# Patent

## Title
Microorganisms and methods for producing sialylated and N-acetylglucosamine-containing oligosaccharides

## Abstract

The invention provides compositions and methods for engineering bacteria to produce sialylated and N-acetylglucosamine-containing oligosaccharides, and the use thereof in the prevention or treatment of infection.

## Background
RELATED APPLICATIONS
This application is a national stage application, filed under 35 U.S.C. §371, of International Application No. PCT/US2014/029804, filed on Mar. 14, 2014, which claims benefit of, and priority to, U.S. Ser. No. 61/782,999, filed on Mar. 14, 2013; the contents of which are hereby incorporated by reference in its entirety.


INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
The contents of the text file named “37847_512001WO_ST25.txt”, which was created on Jun. 9, 2014, and is 144 KB in size, are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
The invention provides compositions and methods for producing purified oligosaccharides, in particular certain N-acetylglucosamine-containing and/or sialylated oligosaccharides that are typically found in human milk.
BACKGROUND OF THE INVENTION
Human milk contains a diverse and abundant set of neutral and acidic oligosaccharides (human milk oligosaccharides, hMOS). Many of these molecules are not utilized directly by infants for nutrition, but they nevertheless serve critical roles in the establishment of a healthy gut microbiome, in the prevention of disease, and in immune function. Prior to the invention described herein, the ability to produce hMOS inexpensively at large scale was problematic. For example, hMOS production through chemical synthesis was limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost. As such, there is a pressing need for new strategies to inexpensively manufacture large quantities of hMOS for a variety of commercial applications.
SUMMARY OF THE INVENTION
The invention described herein features efficient and economical methods for producing N-acetylglucosamine-containing and/or sialylated oligosaccharides.
The invention provides a method for producing an N-acetylglucosamine-containing oligosaccharide in a bacterium comprising the following steps: providing a bacterium that comprises an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and a functional lactose permease; and culturing the bacterium in the presence of lactose. The N-acetylglucosamine-containing oligosaccharide is then retrieved from the bacterium or from a culture supernatant of the bacterium.
The invention further provides a method for producing a sialylated oligosaccharide in a bacterium comprising the following steps: providing a bacterium that comprises an exogenous sialyl-transferase gene, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, and a functional lactose permease gene; and culturing the bacterium in the presence of lactose. The sialylated oligosaccharide is then retrieved from the bacterium or from a culture supernatant of the bacterium. Specifically, a sialic acid synthetic capability comprises expressing exogenous CMP-Neu5Ac synthetase, an exogenous sialic acid synthase, and an exogenous UDP-GlcNAc-2-epimerase, or a functional variant or fragment thereof.
In both methods for producing N-acetylglucosamine-containing and/or sialylated oligosaccharides, it is preferable that the bacterium further comprises the capability for increased UDP-GlcNAc production. By “increased production capability” is meant that the host bacterium produces greater than 10%, 20%, 50%, 100%, 2-fold, 5-fold, 10-fold, or more of a product than the native, endogenous bacterium. Preferably, the bacterium over-expresses a positive endogenous regulator of UDP-GlcNAc synthesis. For example, the bacterium overexpresses the nagC gene of Escherichia coli. Alternatively, the bacterium over-expresses the Escherichia coli glmS (L-glutamine:D-fructose-6-phosphate aminotransferase) gene, or alternatively, over-expresses the Escherichia coli glmY gene (a positive translational regulator of glmS), or, alternatively over-expresses the Escherichia coli glmZ gene (another positive translational regulator of glmS: glmY and glmZ are described in Reichenbach et al Nucleic Acids Res 36, 2570-80 (2008)). Alternatively, the bacterium over-expresses any combination of such approaches. For example, the bacterium over-expresses nagC and glmS. Alternatively, the bacterium over-expresses nagC and glmY. Alternatively, the bacterium over-expresses nagC and glmZ. The methods also further encompass over-expressing any functional variant or fragment of nagC, glmS, glmY and glmZ and any combination thereof. By “overexpression” is meant that the gene transcript or encoded gene product is 10%, 20%, 50%, 2-fold, 5-fold, 10-fold, or more than the level expressed or produced by the corresponding native, naturally-occurring, or endogenous gene.
The invention described herein details the manipulation of genes and pathways within bacteria such as the enterobacterium Escherichia coli K12 (E. coli) leading to high level synthesis of hMOS. Other strains of E. coli for suitable for use in the present invention include E. coli MG1655, E. coli W3110, E. coli DH5aE, E. coli B, E. coli C, and E. coli W. A variety of bacterial species are suitable for use in the oligosaccharide biosynthesis methods, for example Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus are suitable for use, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus are modified using the methods of this invention, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the invention described herein. Also included as part of this invention are strains, modified as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bacteroides (e.g., Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides finegoldii, Bacteroides fragilis, Bacteroides nordii, Bacteroides ovatus, Bacteroides salyersiae, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides xylanisolvens), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Parabacteroides (e.g. Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides johnsonii and Parabacteroides merdae), Prevotella (e.g., Prevotella copri), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa). Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and an N-acetylglucosamine-containing or sialylated oligosaccharide is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. The N-acetylglucosamine-containing or sialylated oligosaccharide is purified for use in therapeutic or nutritional products, or the bacteria are used directly in such products.
The bacterium comprises a deleted or inactivated (i.e., non-functional) endogenous β-galactosidase gene. For example, the β-galactosidase gene comprises an E. coli lacZ gene (e.g., GenBank Accession Number V00296.1 (GI:41901), incorporated herein by reference). The endogenous lacZ gene of the E. coli is deleted or functionally inactivated, but in such a way that expression of the downstream lactose permease (lacY) gene remains intact, i.e. a functional lactose permease gene is also present in the bacterium. By deleted is meant that a portion or the whole coding sequence is absent, such that no gene product is produced. An “inactivated” gene does not produce a gene product that functions as the native, naturally-occuring, or endogenous gene. For example, the functional activity of an inactivated β-galactosidase gene product is reduced to 10%, 20%, 50%, or 100%, 1-fold, 2-fold, 5-fold, or 10-fold less than the functional activity of the native, naturally-occurring, endogenous gene product.
