- fucopentaose I (LNF I), lacto-N-fucopentaose II (LNF II), lacto-N-fucopentaose V (LNF V), lacto-N-difucohexaose I (LDFH I), lacto-N-difucohexaose II (LDFH II), sialyllacto-N- tetraose a (SLNT-a), sialyllacto-N-tetraose b (SLNT-b), disialyllacto-N-tetraose (DSLNT) and sialyllacto-N-fucopentaose II (SLNFP II). FIG. 1 and FIG. 2 diagram synthetic schemes for syntheses of the most abundant neutral and acidic oligosaccharides (respectively) found in human milk. The Type 1 and Type 2 oligosaccharide classification groups are shown in each scheme.

[0030] Type 1 and Type 2 glycan motifs exist not only in human milk oligosaccharides, but also within the structures of certain cell surface glycans in humans comprising antigens recognized under the “Lewis” typing system (Lloyd, 2000; Yuriev et al., 2005) (FIG. 3). [0031] Individuals of Lewis A and Lewis B blood groups carry fucosylated glycans on the surface of red blood cells that comprise the Type 1 core. Lewis X and Lewis Y antigens, which incorporate the Type 2 core structure, are not found on blood cells but do exist on a few other cell types, for example certain epithelial cells such as gastric epithelium. Interestingly, Type 1 and Type 2 motifs, and “human-like” Lewis antigens, are additionally found in carbohydrate structures of the lipopolysaccharide found on the surface of a human bacterial pathogen, Helicobacter pylori, a gram-negative bacterium estimated to have colonized the stomachs of approximately 50% of humanity (Hooi et al., 2017). Helicobacter pylori colonization is usually chronic and typically benign. However sometimes the organism causes significant morbidity, precipitating conditions such as gastritis, stomach or duodenal ulcers, and even cancers (Kusters et al., 2006). One intriguing aspect of H. pylori biology is its avoidance of host immune responses during chronic colonization, and one part of this seems to be its ability to adapt genetically to alter the carbohydrate content of its surface lipopolysaccharide to match/mimic the host’s Lewis antigen type, i.e., to become more like “self’, and thus evade host immune surveillance. One study (Pohl et al., 2009) highlighted genetic changes in a putative and defective pl, 3) galactosyltransferase gene found in the Lewis B negative Helicobacter pylori HP1 as the strain switched to a Lewis B positive phenotype following 8 months of in vivo selection in Lewis B positive transgenic mice. The wild type, putative and defective p(l,3)GT gene of strain HP1 (itself a homolog of a putative and defective, “lipopolysaccharide biosynthesis gene” (JHP0563) from H. pylori strain J99) contained a frameshift that destroyed its reading frame, whereas the Lewis B positive Helicobacter pylori HP1 variant that emerged after in vivo selection (clone 03-270) had mutated (by inserting two nucleotides into the defective JHP0563 variant p(l,3)GT gene) to restore the open reading frame (JHP0563 variant, clone 03-270. SEQ ID NO: 15, (Pohl et al., 2009)).

[0032] Encouraged by this evidence that the restored HP1 p(l,3)GT gene may thus encode an active (3(1,3) galactosyltransferase, we used the JHP0563 protein sequence to probe, using BLAST homology searches (Altschul et al., 1990), several complete Helicobacter pylori genomes located in public DNA sequence databases, looking for full- length, intact, homologs of JHP0563 that might represent authentic wild type (3-1,3- galactosyltransferase genes. Helicobacter pylori strain P12 contained such a homolog. We named this putative p-l,3-galactosyltransferase enzyme “GatA”, whose amino acid sequence is presented as SEQ ID NO: 1. GatA is represented in public sequence databases under accession # ACJ07781.1

[0033] Similar to Helicobacter pylori, lipopolysaccharide (LPS) also comprises the outermost layer of the Escherichia coli cell envelope. The external surface of this envelope LPS in E.coli is decorated with a highly diverse polysaccharide called the “O” antigen, whose precise composition and structure varies dramatically between different E. coli strains. 181 distinct “O” antigen variants have been formally defined (Liu et al., 2020). In contrast to H. pylori, E.coli “O” antigens are usually highly immunogenic, however it is thought that their extreme diversity offers selective advantages in particular niches for individual strain clones (Wang et al., 2010), and thus LPS variants are maintained. The enteropathogenic E. coli O55:H7 strain’s “O” antigen comprises a repeating pentasaccharide structure featuring the familiar Gal(pi-3)GlcNAc motif. The E. coli O55:H7 p-l,3-galactosyltransferase enzyme responsible for formation of this structure, WbgO, has been identified and characterized (Liu et al., 2009), and the amino acid sequence of WbgO (accession # YP_003500090.1) is presented as SEQ ID NO: 2.

[0034] The extraintestinal pathogenic E .coli strain O7:K1 “O” antigen is also a repeating pentasaccharide structure featuring the Gal(pi-3)GlcNAc motif. The E. coli O7:K1 (3-1,3 -galactosyltransferase enzyme responsible for formation of this structure, WbbD, has been identified and characterized (Riley et al., 2005), and the amino acid sequence of WbbD (accession # YP_006144407.1) is presented as SEQ ID NO: 3.

Example 1 : Engineering E. coli to generate host strains for the production of lacto-N- tetraose (LNT).

[0035] The E. coli K12 prototroph, W3110, was chosen as the parent background for LNT biosynthesis. This strain had previously been modified at the ampC locus by the introduction of a tryptophan-inducible VtrpB -cl+ repressor construct (McCoy and Lavallie, 2001), enabling convenient, controllable production of recombinant proteins from the phage X PL promoter (Sanger et al., 1982) through induction with millimolar concentrations of tryptophan (Mieschendahl et al., 1986). The strain GI724, an E. coli W3110 derivative containing the tryptophan-inducible VtrpB -cl+ repressor construct in ampC, was used at the basis for further A. coli strain manipulations

[0036] Biosynthesis of LNT requires the generation of an enhanced cellular pool of lactose. This enhancement was achieved in strain GI724 through several manipulations of the chromosome using X Red recombineering (Court et al., 2002) and generalized Pl phage transduction (Thomason et al., 2007). The ability of the E. coli host strain to accumulate intracellular lactose was first engineered by simultaneous deletion of the endogenous p- galactosidase gene (lacZ) and the lactose operon repressor gene (lad). During construction of this deletion, the constitutive ladq promoter was placed immediately upstream of the lactose permease gene, lacY. The modified strain thus maintains its ability to transport lactose from the culture medium (via LacY), but is deleted for the wild-type copy of the lacZ (p-galactosidase) gene responsible for lactose catabolism. An intracellular lactose pool is therefore created when the modified strain is cultured in the presence of exogenous lactose. [0037] An optional or additional modification useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of LNT) is the incorporation of a lacA mutation. LacA is a lactose acetyltransferase that is only active when high levels of lactose accumulate in the A. coli cytoplasm. High intracellular osmolarity (e.g., caused by a high intracellular lactose pool) can inhibit bacterial growth, and E. coli has evolved a mechanism for protecting itself from high intra cellular osmolarity caused by lactose by “tagging” excess intracellular lactose with an acetyl group using LacA, and then actively expelling the acetyl-lactose from the cell (Danchin, 2009). Production of acetyl-lactose in E. coli engineered to produce human milk oligosaccharides is therefore undesirable: it reduces overall yield. Moreover, acetyl-lactose is a side product that complicates oligosaccharide purification schemes. The incorporation of a lacA mutation resolves these problems, as carrying a deletion of the lacA gene renders the bacterium incapable of synthesizing acetyl-lactose.