The lactose permease gene is an endogenous lactose permease gene or an exogenous lactose permease gene. For example, the lactose permease gene comprises an E. coli lacY gene (e.g., GenBank Accession Number V00295.1 (GI:41897), incorporated herein by reference). Many bacteria possess the inherent ability to transport lactose from the growth medium into the cell, by utilizing a transport protein that is either a homolog of the E. coli lactose permease (e.g., as found in Bacillus licheniformis), or a transporter that is a member of the ubiquitous PTS sugar transport family (e.g., as found in Lactobacillus casei and Lactobacillus rhamnosus). For bacteria lacking an inherent ability to transport extracellular lactose into the cell cytoplasm, this ability is conferred by an exogenous lactose transporter gene (e.g., E. coli lacY) provided on recombinant DNA constructs, and supplied either on a plasmid expression vector or as exogenous genes integrated into the host chromosome.
For the production of N-acetylglucosamine-containing oligosaccharides, the bacterium comprises an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene or a functional variant or fragment thereof. This exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene is obtained from any one of a number of sources, e.g., the LgtA gene described from N. meningitides (SEQ ID NO:16 Genbank protein Accession AAF42258.1, incorporated herein by reference) or N. gonorrhoeae (Genbank protein Accession ACF31229.1). Optionally, an additional exogenous glycosyltransferase gene is co-expressed in the bacterium comprising an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase. For example, a β-1,4-galactosyltransferase gene is co-expressed with the UDP-GlcNAc:Galα/β-R β3-N-acetylglucosaminyltransferase gene. This exogenous β-1,4-galactosyltransferase gene is obtained from any one of a number of sources, e.g., that described from N. meningitidis, the LgtB gene (Genbank protein Accession AAF42257.1), or from H. pylori, the Lex2B gene (SEQ ID NO:17 Genbank protein Accession NP_207619.1, incorporated herein by reference). Optionally, the additional exogenous glycosyltransferase gene co-expressed in the bacterium comprising an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene is a β-1,3-galactosyltransferase gene, e.g., that described from E. coli O55:H7, the WbgO gene (SEQ ID NO:18 Genbank protein Accession YP_003500090.1, incorporated herein by reference), or from H. pylori, the jhp0563 gene (Genbank protein Accession AEZ55696.1). Functional variants and fragments of any of the enzymes described above are also encompassed by the present invention.
In one embodiment, the N-acteylglucosamine-containing oligosaccharides produced by the methods described herein include Lacto-N-triose 2 (LNT2), Lacto-N-tetraose (LNT), Lacto-N-neotetraose (LNnT), Lacto-N-fucopentaose I (LNF I), Lacto-N-fucopentaose II (LNF II), Lacto-N-fucopentaose III (LNF III), Lacto-N-fucopentaose V (LNF V), Lacto-N-difucohexaose I (LDFH I), Lacto-N-difucohexaose II (LDFH II), and Lacto-N-neodifucohexaose II (LFNnDFH II).
For the production of sialyl-oligosaccharides, the bacterium comprises an exogenous sialyl-transferase gene. For example, the exogenous sialyl-transferase gene encodes α(2,3) sialyl-transferase or the exogenous sialyl-transferase gene encodes α(2,6) sialyl-transferase or the exogenous sialyl-transferase gene encodes α(2,8) sialyltransferase. The exogenous sialyl-transferase genes is obtained from any one of a number of sources, e.g., those described from N. meningitidis, N. gonorrhoeae, and from a number of organisms of the genus Photobacterium. Examples of α(2,8) sialyltransferases, useful for the production of polysialic acid for example, are found in Campylobacter jejuni (CstII: ADN52706) and Neisseria meningitides (or siaD:AAA20478).
The bacteria used herein to produce hMOS are genetically engineered to comprise an increased intracellular lactose pool (as compared to wild type) and to comprise UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and/or sialyl-transferase activity. Optionally, they also comprise β-1,4-galactosyltransferase or β-1,3-galactosyltransferase activity, and/or α-1,2-, α-1,3- and/or α-1,4-fucosyltransferase activity. In some cases, the bacterium further comprises a functional, wild-type E. coli lacZ+ gene inserted into an endogenous gene, for example, the lon gene in E. coli or the thyA gene in E. coli. In this manner, the bacterium further comprises a mutation in a lon gene or a mutation in the thyA gene. In these cases, the endogenous lacZ gene of the E. coli is deleted or functionally inactivated, but in such a way that expression of the downstream lactose permease (lacY) gene remains intact. The organism so manipulated maintains the ability to transport lactose from the growth medium, and to develop an intracellular lactose pool for use as an acceptor sugar in oligosaccharide synthesis, while also maintaining a low level of intracellular beta-galactosidase activity useful for a variety of additional purposes. For example, the invention also includes: a) methods for phenotypic marking of a gene locus in a β-galactosidase negative host cell by utilizing a β-galactosidase (e.g., lacZ) gene insert engineered to produce a low but readily detectable level of β-galactosidase activity, b) methods for readily detecting lytic bacteriophage contamination in fermentation runs through release and detection of cytoplasmic β-galactosidase in the cell culture medium, and c) methods for depleting a bacterial culture of residual lactose at the end of production runs. a), b) and c) are each achieved by utilizing a functional β-galactosidase (e.g., lacZ) gene insert carefully engineered to direct the expression of a low, but detectable level of β-galactosidase activity in an otherwise β-galactosidase negative host cell. The bacterium optionally further comprises a mutation in a lacA gene. Preferably, the bacterium accumulates an increased intracellular lactose pool, and produces a low level of beta-galactosidase. An increased intracellular pool is wherein the concentration of lactose in the host bacterium at least 10%, 20%, 50%, 2-fold, 5-fold, or 10-fold higher than that of the native, naturally-occurring bacterium.
In one aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding an UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and an exogenous nucleic acid encoding β-1,4-galactosyltransferase is lacto-N-neotetraose (LNnT). In another aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding a UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and an exogenous nucleic acid encoding β-1,3-galactosyltransferase is lacto-N-tetraose (LNT).
Described herein are compositions comprising a bacterial cell that produces the human milk oligosaccharide LNnT (lacto-N-neotetraose), wherein the bacterial cell comprises an exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase and an exogenous nucleic acid encoding a β-1,4-galactosyltransferase. Preferably, the bacterial cell is E. coli. The exogenous UDP-GlcNAc:Galα/β-R β 3-N-acetylglucosaminyltransferase gene is obtained from any one of a number of sources, e.g., the LgtA gene described from N. meningitides. The exogenous β-1,4-galactosyltransferase gene is obtained from any one of a number of sources, e.g., that described from N. meningitidis, the LgtB gene, or from H. pylori, the jhp0765 gene.
Additionally, the bacterium preferably comprises increased production of UDP-GlcNAc. An exemplary means to achieve this is by over-expression of a positive endogenous regulator of UDP-GlcNAc synthesis, for example, overexpression of the nagC gene of Escherichia coli. In one aspect, this nagC over-expression is achieved by providing additional copies of the nagC gene on a plasmid vector or by integrating additional nagC gene copies into the host cell chromosome. Alternatively, over-expression is achieved by modulating the strength of the ribosome binding sequence directing nagC translation or by modulating the strength of the promoter directing nagC transcription. As further alternatives the intracellular UDP-GlcNAc pool may be enhanced by other means, for example by over-expressing the Escherichia coli glmS (L-glutamine:D-fructose-6-phosphate aminotransferase) gene, or alternatively by over-expressing the Escherichia coli glmY gene (a positive translational regulator of glmS), or alternatively by over-expressing the Escherichia coli glmZ gene (another positive translational regulator of glmS), or alternatively by simultaneously using a combination of approaches. In one preferred embodiment, for example, the nagC (SEQ ID NO:19 Genbank protein Accession BAA35319.1, incorporated herein by reference) and glmS (SEQ ID NO:20 Genbank protein Accession NP_418185.1, incorporated herein by reference) genes which encode the sequences provided herein are overexpressed simultaneously in the same host cell in order to increase the intracellular pool of UDP-GlcNAc. Other components of UDP-GlcNAc metabolism include: (GlcNAc-1-P) N-acetylglucosamine-1-phosphate; (GlcN-1-P) glucosamine-1-phosphate; (GlcN-6-P) glucosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate. Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and lacto-N-neotetraose is retrieved, either from the bacterium itself (i.e., by lysis) or from a culture supernatant of the bacterium.
Also within the invention is an isolated E. coli bacterium as described above and characterized as comprising a deleted or inactivated endogenous β-galactosidase gene, an inactivated or deleted lacA gene, and a functional lactose permease (lacY) gene.
Also described herein are compositions comprising a bacterial cell that produces the human milk oligosaccharide 6′-SL (6′-sialyllactose), wherein the bacterial cell comprises an exogenous sialyl-transferase gene encoding α(2,6)sialyl-transferase. Preferably, the bacterial cell is E. coli. The exogenous sialyl-transferase gene utilized for 6′-SL production is obtained from any one of a number of sources, e.g., those described from a number of organisms of the genus Photobacterium. In yet another aspect, the human milk oligosaccharide produced by engineered bacteria comprising an exogenous nucleic acid molecule encoding an α(2,3) sialyltransferase is 3′-SL (3′-sialyllactose). The exogenous sialyltransferase gene utilized for 3′-SL production is obtained from any one of a number of sources, e.g., those described from N. meningitidis and N. gonorrhoeae. 
Additionally, the bacterium contains a deficient sialic acid catabolic pathway. By “sialic acid catabolic pathway” is meant a sequence of reactions, usually controlled and catalyzed by enzymes, which results in the degradation of sialic acid. An exemplary sialic acid catabolic pathway in Escherichia coli is described herein. In the sialic acid catabolic pathway described herein, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by the enzymes NanA (N-acetylneuraminic acid lyase) and NanK (N-acetylmannosamine kinase) and NanE (N-acetylmannosamine-6-phosphate epimerase), all encoded in the nanATEK-yhcH operon, and repressed by NanR (ecocyc.org/ECOLI). A deficient sialic acid catabolic pathway is engineered in Escherichia coli by way of a mutation in endogenous nanA (N-acetylneuraminate lyase) (e.g., GenBank Accession Number D00067.1 (GI:216588), incorporated herein by reference) and/or nanK (N-acetylmannosamine kinase) genes (e.g., GenBank Accession Number (amino acid) BAE77265.1 (GI:85676015), incorporated herein by reference), and/or nanE (N-acetylmannosamine-6-phosphate epimerase, GI: 947745, incorporated herein by reference). Optionally, the nanT (N-acetylneuraminate transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate. In some preferred embodiments, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated, while nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, while nanK has not been mutated, inactivated or deleted. A mutation is one or more changes in the nucleic acid sequence coding the gene product of nanA, nanK, nanE, and/or nanT. For example, the mutation may be 1, 2, 5, 10, 25, 50 or 100 changes in the nucleic acid sequence. For example, the nanA, nanK, nanE, and/or nanT is mutated by a null mutation. Null mutations as described herein encompass amino acid substitutions, additions, deletions, or insertions that either cause a loss of function of the enzyme (i.e., reduced or no activity) or loss of the enzyme (i.e., no gene product). By deleted is meant that the coding region is removed in whole or in part such that no gene product is produced. By inactivated is meant that the coding sequence has been altered such that the resulting gene product is functionally inactive or encodes a gene product with less than 100%, 80%, 50%, or 20% of the activity of the native, naturally-occurring, endogenous gene product. A “not mutated” gene or protein does not differ from a native, naturally-occurring, or endogenous coding sequence by 1, 2, 5, 10, 20, 50, 100, 200 or 500 more codons, or to the corresponding encoded amino acid sequence.