[0038] A thyA (thymidylate synthase) mutation was introduced by almost entirely deleting the thyA gene and replacing it by an inserted functional, wild-type, but promoter-less E. coli lacZ ⁺ gene carrying the 2.8 ribosome binding site (EthyA::(2.8RBS lacZ ,kan ^r ).

X Red recombineering (Court et al., 2002) was used to perform the construction. FIG. 4 illustrates the new configuration of genes thus engineered at the thyA locus.

[0039] Genomic DNA sequence surrounding the lacZ+ insertion into the thyA region is set forth in SEQ ID NO: 4.

[0040] The thyA defect can be complemented in trans by supplying a wild-type thyA gene on a multicopy plasmid (Belfort et al., 1983). This complementation is used herein as a means of plasmid maintenance (eliminating the need for a more conventional antibiotic selection scheme to maintain plasmid copy number).

[0041] The genotype of strain E680 is given below. E680 incorporates all the changes discussed above and is a host strain suitable for the production of lacto-N-tetraose (LNT). [0042] F’402 proA+B+, PlacIq-ZacT, E(JacI-lacZ)158, ElacA398 / araC, Egpt-mhpC,

AtAy4::(2.8RBS lacZ+, KAN), rpoS+, rph+, ampC..(P\x^ T7gl0 RBS-XcI+, CAT).

Example 2. Production of lacto-N-tetraose (LNT) in E. coli

[0043] The first step in the synthesis (from a lactose precursor) of lacto-N-tetraose (LNT) is the addition of a p(l,3)N-acetylglucosamine residue to lactose, utilizing a heterologous P(l,3)-A-acetylglucosaminyltransferase (pl,3GnT) to form lacto-N-triose 2 (LNT2). FIG. 5 illustrates this reaction.

[0044] The plasmid pG292 (ColEl, thyA+, bla+, Pc-lgtA) (SEQ ID NO: 5, FIG. 6) carries the IgtA |3( l ,3 )-N-acetylglucosaminyl transferase gene of Neisseria meningitidis (Blixt et al., 1999) and can direct the production of LNT2 in E. coli strain E680 under appropriate culture conditions. See SEQ ID NO: 5 pG292.

[0045] FIG. 7 illustrates the conversion of LNT2 to LNT by a (l,3)galactosyltransferase, for example WbgO.

[0046] pG221 (ColEl, thyA+, bla+, Vc-lgtA-wbgO) (SEQ ID NO: 6, FIG. 8) is a derivative of pG292 that carries both the IgtA p(l,3)-A-acetylglucosaminyltransferase gene of N meningitidis and the wbgO p(l,3)-galactosyltransferase gene of A. coli O55:H7 (arranged on the plasmid as a two-gene operon). pG221 directs the production of LNT in E. coli strain E680 under appropriate culture conditions. See SEQ ID NO: 6 pG221.

[0047] The addition of tryptophan to lactose-containing growth medium of cultures of either of the E680-derivative strains transformed with plasmids pG292 or pG221 leads, for each particular E680/plasmid combination, to activation of the host E. coli tryptophan utilization repressor TrpR, subsequent repression of PtrpB, and a consequent decrease in cytoplasmic cl levels, which results in a de-repression of PL, expression of IgtA or IgtA+wbgO respectively, and production of LNT2 or LNT2 and LNT, respectively.

[0048] For LNT2 or LNT production in small scale laboratory cultures (<100ml), strains were grown at 30°C to early exponential phase in IMC medium (M9 salts, 0.5% glucose, 0.4% casaminoacids, and lacking both thymidine and tryptophan). Lactose was then added to a final concentration of 0.5 or 1%, along with tryptophan (200 pM final) to induce expression of the respective glycosyltransferases, driven from the PL promoter. At the end of the induction period (~24 h), TLC analysis was performed on aliquots of cell-free culture medium. FIG. 9 shows a thin layer chromatogram of culture medium samples taken from small scale A. coli cultures, and demonstrating synthesis of LNT2 and LNT (utilizing induced, lactose-containing cultures of E680 transformed with pG292 or pG221, respectively).

Example 3. Comparing known p-l,3-galactosyltransferase enzymes WgbO and WbbD with the putative p-l,3-galactosyltransferase “GatA” for production of lacto-N-tetraose (LNT) in E. coli

[0049] To compare the ability of putative p-l,3-galactosyltransferase “GatA” (from Helicobacter pylori Pl 2) with known p-1, 3 -galactosyltransferases WbgO (from E. coli O55:H7) and WbbD (from E.coli O7:K1) for the synthesis of LNT in engineered E.coli K-12 host strain E680, two additional plasmids were constructed; pG293 (SEQ ID NO: 7) and pG294 (SEQ ID NO: 8). In these two plasmids, the WbgO coding sequence present in plasmid pG221 was replaced precisely by DNA sequences encoding WbbD and GatA, respectively. See SEQ ID NO: 7 pG293 and SEQ ID NO: 8 pG294.

[0050] For LNT production at small scale (5ml), cultures comprising host strain E680 transformed with either pG221 (WbgO), pG293 (WbbD) or E294 (GatA) were grown at 30°C to early exponential phase in IMC medium (M9 salts, 0.5% glucose, 0.4% casaminoacids, and lacking both thymidine and tryptophan). Lactose was then added to a final concentration of 0.5%, along with tryptophan (200 pM final) to induce expression of p(l,3)-A- acetylglucosaminyltransf erase LgtA along with the respective p-1, 3 -galactosyltransferase, both driven from the PL promoter. At the end of the induction period (~24 h), TLC analysis was performed on aliquots of cell-free culture medium. FIG. 10 shows a thin layer chromatogram of culture medium samples taken from small scale E. coli cultures and demonstrating synthesis of both LNT2 and LNT (utilizing induced, lactose-containing cultures of E680 transformed with pG221, pG293 and pG294). As can be seen pG221, expressing WbgO the known p-l,3-galactosyltransferase control, produced LNT as expected. pG294 expressing GatA, the putative p-1, 3 -galactosyltransferase from H. pylori P12, also produced LNT, for the first time confirming that GatA is indeed a p-1,3 - galactosyltransferase. Interestingly the conversion of LNT2 to LNT looked more complete with GatA than it did with WbgO. Unexpectedly, pG293 expressing WbbD, produced only a trace of LNT, if any at all.

Example 4. Searching public DNA sequence databases for additional candidate [3-1,3- galactosyltransferase enzymes. [0051] We used the amino acid sequence of GatA as a query for the database search algorithm PSI-BLAST (Position Specific Iterated Basic Local Alignment Search Tool) in an effort to identify additional candidate p-1, 3 -galactosyltransferase enzymes. To execute a PSI-BLAST search, a list of closely related proteins is created based on a query sequence. These proteins are then combined into a general profile sequence, which summarizes significant motifs present in these sequences. This profile is then used as a query to identify a larger group of proteins, and the process is repeated to generate an even larger group of candidates (Altschul et al., 1990; Altschul et al., 1997).