Moreover, the bacterium (e.g., E. coli) also comprises a sialic acid synthetic capability. For example, the bacterium comprises a sialic acid synthetic capability through provision of an exogenous UDP-GlcNAc 2-epimerase (e.g., neuC of Campylobacter jejuni (SEQ ID NO: 13, GenBank AAK91727.1; GI:15193223, incorporated herein by reference) or equivalent (e.g. E. coli S88 neuC GenBank YP_002392936.1; GI: 218560023), a Neu5Ac synthase (e.g., neuB of C. jejuni (SEQ ID NO:14 AAK91726.1GenBank GI:15193222, incorporated herein by reference) or equivalent, (e.g. Flavobacterium limnosediminis sialic acid synthase, GenBank GI:559220424), and/or a CMP-Neu5Ac synthetase (e.g., neuA of C. jejuni (SEQ ID NO: 15 GenBank AAK91728.1; GI:15193224, incorporated herein by reference) or equivalent, (e.g. Vibrio brasiliensis CMP-sialic acid synthase, GenBank GI: 493937153). Functional variants and fragments are also disclosed herein.
Additionally, the bacterium comprising a sialic acid synthetic capability preferably increased production of UDP-GlcNAc. An exemplary means to achieve this is by over-expression of a positive endogenous regulator of UDP-GlcNAc synthesis, for example, simultaneous overexpression of the nagC and glmS genes of Escherichia coli. This nagC and glmS over-expression is achieved by providing additional copies of the nagC and glmS genes on a plasmid vector, or by integrating additional nagC and glmS gene copies into the host cell chromosome. Alternatively, over-expression is achieved by modulating the strength of the ribosome binding sequence directing nagC (described by Sleight et al, Nucleic Acids Res. May 2010; 38(8): 2624-2636) and/or glmS translation, or by modulating the strength of the promoter/s directing nagC and glmS transcription (Sleight et al, Nucleic Acids Res. May 2010; 38(8): 2624-2636)
Bacteria comprising the characteristics described herein are cultured in the presence of lactose, and, in the instance where cells comprise an α(2,6) sialyltransferase (e.g. Photobacterium spp JT-ISH-224 (SEQ ID NO:21 Genbank protein Accession BAF92026.1, incorporated herein by reference), 6′-sialyllactose is retrieved, either from the bacterium itself or from a culture supernatant of the bacterium. In the instance where cells comprise an α(2,3) sialyltransferase, (e.g. Neisseria meningitidis 1st (Genbank protein Accession NP273962.1) 3′-sialyllactose is recovered either from the bacterium itself (e.g., by lysis of the bacterium) or from a culture supernatant of the bacterium.
Also within the invention is an isolated E. coli bacterium as described above and characterized as comprising a deleted or inactivated endogenous β-galactosidase gene, an exogenous sialyl-transferase gene, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, a deleted lacA gene, and a functional lactose permease (lacY) gene.
A purified N-acetylglucosamine-containing or sialylated oligosaccharide produced by the methods described above is also within the invention. A purified oligosaccharide, e.g., 6′-SL, is one that is at least 90%, 95%, 98%, 99%, or 100% (w/w) of the desired oligosaccharide by weight. Purity is assessed by any known method, e.g., thin layer chromatography or other electrophoretic or chromatographic techniques known in the art. The invention includes a method of purifying an N-acetylglucosamine-containing or sialylated oligosaccharide produced by the genetically engineered bacteria described above, which method comprises separating the desired N-acetylglucosamine-containing or sialylated oligosaccharide (e.g., 6′-SL) from contaminants in a bacterial cell extract or lysate, or bacterial cell culture supernatant. Contaminants include bacterial DNA, protein and cell wall components, and yellow/brown sugar caramels sometimes formed in spontaneous chemical reactions in the culture medium.
The oligosaccharides are purified and used in a number of products for consumption by humans as well as animals, such as companion animals (dogs, cats) as well as livestock (bovine, equine, ovine, caprine, or porcine animals, as well as poultry). For example, a pharmaceutical composition comprising purified 6′-sialyllactose (6′-SL) and an excipient is suitable for oral administration. Large quantities of 6′-SL are produced in bacterial hosts, e.g., an E. coli bacterium comprising a heterologous sialyltransferase, e.g., a heterologous α(2,6)sialyltransferase. An E. coli bacterium comprising an enhanced cytoplasmic pool of each of the following: lactose and CMP-Neu5Ac, is useful in such production systems. In the case of lactose, endogenous E. coli metabolic pathways and genes are manipulated in ways that result in the generation of increased cytoplasmic concentrations of lactose, as compared to levels found in wild type E. coli. For example, the bacteria contain at least 10%, 20%, 50%, 2×, 5×, 10× or more of the levels in a corresponding wild type bacteria that lacks the genetic modifications described above. In the case of CMP-Neu5Ac, endogenous Neu5Ac catabolism genes are inactivated and exogenous CMP-Neu5Ac biosynthesis genes introduced into E. coli resulting in the generation of a cytoplasmic pool of CMP-Neu5Ac not found in the wild type bacterium.