[0052] We used the GatA amino acid sequence as a query for three search iterations in an initial PSI-BLAST screen. This approach yielded a group of several hundred candidates that was winnowed down by removing all hits to eukaryotes and archaea, hits with alignment lengths to GatA of less than 200 amino acids, hits to Helicobacter pylori sequences less than 350 amino acids in alignment length, hits to candidates with % identity to GatA of less than 13%, and by focusing on hits from pathogenic species. We selected 6 predicted p(l,3)GT candidates from this first PSI-BLAST screen, with homologies to GatA ranging from 13-81% at the amino acid level, for experimental validations. FIG. 11 presents a table of pairwise amino acid sequence identity comparisons to GatA for these six p(l,3)GT candidates.

Species source, accession number, SEQ ID NO, and a candidate identifier for each are included in Table 1.

Table 1 [0053] Coding regions for each of the 6 candidate p(l,3)GT genes were cloned by standard molecular biological techniques (Green et al., 2012) into expression plasmid pG221, with the WbgO coding sequence in pG221 being precisely replaced with the coding sequence of each candidate.

[0054] E680-derived E. coll strains harboring the six p(l,3)GT candidate gene expression plasmids were analyzed (in duplicate) in small-scale experiments. Strains were grown in IMC media (M9 salts containing glucose at 0.5% and casamino acids at 0.4%, and lacking thymidine), to early exponential phase at 30°C. Lactose was then added to a final concentration of 0.5%, and tryptophan (200 pM) was added to induce expression of each candidate from the PL promoter. At the end of the induction period (~23 h) aliquots of clarified media from each strain culture were analyzed for the presence of LNT2 and LNT by thin layer chromatography (TLC). As shown in FIG. 12, a control strain expressing LgtA and GatA showed, as expected, biosynthesis of both LNT2 and LNT. Each of the p(l,3)GT candidate cultures also showed production of LNT2. However, the Hcl p(l,3)GT candidate culture also produced LNT, indicating for the first time that Helicobacter cetorum WP_104713491.1 is a p-l,3-galactosyltransferase. The fact that only one of the six candidates tested was able to synthesize LNT in our engineered E. coli strain indicates the novelty and uniqueness of our findings.

Example 5. Searching public DNA sequence databases for additional candidate [3-1,3- galactosyltransferase enzymes.

[0055] We conducted a second PSI-BLAST screen looking for additional candidate p- 1,3-galactosyltransferases. For this query in this second screen, we used a profile that was derived from a multiple sequence alignment of four known p-1, 3 -galactosyltransferase enzymes, i.e.\

1. GatA (SEQ ID NO : 1 from this study, AC J07781.1)

2. Hcl (SEQ ID NO: 10 from this study, WP 104713491.1)

3. jhpO563 from Helicobacter pylori strain 03-270 (from (Pohl et al., 2009), JQ002580.1, SEQ ID NO: 15)

4. Sequence 1 Helicobacter pylori (strain unknown) p (1,3)GT from patent US6974687, SEQ ID NO: 16

[0056] We used the above profile as the query for four search iterations in this second PSI-BLAST screen. The search yielded a group of several hundred candidates that was winnowed down again by removing all hits to eukaryotes and archaea, hits with alignment lengths less than 200 amino acids, hits to Helicobacter pylori sequences less than 325 amino acids in alignment length, hits to candidates with % identity to GatA less than 15%, and by focusing on hits from pathogenic species. We selected just two predicted p(l,3)GT candidates from this screen. FIG. 13 presents a table of pairwise amino acid sequence identity comparisons to GatA of these two p(l,3)GT candidates. Species source, accession number, SEQ ID NO, and a candidate identifier for each are included in Table 2.

Table 2

[0057] Coding regions for the 2 additional candidate p(l,3)GT genes (Hp3 and Hc2) were cloned by standard molecular biological techniques (Green et al., 2012) into expression plasmid pG221, with the WbgO coding sequence in pG221 being precisely replaced with the coding sequence of each candidate.

[0058] E680-derived E. coli strains harboring the 2 additional p(l,3)GT candidate gene expression plasmids were analyzed (in duplicate) in small-scale experiments. Strains were grown in a mineral salts selective media (containing glucose at 1%, but lacking thymidine), to early exponential phase at 30°C. Lactose was then added to a final concentration of 0.5%, and tryptophan (200 pM) was added to induce expression of each candidate from the PL promoter. At the end of the induction period (~24 h) aliquots of clarified media from each strain culture were analyzed for the presence of LNT2 and LNT by thin layer chromatography (TLC). The presence of LNT2 and LNT inside the cells was also examined by additionally running aliquots of soluble heat extracts of candidate strain cell pellets on the TLC (treatment at 95°C, 10 minutes). The new candidates were compared on the TLC with a strain containing WbgO, a strain containing GatA, and a strain containing Hcl from the first PSI-BLAST screen. As shown in FIG. 14, the control strains expressing LgtA and GatA, and LgtA and Hcl showed, as expected, biosynthesis of both LNT2 and LNT. Each of the cultures expressing the two new p(l,3)GT candidates (Hp3 and Hc2) also showed production of both LNT2 and LNT, for the first time showing that both of these two enzymes are indeed p-l,3-galactosyltransferases. Hp3 utilized the available LNT2 better than all other enzymes tested, and Hc2 produced the lowest level of LNT overall.

[0059] In summary, we have used a directed screening approach to identify and characterize four new bacterial LNT2-accepting p-l,3-galactosyltransferases. We named these enzymes GatA, GatB, GatC and GatD. Table 3 lists these names along with previous candidate identifiers, source organisms and strains, database accession numbers, and SEQ ID NOs.

Table 3

[0060] FIG. 15 shows a pairwise amino acid identity comparison of the four newly discovered LNT2-accepting p-1, 3 -galactosyltransferases of this work, GatA, GatB, GatC and GatD, with previously identified p-1, 3 -galactosyltransferases mentioned above.

[0061] We have shown that these newly discovered p-1, 3 -galactosyltransferases are useful in the production of LNT in small scale microbial cultures, and thus they will be useful in the production at large scale of LNT and a variety of other Type 1 human milk oligosaccharides to supply demand for these important molecules as nutritional supplements and therapeutics.

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B-L3-galactosyltransferase sequences SEQ ID NO: 1 H. pylori P12 GatA (3GalT) ACJ07781

>GatA_ (3GalT) _ACJ07781 . 1 lipopolysaccharide biosynthesis protein [Helicobacter pylori P12 ] .

MIGVYI ISLKESQRRLDTEKLVSESNEKFKGRCVFQIFDAISPKHEDFEKFVQELYDAQS

MLKSDWFHSDYCYQELLPREFGCYLGHYFLWKECVKTNQPWILEDDVALESNFMQAL ED

CLKSPFDFVRLYGHYWGGHKTNLCALPIYTEAEVPIENHEVTPPPPNPARDTQQDFI IET

QQDPKEPSDPCKIAPQKISFNQWFKKIKRKLNRFIGSILARTEVYKNWAKYDDLTKK Y

DDLTKKYDELTGKYESLLAKETNIKETFWERRADNEKEALFLEHFYLTSVYVATTAG YYL

TPKGAKTFIEATERFKI IEPVDMFMNNPTYHDVANFTYLPCPVSLNKHAFNSTIQNAKKP DISLKSPKKSYFDNLFYDQLNTKKCLRAFHKYSKQYAPLKTPKEI

SEQ ID NO: 2 E. coli WbgO YP_003500090

>WbgO_YP_003500090 putative glycosyltransferase WbgO [Escherichia coli 055 : H7 str . CB9615 ] .

MI IDEAESAESTHPWSVILPVNKKNPFLDEAINS ILSQTFSSFEI I IVANCCTDDFYNE

LKHKVNDKIKLIRTNIAYLPYSLNKAIDLSNGEFIARMDSDDISHPDRFTKQVDFLK NNP

YVDWGTNAIFIDDKGREINKTKLPEENLDIVKNLPYKCCIVHPSVMFRKKVIASIGG YM

FSNYSEDYELWNRLSLAKIKFQNLPEYLFYYRLHEGQSTAKKNLYMVMVNDLVIKMK CFF

LTGNINYLFGGIRTIASFIYCKYIK

SEQ ID NO: 3 E. coli WbbD YP_006144407

>WbbD_YP_006144407 UDP-Gal : GlcNAc alpha-pyrophosphate -R beta 1 , 3 - galactosyltransf erase [Escherichia coli 07 : KI str . CE10 ] .

MSDDTPKFSVLMAIYIKDSPLFLSEALQSIYKNTVAPDEVI I IRDGKVTSELNSVIDSWR

RYLNIKDFTLEKNMGLGAALNFGLNQCMHDLVIRADSDDINRTNRFECILDFMTKNG DVH

ILSSWVEEFEFNPGDKGI IKKVPSRNSILKYSKNRSPFNHPAVAFKKCEIMRVGGYGNEY

L YED YAL WL KS L ANGCNGDN I QQVL VDMRF S KETAKRRGG I KYA I S E I KAQYHF YRANY I SYQDFI INI ITRIFVRLLPTSFRGYIYKKVIRRFL

SEQ ID NO: 4 thyA_2.8RBS lacZ

>E680 thyA 2 . 8RBS lacZ , KAN Escherichia coli str . K- 12

TCACAGGTTGAATCCTGTCACGCTATAGCTGGCATTCACCACGGTTTGCGGTTCAGA CTT

ACTGGCAGCACGCATATTAACCGTCAACACCGGCGAGAAGCCGCTGACATCCTGACG TAC

GACCTGAAAAGTGTCGATAATGATGGCATCCGGATTAGTGACTTTATCCCAGCCCTT ACC

TTCACAGGATGTCGCACCGCGTAGCGTTTCCAGCACATGCTCCTTCAGACGAAATCC AAT

CTGGTCGGACTCTTTTACCGGTTCGCGATCCCAGATACCGTTACTGTTCGCATCCCA CTG

CACAATGACACAGTCACCCTGTCCGACAATTTCCAGCCCTTCGCCGGTACAGATGCC ATG

ACAATAACCCGCCCTCTGGAGATGCTTCGCGACGGTAAATACCCGCAGCCAGATTTC ATC

TTCCAGCGCCAGCTTACGGGTGCTCGTTAAACTTTCACGCTGTAACGCAGGCAGAAA GCG

TGCCGCCCCCAGCAACAATACGCTACTGATCGCCATAGCAATCAACACTTCCAGCAG AGA

AAAACCTTGCTCTTTTACAGGCATCCTTCTGTTTCTCCTTGCTGACAAAGCCGGAGT CTT

CCCCACGGCGAAACCACCAGCCACCACTCGCCCGTTGAGTTTTTGAAGCGAATATGC CCG

GCCCATGCGGTATTGCGCAGGCCAAAGAAAGCAAGCGAAGGTGTCAGGTCGCTCATT TCG

ACTTCGGGCCAGCGTGGCACAAAGACCAATGGTGAACTGCCATGACAGGTATTGGCC CCA

GCAGCGGAACTCACAAGGCACCATAACGTCCCCTCCCTGATAACGCTGATACTGTGG TCG

CGGTTATGCCAGTTGGCATCTTCACGTAAATAGAGCAAATAGTCCCGCGCCTGGCTG GCG

GTTTGCCATAGCCGTTGCGACTGCTGCCAGTATTGCCAGCCATAGAGTCCACTTGCG CTT

AGCATGACCAAAATCAGCATCGCGACCAGCGTTTCAATCAGCGTATAACCACGTTGT GTT

TTCATGCCGGCAGTATGGAGCGAGGAGAAAAAAAGACGAGGGCCAGTTTCTATTTCT TCG

GCGCATCTTCCGGACTATTTACGCCGTTGCAGGACGTTGCAAAATTTCGGGAAGGCG TCT

CGAAGAATTTAACGGAGGGTAAAAAAACCGACGCACACTGGCGTCGGCTCTGGCAGG ATG

TTTCGTAATTAGATAGCCACCGGCGCTTTattaaacctactATGACCATGATTACGG ATT

CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTA ATC

GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCG ATC

GCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGG CAC

CAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCG TCG

TCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCT ATC CCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCA

CATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATG GCG

TTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACA GTC

GTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCG CGG

TGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGA TGA

GCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATT TCC

ATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTC AGA

TGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAA CGC

AGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTT ATG

CCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAA TCC

CGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAG CAG

AAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGA ACG

GCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTC AGG

TCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTA ACG

CCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCT ACG

GCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATC GTC

TGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGC AGC

GCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACG GCG

CTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGC AGT

ATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGC GCG

TGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTT CGC

TACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACA GTC

TTGGCGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCG GCT

TCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGT GGT

CGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACG GTC

TGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGC AGT

TTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCC GTC

ATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGTAAGCCGCTGGCAA GCG

GTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATTGAACTGCCTGAAC TAC

CGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACG CGA

CCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAAA ACC

TCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCACCAGCGAAA TGG

ATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTC TTT

CACAGATGTGGATTGGCGATAAAAAACAACTGtTGACGCCGCTGCGCGATCAGTTCA CCC

GTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACG CCT

GGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGT GCA

CGGCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATC AGG

GGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGG CGA

TTACCGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGA ACT

GCCAGCTGGCGCAGGTAGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAGAAA ACT

ATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACA TGT

ATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGA ATT

ATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAAC AGC

AACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGA ATA

TCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGG CGG

AATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAAG CGG

CCGCtTTATGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAA CTT

CGGAATAGGAACTTCAAGATCCCCTTATTAGAAGAACTCGTCAAGAAGGCGATAGAA GGC

GATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCA TTC

GCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTC CGC

CACACCCAGCCGGCCACAGTCGATGAATCCtGAAAAGCGGCCATTTTCCACCATGAT ATT

CGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGC CTT

GAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATC CTG

ATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTG GTG

GTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCAT GAT

GGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTC GCC

CAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGG AAC

GCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGC ACC

GGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACAC GGC

GGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCAC CCA

AGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCA TCC TGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGC7\AG7U\AGCC AT CCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGG TTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGC TACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATT CATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCA GCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAGCTTCAAAAGCGCTCTGAAGTTCCTAT ACTTTCTAGAGAATAGGAACTTCGAACTGCAGGTCGACGGATCCCCGGAATCATGGTTCC TCAGGAAACGTGTTGCTGTGGGCTGCGACGATATGCCCAGACCATCATGATCACACCCGC GACAATCATCGGGATGGAAAGAATTTGCCCCATGCTGATGTACTGCACCCAGGCACCGGT AAACTGCGCGTCGGGCTGGCGGAAAAACTCAACAATGATGCGAAACGCGCCGTAACCAAT CAGGAACAAACCTGAGACAGCTCCCATTGGGCGTGGTTTACGAATATACAGGTTGAGGAT AATAAACAGCACCACACCTTCCAGCAGCAGCTCGTAAAGCTGTGATGGGTGGCGCGGCAG CACACCGTAAGTGTCGAAAATGGATTGCCACTGCGGGTTGGTTTGCAGCAGCAAAATATC TTCTGTACGGGAGCCAGGGAACAGCATGGCAAACGGGAAGTTCGGGTCAACGCGGCCCCA C AATTC AC CGTTAATAAAGTTGC C C AGACGC C CGGC AC C AAGAC C AAACGGAATGAGTGG TGCGATAAAATCAGAGACCTGGAAGAAGGAACGTTTAGTACGGCGGGCGAAGATAATCAT CACCACGATAACGCCAATCAGGCCGCCGTGGAAAGACATGCCGCCGTCCCAGACACGGAA CAGATACAGCGGATCGGCCATAAACTGCGGGAAATTGTAGAACAGAACATAACCAATACG TCCCCCGAGGAAGACGCCGAGGAAGCCCGCATAGAGTAAGTTTTCAACTTCATTTTTGGT CCAGCCGCTGCCCGGACGATTCGCCCGTCGTGTTGCCAGCCACATTGCAAAAATGAAACC CACCAGATACATCAGGCCGTACCAGTGAAGCGCCACGGGTCCTATTGAGAAAATGACCGG ATCAAACTCCGGAAAATGCAGATAGCTACTGGTCATCTGTCACCACAAGTTCTTGTTATT TCGCTGAAAGAGAACAGCGATTGAAATGCGCGCCGCAGGTTTCAGGCGCTCCAAAGGTGC

GAATAATAGCACAAGGGGACCTGGCTGGTTGCCGGATACCGTTAAAAGATATGTATA

SEQ ID NO: 5 pG292

>pG292 , complete sequence .

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGG TCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGCTCGAGTCAACGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGCCTCAA GGTAAACAGCCGCCGCATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCGGGCAG CGTGTCCGTCCGTTTGAAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCTTCTTC CGGCAAATGTTTCTCCAGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCAAGGCT GTCGAACCGGGTTTTAAAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTTTGGAT GCCTTGCGCGATTTCGTGTTGGCGGATGCTGTATTTGGATG7U\ACCTGATTGGCGTG7\ AG GCGGTATTTGACCAAGGCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACATCGTA CCAAAATTGGTAATCTTCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCGTCAAT GACGCTGCGCCTCATAATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAAAAGTC GGCAATGTCTTCGTGTCGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGCCGCGC CAGCCGGTTGCCGTCCTTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCGATGAT GCTGCGGTCTTTTTCCATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCAATATC GTCTGCATCGGTGCGCGCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCATCCAG CCCGATGTTTAAAGAGGGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGGATGCG GCCGTCCTGTTCTTGGAAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCGTCATC GACAATCAAAATATCCAAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGATTGGGC GAAATATTTTTCTACGTTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAATTTATT CTCCCGATAGGCACGATGCCGTCTGAAGGCTTCAGACGGCATATGtatatctccttcttg aaTTCTAACAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAAT TTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTT TTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTT AAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTT CCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGAT TCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCG CGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTAT CACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGA GCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGT ATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTT ATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATT AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGC TCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTA GCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA GAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCT CACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTG GTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCA GAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCT GAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG CGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAAC TCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTG ACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGC CCTTTCGTC

SEQ ID NO: 6 pG221 >pG221 , complete sequence .

TCGCGCGTTTCGGTGATGACGGTGZ*AAACCTCTGACACATGCAGCTCCCGGAGACG GTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGCTCGAGTTATTATTTAATATATTTACAATAGATGAAGGACGCAATCGTACGGAT ACCGCCGAACAGGTAGTTAATGTTACCGGTCAGGAAGAAGCACTTCATTTTGATAACCAG GTCGTTAACCATCACCATGTACAGGTTTTTTTTTGCGGTAGACTGACCTTCGTGCAGGCG GTAGTAGAACAGGTATTCCGGCAGGTTTTGGAACTTGATTTTTGCCAGGCTCAGACGGTT CCACAGCTCGTAATCTTCGGAGTAGTTAGAAAACATATAACCACCGATGCTCGCGATGAC TTTTTTACGAAACATTACGCTCGGGTGAACAATACAACACTTATACGGCAGGTTTTTAAC GATGTCCAGGTTCTCTTCCGGCAGTTTGGTCTTGTTGATTTCACGACCTTTGTCGTCAAT AAAGATTGCGTTGGTACCCACAACATCTACGTACGGATTGTTCTTCAGGAAGTCAACCTG TTTAGTAAAACGGTCCGGGTGAGAGATGTCGTCAGAGTCCATACGGGCAATAAATTCGCC GTTGCTCAGGTCGATCGCTTTGTTCAGGGAGTACGGCAGGTAAGCGATGTTAGTGCGGAT CAGTTTGATTTTGTCGTTAACTTTGTGTTTCAGTTCGTTATAGAAGTCGTCAGTGCAGCA GTTCGCAACGATGATGATTTCGAAGCTGCTGAAGGTCTGAGACAGGATGCTGTTGATCGC TTCGTCCAGAAAAGGGTTTTTCTTGTTAACAGGCAGGATAACGCTCACAACCGGGTGGGT AGATTCCGCGGATTCCGCTTCATCGATGATCATATGTATATCTCCTTCTTCTCGAGTCAA CGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGCCTCAAGGTAAACAGCCGCCGC ATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCGGGCAGCGTGTCCGTCCGTTTG AAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCTTCTTCCGGCAAATGTTTCTCC AGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCAAGGCTGTCGAACCGGGTTTTA AAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTTTGGATGCCTTGCGCGATTTCG TGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCGTGAAGGCGGTATTTGACCAAG GCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACATCGTACCAAAATTGGTAATCT TCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCGTCAATGACGCTGCGCCTCATA ATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAAAAGTCGGCAATGTCTTCGTGT CGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGCCGCGCCAGCCGGTTGCCGTCC TTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCGATGATGCTGCGGTCTTTTTCC ATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCAATATCGTCTGCATCGGTGCGC GCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCATCCAGCCCGATGTTTAAAGAG GGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGGATGCGGCCGTCCTGTTCTTGG AAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCGTCATCGACAATCAAAATATCC AAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGATTGGGCGAAATATTTTTCTACG TTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAATTTATTCTCCCGATAGGCACGA TGCCGTCTGAAGGCTTCAGACGGCATATGtatatctccttcttgaaTTCTAACAATTGAT TGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCA GGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGG GCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGA ACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTC TCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGC TTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGC AGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCC TTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGT GGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCG CCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTT TGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACG CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT ATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTAC CGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGT ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTT ACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG TATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGT AAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAAC TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGG AATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAG CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAA ACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

SEQ ID NO: 7 pG293

>pG293 , complete sequence .