A method of producing a pharmaceutical composition comprising a purified hMOS is carried out by culturing the bacterium described above, purifying the hMOS produced by the bacterium, and combining the hMOS with an excipient or carrier to yield a dietary supplement for oral administration. These compositions are useful in methods of preventing or treating enteric and/or respiratory diseases in infants and adults. Accordingly, the compositions are administered to a subject suffering from or at risk of developing such a disease using known methods of clinical therapy.
The invention also provides for increasing, in E. coli, the intracellular concentration of the nucleotide sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). This is achieved by over-expressing the bi-functional endogenous positive regulator of UDP-GlcNac synthesis and repressor of glucosamine and N-acetylglucosamine catabolism, nagC, simultaneously with the gene encoding L-glutamine:D-fructose-6-phosphate aminotransferase, glmS.
The invention also provides for increasing the intracellular concentration of lactose in E. coli, for cells grown in the presence of lactose, by using manipulations of endogenous E. coli genes involved in lactose import, export, and catabolism. In particular, described herein are methods of increasing intracellular lactose levels in E. coli genetically engineered to produce a human milk oligosaccharide by incorporating a lacA mutation into the genetically modified E. coli. The lacA mutation prevents the formation of intracellular acetyl-lactose, which not only removes this molecule as a contaminant from subsequent purifications, but also eliminates E. coli's ability to export excess lactose from its cytoplasm, thus greatly facilitating purposeful manipulations of the E. coli intracellular lactose pool.
Also described herein are bacterial host cells with the ability to accumulate a intracellular lactose pool while simultaneously possessing low, functional levels of cytoplasmic β-galactosidase activity, for example as provided by the introduction of a functional recombinant E. coli lacZ gene, or by a β-galactosidase gene from any of a number of other organisms (e.g., the lac4 gene of Kluyveromyces lactis (e.g., GenBank Accession Number M84410.1 (GI:173304), incorporated herein by reference). Low, functional levels of cytoplasmic β-galactosidase include β-galactosidase activity levels of between 0.05 and 200 units, e.g., between 0.05 and 5 units, between 0.05 and 4 units, between 0.05 and 3 units, or between 0.05 and 2 units (for standard definition see: Miller J H, Laboratory CSH. Experiments in molecular genetics. Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.; 1972; incorporated herein by reference). This low level of cytoplasmic β-galactosidase activity, while not high enough to significantly diminish the intracellular lactose pool, is nevertheless very useful for tasks such as phenotypic marking of desirable genetic loci during construction of host cell backgrounds, for detection of cell lysis due to undesired bacteriophage contaminations in fermentation processes, for the facile removal of undesired residual lactose at the end of fermentations, or for in-process fermentation QC purposes (i.e. as a non-standard phenotype the provision of a weak lacZ phenotype aids in culture purity assessments).
Methods of purifying a N-acetylglucosamine-containing or sialylated oligosaccharide produced by the methods described herein are carried out by binding the oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of the bacterium to a carbon column, and subsequently eluting it from the column. Purified N-acetylglucosamine-containing or sialylated oligosaccharides are produced by the methods described herein.
Optionally, the invention features a vector, e.g., a vector containing a nucleic acid. The vector can further include one or more regulatory elements, e.g., a heterologous promoter. The regulatory elements can be operably linked to a protein gene, fusion protein gene, or a series of genes linked in an operon in order to express the fusion protein. To maintain the plasmid vector stably within the cell a selectable marker is included within its sequence, such as an antibiotic resistance gene or a gene that complements a nutritional auxotrophy of the host bacterium. For example, in E. coli, a thymidine deficiency caused by a chromosomal defect in the thymidylate synthase gene (thyA) can be complemented by a plasmid borne wild type copy of the thyA (M. Belfort, G. F. Maley, F. Maley, Proceedings of the National Academy of Sciences 80, 1858 (1983)) gene. Alternatively an adenine deficiency caused by a chromosomal deficiency in the adenylosuccinate synthetase (purA) gene (S. A. Wolfe, J. M. Smith, J Biol Chem 263, 19147-53 (1988)) can be complemented by a plasmid borne wild type copy of purA. Two plasmid vectors may be utilized simultaneously within the same bacterial cell by employing separate selectable markers, for example one plasmid utilizing thyA selection and one utilizing purA selection, and by utilizing two compatible plasmid replicons, for example in E. coli two such compatible replicons comprise the ColE1 (pUC) replicon and the p15A (pACYC) replicon (R. E. Bird, J Bacteriol 145, 1305-9 (1981)). In yet another aspect, the invention comprises an isolated recombinant cell, e.g., a bacterial cell containing aforementioned nucleic acid molecule/s or vector/s. The nucleic acid sequences can be optionally integrated into the genome.
The invention provides a method of treating, preventing, or reducing the risk of infection in a subject comprising administering to said subject a composition comprising a human milk oligosaccharide, purified from a culture of a recombinant strain of the current invention, wherein the hMOS binds to a pathogen and wherein the subject is infected with or at risk of infection with the pathogen. In one aspect, the infection is caused by a Norwalk-like virus or Campylobacter jejuni. The subject is preferably a mammal in need of such treatment. The mammal is, e.g., any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, or a pig. In a preferred embodiment, the mammal is a human. For example, the compositions are formulated into animal feed (e.g., pellets, kibble, mash) or animal food supplements for companion animals, e.g., dogs or cats, as well as livestock or animals grown for food consumption, e.g., cattle, sheep, pigs, chickens, and goats. Preferably, the purified hMOS is formulated into a powder (e.g., infant formula powder or adult nutritional supplement powder, each of which is mixed with a liquid such as water or juice prior to consumption) or in the form of tablets, capsules or pastes or is incorporated as a component in dairy products such as milk, cream, cheese, yogurt or kefir, or as a component in any beverage, or combined in a preparation containing live microbial cultures intended to serve as probiotics, or in prebiotic preparations intended to enhance the growth of beneficial microorganisms either in vitro or in vivo. For example, the purified sugar (e.g., LNnT or 6′-SL) can be mixed with a Bifidobacterium or Lactobacillus in a probiotic nutritional composition. (i.e. Bifidobacteria are beneficial components of a normal human gut flora and are also known to utilize hMOS for growth.