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGG TCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGGTGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGCTCGAGTTACTATAAAAATCTCCTGATAACTTTTTTATATATATAGCCACGAAA ACTAGTGGGAAGAAGTCTAACAAATATCCTTGTGATAATATTTATTATAAAGTCTTGATA TGATATATAATTTGCACGATAAAAATGATACTGAGCTTTAATTTCTGAAATGGCATATTT TATTCCACCTCGTCTTTTTGCTGTTTCCTTTGAAAATCTCATATCAACTAAAACTTGTTG AATATTATCACCATTACATCCATTAGCTAAAGATTTCAACCAAAGGGCATAATCTTCATA TAAGTACTCATTTCCATAACCGCCGACGCGCATTATTTCACACTTTTTAAATGCAACTGC AGGGTGATTAAAAGGAGATCTGTTTTTTGAATATTTAAGTATAGAATTCCGACTTGGTAC TTTTTTTATTATGCCCTTATCTCCTGGATTGAATTCGAACTCTTCAACCCAAGAGCTAAG AATATGAACATCACCATTCTTAGTCATAAAATCAAGTATACATTCGAATCGATTTGTTCT ATTTATATCATCAGAATCAGCACGTATTACTAAATCATGCATACATTGATTCAACCCAAA ATTTAACGCTGCCCCCAACCCCATATTTTTTTCAAGTGTGAAATCTTTTATATTTAAATA TCTTCTCCAACTATCAATAACAGAATTGAGTTCAGATGTGACCTTACCATCACGAATAAT AATAACTTCATCTGGGGCAACCGTATTTTTATAAATTGATTGTAAAGCCTCAGAGAGAAA TAGGGGAGAATCCTTGATGTATATAGCCATCAAAACAGAAAACTTTGGAGTGTCATCTGA CATATGTATATCTCCTTCTTCTCGAGTCAACGGTTTTTCAGCAATCGGTGCAAAATGCCG AAGTATTGCCTCAAGGTAAACAGCCGCCGCATCCTGCCGTCTGCCGCAAAATCCAGCCAC GCGCCGGCGGGCAGCGTGTCCGTCCGTTTGAAGCATTGGTACAAAAACCGGCGGGCGCGT TCAAAATCTTCTTCCGGCAAATGTTTCTCCAGCAATTCATACGCTACTGCTTTTATTTGG CGGTATTCAAGGCTGTCGAACCGGGTTTTAAAACCCATAGACTGCAAAAAATCGTTTCTG GCGGTTTTTTGGATGCCTTGCGCGATTTCGTGTTGGCGGATGCTGTATTTGGATGAAACC TGATTGGCGTGAAGGCGGTATTTGACCAAGGCTTCGGGATAATAAGCCAGCCTGCCCAAT TTGCTGACATCGTACCAAAATTGGTAATCTTCCGCCCAATCCCGCTCGGTGTTGTAACGC AAACCGCCGTCAATGACGCTGCGCCTCATAATCATCGTGTTGTTGTGTATGGGGTTGCCG AAAGGGAAAAAGTCGGCAATGTCTTCGTGTCGGGTCGGTTTTTTCCAAATTTTGCCGTGT TCGTGGTGCCGCGCCAGCCGGTTGCCGTCCTTTTCTTCCGACAAAACTTCCAGCCACGCA CCCATCGCGATGATGCTGCGGTCTTTTTCCATCTCACCCACGATTTTCTCAATCCAGTCG GGGGCGGCAATATCGTCTGCATCGGTGCGCGCAATATATTCCCCCCCCCCCCCCGACTTT GCCAATTCATCCAGCCCGATGTTTAAAGAGGGAATCAGACCGGAATTGCGCGGCTGCGCG AGGATGCGGATGCGGCCGTCCTGTTCTTGGAAACGCTGGGCAATGGCAAGCGTACCGTCC GTCGAGCCGTCATCGACAATCAAAATATCCAAGTTGCGCCAAGTTTGATTCACGACGGCG GCTAATGATTGGGCGAAATATTTTTCTACGTTGTAGGCGCAAATCAATACGCTGACTAAA GGCTGCAATTTATTCTCCCGATAGGCACGATGCCGTCTGAAGGCTTCAGACGGCATATGt atatctccttc t tgaaTTCTAACAATTGATTGAATGTATGCAAATAAATGCATACACCAT AGGTGTGGTTTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTT ATGCTGTTGTTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCAT CAGCGTTTATAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGG GGATTTGCTGCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGT GCATCCATCTGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGA ACGAAAACCCCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAAT GCTTCGTTTCGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGG CTTAATTTTTAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTA TCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTA TCTGTATGTTTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAG ATCAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCG CTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAA GAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG GTGGCG7U\ACCCGACAGGACTAT7U\AGATACCAGGCGTTTCCCCCTGG7\AGCTCCCT CGT GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGG AAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG CTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GCCTAACTACGGCTACACTAGAAGaACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGG TGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCC TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTT TAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAG TGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGT CGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGC CGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACG

ATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGG TCC TCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACT GCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTC AACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAAT ACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTC TTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCAC TCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACT CATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAG GCGTATCACGAGGCCCTTTCGTC

SEQ ID NO: 8 pG294

>pG294 , complete sequence .

TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGG TCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAggcg ccTCCTCAACCTGTATATTCGTAAACCACGCCCAATGGGAGCTGTCTCAGGTTTGTTCCT GATTGGTTACGGCGCGTTTCGCATCATTGTTGAGTTTTTCCGCCAGCCCGACGCGCAGTT TACCGGTGCCTGGGTGCAGTACATCAGCATGGGGCAAATTCTTTCCATCCCGATGATTGT CGCGGGTGTGATCATGATGGTCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTG AGGAACCATGAAACAGTATTTAGAACTGATGCAAAAAGTGCTCGACGAAGGCACACAGAA AAACGACCGTACCGGAACCGGAACGCTTTCCATTTTTGGTCATCAGATGCGTTTTAACCT GCAAGATGGATTCCCGCTGGTGACAACTAAACGTTGCCACCTGCGTTCCATCATCCATGA ACTGCTGTGGTTTCTGCAGGGCGACACTAACATTGCTTATCTACACGAAAACAATGTCAC CATCTGGGACGAATGGGCCGATGAAAACGGCGACCTCGGGCCAGTGTATGGTAAACAGTG GCGCGCCTGGCCAACGCCAGATGGTCGTCATATTGACCAGATCACTACGGTACTGAACCA GCTGAAAAACGACCCGGATTCGCGCCGCATTATTGTTTCAGCGTGGAACGTAGGCGAACT GGATAAAATGGCGCTGGCACCGTGCCATGCATTCTTCCAGTTCTATGTGGCAGACGGCAA ACTCTCTTGCCAGCTTTATCAGCGCTCCTGTGACGTCTTCCTCGGCCTGCCGTTCAACAT

TGCCAGCTACGCGTTATTGGTGCATATGATGGCGCAGCAGTGCGATCTGGAAGTGGG TGA TTTTGTCTGGACCGGTGGCGACACGCATCTGTACAGCAACCATATGGATCAAACTCATCT GCAATTAAGCCGCGAACCGCGTCCGCTGCCGAAGTTGATTATCAAACGTAAACCCGAATC CATCTTCGACTACCGTTTCGAAGACTTTGAGATTGAAGGCTACGATCCGCATCCGGGCAT TAAAGCGCCGGTGGCTATCTAATTACGAAACATCCTGCCAGAGCCGACGCCAGTGTGCGT CGGTTTTTTTACCCTCCGTTAAATTCTTCGAGACGCCTTCCCGAAggcgccATTCGCCAT TCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT CACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAAAAAGGCACGT CATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGTCGACTCTAGA TGCATGCTCGAGTTATTAGATTTCCTTCGGCGTCTTCAGCGGGGCATACTGCTTAGAATA CTTATGGAATGCACGCAGACATTTCTTGGTATTCAGCTGGTCGTAGAATAAGTTGTCGAA GTAGCTCTTCTTCGGGCTTTTCAGGCTGATATCTGGTTTCTTAGCGTTCTGGATCGTAGA GTTAAACGCGTGTTTATTCAGGCTTACCGGACACGGTAGGTAAGTGAAGTTCGCAACGTC GTGATAGGTCGGATTATTCATAAACATGTCAACAGGTTCGATAATCTTGAATCTTTCGGT CGCCTCGATGAAAGTCTTTGCACCTTTCGGAGTCAAATAATAACCAGCGGTCGTTGCAAC GTAGACAGAAGTCAAATAGAAGTGTTCCAGGAACAAAGCCTCCTTCTCATTGTCCGCTCT GCGCTCCCAGAAGGTTTCCTTAATATTGGTTTCCTTCGCTAGCAGACTCTCATACTTACC CGTtAACTCGTCATACTTCTTcGTtAAaTCGTCATACTTCTTaGTtAAATCGTCATACTT CGCAACCACGTTCTTGTAGACTTCGGTACGCGCCAGGATGGAGCCGATAAAACGGTTCAG TTTGCGTTTGATTTTTTTGAAAACTACCTGGTTGAAGCTAATTTTCTGCGGCGCGATCTT ACACGGGTCGCTCGGTTCCTTCGGATCCTGCTGAGTCTCGATGATGAAGTCCTGCTGGGT GTCCCTGGCCGGGTTCGGCGGCGGTGGAGTTACCTCGTGATTCTCGATCGGTACCTCCGC TTCAGTGTAGATCGGCAACGCACATAGGTTCGTCTTGTGACCACCCCAGTAGTGGCCATA CAGGCGAACGAAGTCGAACGGAGACTTTAGACAGTCCTCCAGAGCCTGCATAAAGTTGCT TTCCAGAGCGACGTCGTCCTCCAGGATGACAACTGGCTGATTAGTCTTTACACACTCCTT CCATAGGAAGTAGTGACCCAGGTAACAACCGAACTCACGCGGTAGCAGCTCCTGGTAGCA GTAGTCGCTGTGAAACCAGTCACTCTTCAGCATGGACTGGGCGTCGTACAATTCCTGGAC GAACTTCTCGAAGTCCTCATGCTTCGGAGAGATCGCATCGAAAATCTGGAATACACATCT ACCCTTGAATTTCTCGTTACTCTCACTGACCAACTTCTCGGTGTCTAGCCTACGCTGGGA CTCCTTCAGGCTGATAATGTATACGCCGATCATATGTATATCTCCTTCTTCTCGAGTCAA CGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGCCTCAAGGTAAACAGCCGCCGC ATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCGGGCAGCGTGTCCGTCCGTTTG AAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCTTCTTCCGGCAAATGTTTCTCC AGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCAAGGCTGTCGAACCGGGTTTTA AAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTTTGGATGCCTTGCGCGATTTCG TGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCGTGAAGGCGGTATTTGACCAAG GCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACATCGTACCAAAATTGGTAATCT TCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCGTCAATGACGCTGCGCCTCATA ATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAAAAGTCGGCAATGTCTTCGTGT CGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGCCGCGCCAGCCGGTTGCCGTCC TTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCGATGATGCTGCGGTCTTTTTCC ATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCAATATCGTCTGCATCGGTGCGC GCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCATCCAGCCCGATGTTTAAAGAG GGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGGATGCGGCCGTCCTGTTCTTGG AAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCGTCATCGACAATCAAAATATCC AAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGATTGGGCGAAATATTTTTCTACG TTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAATTTATTCTCCCGATAGGCACGA TGCCGTCTGAAGGCTTCAGACGGCATATGtatatctccttcttgaaTTCTAACAATTGAT TGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGATGCCCTTTTTCA GGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTTACTCGGGAAGG GCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAAAATCTTTCGGA ACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATTGAGCCTGTTTC TCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCCTGTCAGTTAGC TTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATTGGCACATTGGC AGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACACACCCCAAAGCC TTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTCACCTTCATGGT GGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCG CCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATGAATTTATTTTT TGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGAATCGGCCAACG CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCT GCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT ATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTAC CGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCC CGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAG ACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGaACAGT ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTG ATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATT TCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTT ACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG TATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTT GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGT AAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAAC TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGG AATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAG CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAA ACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCAT TATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

SEQ ID NO: 9 Hp2 WP 033756231.1

>Hp2 Helicobacter_pylori_SA173C_WP_033756231 . 1 LPS biosynthesis protein [Helicobacter pylori ]

MIGVYI ISLKESQRRLDTEKLILESNEKFKGRCVFQIFDAISPKHEDFEKFVQELYDAQS

MLKSDWFHSDWCRGELLPQEFGCYLSHYLLWKECVKLNQPWILEDDVALESNFMQAL ED

CLKSPFDFVKLFGWYWNFHKTNLRTLPLERDAVESVGETPIEDHVKTEAPETPIENH EVT

PPPNPARDAQQDFI IETQQEELSEPCKIAPQKISFNQWFKKIKRKLNHFIGNILARTEV

YKKLTGKYDELTGKYDELTGKYDELTGKYDELTGKYESLLAKETNIKETFWERRADS EEE

AFFLEHFYLTSVYVASTAGYYITPKGAKTFIEATERFKI IEPVDMFINNPTYHDVATLTY

LPLPVSLNKHCKISTIQNLKKSDISLSGPKKSYLDNLLYDQLNTRKCLKAFHKYSKQ YAP

LKTPKEI

SEQ ID NO: 10 Hcl WP_104713491.1

> Hcl Helicobacter_cetorum_GatC_WP_104713491 . 1 lipopolysaccharide biosynthesis protein [Helicobacter cetorum]

MTQVYI ISLKDSKRRLDTEELVSQANIDFEGHCAFHIFDAISPKHKDFEELVREFYEPKS

LLKSDWFHSDCCNGGLLPQELGCFLSHYFLWKKCLELNEPI I ILEDDVALEPNFIQALKD

CLKSPFEFVRFCGDYWGYHHTYLNALPIYDNGITPPPPNEESQPIQGSFLAHMVHRV LYF

I IYKIFNRIFHLSLYSIVYRFSRI IKNLQRSHYKKYEKETFFLEHFYLTSVYVGRTAGYY

LTPKGAKAFVDATRNFKMIEPVDMFMDNPAYTDIASITYIPCALSLNEHSLNSTIAN QKP

ELLKSYALPKAPKKSYFKNLFYYALNARKRQKAFKKFYEKYAYLKSCKDF

SEQ ID NO: 11 Hfl WP 023949252.1

>Hf 1 Helicobacter_f enneliae_WP_023949252 . 1 beta- 1 , 4 - galactosyl transferase [Helicobacter fennelliae]