All genes described herein also include a description of the corresponding encoded gene products. As such, the uses of exogenous genes as described herein encompass nucleic acids that encode the gene product sequences disclosed herein. The person skilled in the art could readily generate nucleic acid sequences that encode the protein sequences described herein and introduce such sequences into expression vectors to carry out the present invention.
The term “substantially pure” in reference to a given polypeptide, polynucleotide or oligosaccharide means that the polypeptide, polynucleotide or oligosaccharide is substantially free from other biological macromolecules. The substantially pure polypeptide, polynucleotide or oligosaccharide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate calibrated standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, thin layer chromatography (TLC) or HPLC analysis.
Polynucleotides, polypeptides, and oligosaccharides of the invention are purified and/or isolated. Purified defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. Specifically, as used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, protein or oligosaccharide, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. For example, purified hMOS compositions are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate calibrated standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, thin layer chromatography (TLC) or HPLC analysis. For example, a “purified protein” refers to a protein that has been separated from other proteins, lipids, and nucleic acids with which it is naturally associated. Preferably, the protein constitutes at least 10, 20, 50 70, 80, 90, 95, 99-100% by dry weight of the purified preparation.
By “isolated nucleic acid” is meant a nucleic acid that is free of the genes that flank it in the naturally-occurring genome of the organism from which the nucleic acid is derived. The term covers, for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule, but is not flanked by both of the nucleic acid sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner, such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the present invention further include molecules produced synthetically, as well as any nucleic acids that have been altered chemically and/or that have modified backbones. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide.
A “heterologous promoter”, when operably linked to a nucleic acid sequence, refers to a promoter which is not naturally associated with the nucleic acid sequence.
The term “over-express” as used herein refers to gene transcript or encoded gene product is 10%, 20%, 50%, 2-fold, 5-fold, 10-fold, or more than the level expressed or produced by a native, naturally-occurring, or endogenous gene in a bacterium in which it naturally occurs. For example, the host bacterium described herein are engineered to over-express an exogenous gene transcript or encoded gene product of UDP-GlcNAc:Galα/β-R β3-N-acetylglucosaminyltransferase, nagC, glmS, glmY, glmZ, a sialyl-transferase, a β-galactosyltransferase, an α-fucosyltransferase, CMP-Neu5Ac synthetase, a sialic acid synthase, or a UDP-GlcNAc 2-epimerase, i.e., a gene or gene product with a sequence corresponding to that of a bacterium other than the host bacterium.
The terms “treating” and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and/or frequency of symptoms, eliminate the symptoms and/or their underlying cause, and/or facilitate improvement or remediation of damage. The terms “preventing” and “prevention” refer to the administration of an agent or composition to a clinically asymptomatic individual who is susceptible to a particular adverse condition, disorder, or disease, and thus relates to the prevention of the occurrence of symptoms and/or their underlying cause.
By the terms “effective amount” and “therapeutically effective amount” of a formulation or formulation component is meant a nontoxic but sufficient amount of the formulation or component to provide the desired effect.
The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
Other features and advantages of the invention 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. 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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic demonstrating metabolic pathways and the changes introduced into them to engineer 2′-fucosyllactose (2′-FL) synthesis in Escherichia coli (E. coli). Specifically, the lactose synthesis pathway and the GDP-fucose synthesis pathway are illustrated. In the GDP-fucose synthesis pathway: manA=phosphomannose isomerase (PMI), manB=phosphomannomutase (PMM), manC=mannose-1-phosphate guanylyltransferase (GMP), gmd=GDP-mannose-4,6-dehydratase, fcl=GDP-fucose synthase (GFS), and ΔwcaJ=mutated UDP-glucose lipid carrier transferase.
FIG. 2 is a schematic demonstrating metabolic pathways involved in the synthesis of UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) and catabolism of glucosamine and N-acetylglucosamine in E. coli. In the schematic: (GlcNAc-1-P) N-acetylglucosamine-1-phosphate; (GlcN-1-P) glucosamine-1-phosphate; (GlcN-6-P) glucosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; and (Fruc-6-P) Fructose-6-phosphate; glmS (L-glutamine:D-fructose-6-phosphate aminotransferase), glmM (phosphoglucosamine mutase), glmU (fused N-acetyl glucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyl transferase), nagC (bifunctional transcriptional activator/repressor protein), nagA (N-acetylglucosamine-6-phosphate deacetylase) and nagB (glucosamine-6-phosphate deaminase), nagE (N-acetylglucosamine transporter] and manXYZ [glucosamine transporter).
FIG. 3 is a schematic demonstrating metabolic pathways and one example (utilizing nanT, nanA and nanK deletions) of the changes introduced into them to engineer 6′-sialyllactose (6′-SL) synthesis in E. coli. Abbreviations include: (Neu5Ac) N-acetylneuraminic acid, sialic acid; (ΔnanT) mutated N-acetylneuraminic acid transporter; (ΔnanA) mutated N-acetylneuraminic acid lyase; (ManNAc) N-acetylmannosamine; (ΔnanK) mutated N-acetylmannosamine kinase; (nanE) wild-type N-acetylmannosamine-6-phosphate epimerase; (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; (Fruc-6-P) Fructose-6-phosphate; (neuA), CMP-N-acetylneuraminic acid synthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; (neuB), N-acetylneuraminic acid synthase; (neuC) UDP-GlcNAc-2-epimerase; and (UDP-GlcNAc) uridine diphosphate N-acetylglucosamine.