MFHIFI ISLQNSPRRAFMQEQCTHLDRGICQVHFFDAIDFRTNAYPALNSKIKPLWNRIY

WGRELSISELGCFGSHYSLWEKCIELNAPI IVLEDDVKLESFFMQGLQEIDQSGFEYVRL

MGLFDVKIEPIKTKSAESKLAESTTKTQHFFKTTDQIAGTQGYYLTPNAAKKFIAKL HSF

CMPVDDYMDCFFIHKVGNILYKPYLIAPAELESTISGRIKQPFSVFKITRECFRLWR KLR

RLLHCL

SEQ ID NO: 12 Cjl OEV48919.1

>Cj l Campylobacter_j e j uni_OEV48919 . 1 lipooligosaccharide biosynthesis glycosyltransferase [Campylobacter j ej uni ]

MKVFI INLERSLDRKKHMQKQIQKLFEKNPSLKNKLEFIFFKAIDAKNKEHLEFKDHFSW

WGSWILGRELSDGEKACFASHYKLWQECVKLDEPI I ILEDDVEFSDEFLNNGIEYIDELL KSKYEYIRLCYLFDKRLYFLSEGGYYLSFEKLAGTQGYVLQVSAAKKFLKCAKNWIYAVD

DYMDMFYKHNVLNIVKRPLFLKQANFSSVIVEYGRKFSIKLILYKKIAREIFRFYSN ILR LLSIVYIKNRLKLK

SEQ ID NO: 13 Vcl WP_002023705.1

>Vcl Vibrio_cholerae_WP_002023705 . 1 glycosyl transferase [Vibrio cholerae]

MKIYVISLKNSLDRRASIEQQMTSHGLKFEFFDAIDGRIDPPHPLFANYDYIKRLWL TSG

KMPMRGELGCYASHYLLWQKCVELNAP I WLEDDVI INENFSQYLS I I KDKTNEYGFLRL

EPEVGKCSLFSKESKENYSIAFMDNNWGGTRAYSISPDSARKLILGSQKWSMAVDNY IGC

TYIHKMPSYIFSPSMVEHGVEFETTFQNEKRIRVPLYRKPTREIYSVYKKIRIMMFA NEY KK

SEQ ID NO: 14 Gal WP 018346553.1

>Gal Gallibacterium_anatis_WP_018346553 . 1 hypothetical protein [Gallibacterium anatis]

MLPIYVIHIDSATERADSIRQQFDNLKIEFEFFPAINAKKTPNHPLFSHYNAKKHFQ RKG

RNLSSGELGCYASHYSTWKKCLELNQPI IVLEDDVTILENFKDIYTNAERI IQKYDFVWL

HKNHRSDDKVIVESIDAFSIAKFYRDYFCAQGYLITPKAAKQLLTYCEEWIYPVDDQ MGR FYENKIENYAIYPACIDHIASMESLIGDDRRGKKKLSFTSKIRREYFNLKDHCRRAWYNF CFKLGAEVD

SEQ ID NO: 15 03-270 JQ002580 ON_ translation

>03 - 270_JQ002580_GN translation

MVECQRIPYLGVHLIQVYI ISLKESQRRLDTEKLVLESNEKFKGRCVFQIFDAISPKHQD

FEKFVQELYDAQSMLKSDWFHSDYCYQELLPQELGCYLSHYLLWKECVKTDQPIVIL EDD

VALESNFMQALEDCLKSPFDFVRLYGHYWGGHKTNLCALPIYTEAEETDYIETEAPI ENH

EVTPPPPNPAQDTQQDLINETQQKEPSEPCKIAPPKISFNQWFKKIKRKLNHFIGNI LA

RTEVHKKLVAKYDELTGKYDELTGKYDELTGKYDELTGKYDELTGKYDELTGKYDEL TGK

YESLLAKESNIKETFWERRADSEKEAFFLEHFYLTSVYVSTTAGYYLTPKGAKTFIE ATE

RFKI IEPVDMFINNPTYHDIANFTYVPCPVSLNKHAFNSTIQNAKKPDISLKPPKKSYFD NLFYNQLNTRKCLRAFHKYSKQYAPLKTPKEV

SEQ ID NO: 16 US6974687 1

>US6974687_1 Sequence 1 from Patent US 6974687 inClaims gi : 91123855

MISVYI ISLKESQRRLDTEKLVLESNEKFKGRCVFQIFDAISPKHEDFEKLLQELYDSSN

LLKSDWFHSDYCYQELLPQEFGCYLSHYLLWKECVKTNQPWILEDDIALESNFMQAL ED

CLKSPFDFVRLYGHYWGGHKTNLCALPIYTENENEEVEVPMENHAETEASMEKTPIE NHE

VTPPPPNPTQDAQQDCI IETQQDPKELSEPCKIAPQKTSFNPWFRKIKRKLNRFIGNIL

ARTEVYKNLVSKYDELTGKYDELTGKYDELTGKYDELTGKYDELTGKYDELTGKYDE LTG

KYDELTGKYDELTGKYDELTGKYDELTGKYESLLAKEVNIKETFWESRADSEKEALF LEH

FYLTSVYVATTAGYYLTPKGAKTFIEATERFKI IEPVDMFINNPTYHDVANFTYLPCPVS LNKHAFNSTIQNAKKPDISLKPPKKSYFDNLFYHKFNAQKCLKAFHKYSKQYAPLKTPKE V

SEQ ID NO: 17 H. pylori GatB WP 075667830.1

>Hp3 Helicobacter pylori_GatB_WP_075667830 . 1 glycosyltransferase family 25 protein [Helicobacter pylori ] .

MIQVYI ISLKESQRRLDTEKLVLESNEKFKGRCVFQIFDAISPKHQDFEKFVQELYDAQS

MLKSDWFHSDYCYQELLPREFGCYLSHYLLWKECVKTNQPWILEDDVALESNFMQAL ED

CLKSPFDFVRLYGHYWGGHKTNLCALPIYTEIEETDYTEIEEAEAPIENHEVPPPPP NST

QDTQQDLINETQQNPKEPSNPCKIAPQKVSFNQWFKKIKRKLNHFIGNILARTEVYK KL

VAKYDDLTGKYDELTGKYDELTGKYDELTGKYDELTGKYDELTGKYDELTGKYESLL AKE

ANIKETFWERRADSEKEAFFLEHFYLTSVYVSTTAGYYITPKGAKTFIEATERFKI IEPV

DMFINNPTYHDIANFTYVPCPISLNKHAFNSTIQNAKKPDISLKPPKKSYWDNLFYN QLN TKKCLRAFHKYSKQYDHLKTPKEV

SEQ ID NO: 18 H. cetorum GatD WP 014659558.1 >Hc2 Helicobacter cetorum_GatD_WP_014659558 . 1 LPS biosynthesis protein [Helicobacter cetorum] .

MISVYI ISLKDSKRRLDTEKLVLESNEKFRGHCVFHIFDAISPKHEDFEKLVKELYDASS LLQSDWFCSSVGNGLSLPELGCYLSHYFLWEECAKLNQPVIVLEDDVALESNFIQALEDC L KS P FD F VRL YGD YWYFHS TD FNTL FTQTANTE KNF KYY I KS RL KNL FKSIPLSQI I IRI PTKTAELFQRKYFSKREKEALFLEHFYLTSVYVATTAGYYLTPKGAKTFIDATKKFKI IE PVDMFMDNPTYHDVASLTYVPCALSLNGHSFNSTIQSQHQGNKKENKKRYKIVLPTPPRK AYLKRLFSYATNAKKRLKAFQQFYEKYAHLESHT

[0062] Other features and advantages of the disclosure will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

[0063] All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.