FIG. 4 is a schematic that illustrates the new configuration of genes engineered at the Escherichia coli thyA locus in strains used to produce N-acetylglucosamine-containing oligosaccharides.
FIG. 5 is a plasmid map of pG292, which expresses the N. meningitidis β(1,3)-N-acetylglucosaminyltransferase gene lgtA.
FIG. 6 is a plasmid map of pG221, which expresses, as an operon, the N. meningitidis β(1,3)-N-acetylglucosaminyltransferase gene lgtA and the E. coli O55:H7 wbgO β(1,3)-galactosyltransferase gene.
FIG. 7 is a plasmid map of pG222, which expresses, as an operon, the N. meningitidis β(1,3)-N-acetylglucosaminyltransferase gene lgtA and the H. pylori 4GalT (jhp0765) β(1,4)-galactosyltransferase gene.
FIG. 8 illustrates schematically the enzymatic reactions necessary to produce from lactose, via the intermediate trisaccharide lacto-N-triose 2 (LNT2), the two human milk oligosaccharides: Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT). A thin layer chromatogram (on left) is presented of culture medium samples taken from small scale E. coli cultures and demonstrating synthesis of LNT2, LNT and LNnT. A second thin layer chromatogram (on right) is presented of culture medium samples taken from a 15 L E. coli bioreactor culture-demonstrating synthesis of LNnT.
FIG. 9 is a plasmid map of pG317, a low-copy vector which expresses as an operon, under the control of the E. coli lac promoter, the Campylobacter jejuni ATCC43438 neuB, neuC and neuA genes, encoding N-acetylneuraminate synthase, UDP-N-acetylglucosamine 2-epimerase, and N-acetylneuraminate cytidylyltransferase, respectively.
FIG. 10 is a plasmid map of pG315, a multi-copy vector which expresses a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224, under the control of the E. coli lac promoter.
FIG. 11 is a photograph of a thin layer chromatogram showing 6′-SL in culture medium produced by E. coli strain E547 (ΔnanRATEK), containing plasmids expressing a bacterial α(2,3)sialyltransferase and neuA, neuB and neuC. FIG. 11 also shows a TLC analysis of culture supernatants from two fermentations producing 6′-sialylactose (6′-SL). Samples to the left of the figure are taken from a fermentation of an E. coli strain containing pG315 (carrying a strong RBS in front of the α(2,6)sialyltransferase gene in the vector). Samples on the right of the figure are taken from a fermentation of an E. coli strain containing a close variant of pG315 that carries a weaker RBS in front of the α(2,6)sialyltransferase gene.
FIG. 12 is a plasmid map of pG345, a multi-copy vector which expresses a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224, under the control of a weaker ribosomal binding site (SEQ ID NO:8) and the E. coli lac promoter.
FIG. 13 is a schematic demonstrating metabolic pathways and a second example (utilizing nanT, nanA and nanE deletions) of the changes introduced into them to engineer 6′-sialyllactose (6′-SL) synthesis in E. coli. Abbreviations include: (Neu5Ac) N-acetylneuraminic acid, sialic acid; (ΔnanT) mutated N-acetylneuraminic acid transporter; (ΔnanA) mutated N-acetylneuraminic acid lyase; (ManNAc) N-acetylmannosamine; (nanK) wild-type N-acetylmannosamine kinase; (ΔnanE) mutated N-acetylmannosamine-6-phosphate epimerase; (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6-P) Glucosamine-6-phosphate; (Fruc-6-P) Fructose-6-phosphate; (neuA), CMP-N-acetylneuraminic acid synthetase; (CMP-Neu5Ac) CMP-N-acetylneuraminic acid; (neuB), N-acetylneuraminic acid synthase; (neuC) UDP-GlcNAc-2-epimerase; and (UDP-GlcNAc) uridine diphosphate N-acetylglucosamine.
FIG. 14 illustrates the TLC analysis of cell pellets and or supernatants from a three pilot scale fermentation experiments using three E. coli strains carrying various combinations of nan mutations
FIG. 15 is a schematic illustrating the location of the gene deletion made within the E. coli nan operon to generate the [nanR+, nanA, nanT, nanE, nanK+] mutant locus of strains E1017 and E1018.
FIG. 16 is a cell density growth curve plot of four cultures of E680 transformed with pG292, induced or un-induced by tryptophan addition, and in the presence or absence of lactose in the growth medium. Abundant cell lysis is seen in the lactose-containing cultures.
FIG. 17 is a plasmid map of pG356, which expresses, as an operon, the E. coli glmS and nagC genes. pG356 carries a p15A replication origin and both ampC and purA selectable markers.
FIG. 18 is a fementation parameter trace and TLC culture supernatant analysis (for LNnT production) of a 1.5 L bioreactor culture of E796 transformed with pG222.
FIG. 19 is a fementation parameter trace and TLC culture supernatant analysis (for LNnT production) of a 1.5 L bioreactor culture of E866 transformed with both pG222 and pG356.



DETAILED DESCRIPTION OF THE INVENTION
Described herein are genetic constructs and methods for production of N-acetylglucosamine-containing human milk oligosaccharides (hMOS) and sialyloligosaccharides. In order to make both N-acetylglucosamine-containing and sialyl-containing hMOS, one needs to tap into the cellular UDP-GlcNAc pool. Doing so can be challenging, since UDP-GlcNAc is an essential metabolite for bacteria (used to make the cell wall). The constructs, compositions, and methods of the invention overcome difficulties of the past by enhancing the UDP-GlcNAc pool, a strategy that represents an advantage in the production of both classes of hMOS. Other distinctions over earlier approaches represent improvements and/or confer advantages over those earlier strategies.
hMOS
Human milk glycans, which comprise both oligosaccharides (hMOS) and their glycoconjugates, play significant roles in the protection and development of human infants, and in particular the infant gastrointestinal (GI) tract. Milk oligosaccharides found in various mammals differ greatly, and their composition in humans is unique (Hamosh M., 2001 Pediatr Clin North Am, 48:69-86; Newburg D. S., 2001 Adv Exp Med Biol, 501:3-10). Moreover, glycan levels in human milk change throughout lactation and also vary widely among individuals (Morrow A. L. et al., 2004 J Pediatr, 145:297-303; Chaturvedi P et al., 2001 Glycobiology, 11:365-372). Previously, a full exploration of the roles of hMOS was limited by the inability to adequately characterize and measure these compounds. In recent years sensitive and reproducible quantitative methods for the analysis of both neutral and acidic hMOS have been developed (Erney, R., Hilty, M., Pickering, L., Ruiz-Palacios, G., and Prieto, P. (2001) Adv Exp Med Biol 501, 285-297. Bao, Y., and Newburg, D. S. (2008) Electrophoresis 29, 2508-2515). Approximately 200 distinct oligosaccharides have been identified in human milk, and combinations of a small number of simple epitopes are responsible for this diversity (Newburg D. S., 1999 Curr_Med Chem, 6:117-127; Ninonuevo M. et al., 2006 J Agric Food Chem, 54:7471-74801). hMOS are composed of 5 monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc), and sialic acid (N-acetyl neuraminic acid, Neu5Ac, NANA). hMOS are usually divided into two groups according to their chemical structures: neutral compounds containing Glc, Gal, GlcNAc, and Fuc, linked to a lactose (Galβ1-4Glc) core, and acidic compounds including the same sugars, and often the same core structures, plus NANA (Charlwood J. et al., 1999 Anal_Biochem, 273:261-277; Martín-Sosa et al., 2003 J Dairy Sci, 86:52-59; Parkkinen J. and Finne J., 1987 Methods Enzymol, 138:289-300; Shen Z. et al., 2001 J Chromatogr A, 921:315-321). Approximately 70-80% of oligosaccharides in human milk are fucosylated. A smaller proportion of the oligosaccharides in human milk are sialylated, or are both fucosylated and sialylated.
Interestingly, hMOS as a class, survive transit through the intestine of infants very efficiently, a function of their being poorly transported across the gut wall and of their resistance to digestion by human gut enzymes (Chaturvedi, P., Warren, C. D., Buescher, C. R., Pickering, L. K. & Newburg, D. S. Adv Exp Med Biol 501, 315-323 (2001)). One consequence of this survival in the gut is that hMOS are able to function as prebiotics, i.e. they are available to serve as an abundant carbon source for the growth of resident gut commensal microorganisms (Ward, R. E., Niñonuevo, M., Mills, D. A., Lebrilla, C. B., and German, J. B. (2007) Mol Nutr Food Res 51, 1398-1405). Recently, there is burgeoning interest in the role of diet and dietary prebiotic agents in determining the composition of the gut microflora, and in understanding the linkage between the gut microflora and human health (Roberfroid, M., Gibson, G. R., Hoyles, L., McCartney, A. L., Rastall, R., Rowland, I., Wolvers, D., Watzl, B., Szajewska, H., Stahl, B., Guarner, F., Respondek, F., Whelan, K., Coxam, V., Davicco, M. J., Léotoing, L., Wittrant, Y., Delzenne, N. M., Cani, P. D., Neyrinck, A. M., and Meheust, A. (2010) Br J Nutr 104 Suppl 2, S1-63).
A number of human milk glycans possess structural homology to cell receptors for enteropathogens, and serve roles in pathogen defense by acting as molecular receptor “decoys”. For example, pathogenic strains of Campylobacter bind specifically to glycans in human milk containing the H-2 epitope, i.e., 2′-fucosyl-N-acetyllactosamine or 2′-fucosyllactose (2′-FL); Campylobacter binding and infectivity are inhibited by 2′-FL and other glycans containing this H-2 epitope (Ruiz-Palacios, G. M., Cervantes, L. E., Ramos, P., Chavez-Munguia, B., and Newburg, D. S. (2003) J Biol Chem 278, 14112-14120). Similarly, some diarrheagenic E. coli pathogens are strongly inhibited in vivo by hMOS containing 2′-linked fucose moieties. Several major strains of human caliciviruses, especially the noroviruses, also bind to 2′-linked fucosylated glycans, and this binding is inhibited by human milk 2′-linked fucosylated glycans. Consumption of human milk that has high levels of these 2′-linked fucosyloligosaccharides has been associated with lower risk of norovirus, Campylobacter, ST of E. coli-associated diarrhea, and moderate-to-severe diarrhea of all causes in a Mexican cohort of breastfeeding children (Newburg D. S. et al., 2004 Glycobiology, 14:253-263; Newburg D. S. et al., 1998 Lancet, 351:1160-1164). Several pathogens are also known to utilize sialylated glycans as their host receptors, such as influenza (Couceiro, J. N., Paulson, J. C. & Baum, L. G. Virus Res 29, 155-165 (1993)), parainfluenza (Amonsen, M., Smith, D. F., Cummings, R. D. & Air, G. M. J Virol 81, 8341-8345 (2007), and rotoviruses (Kuhlenschmidt, T. B., Hanafin, W. P., Gelberg, H. B. & Kuhlenschmidt, M. S. Adv Exp Med Biol 473, 309-317 (1999)). The sialyl-Lewis X epitope is used by Helicobacter pylori (Mandavi, J., Sondén, B., Hurtig, M., Olfat, F. O., et al. Science 297, 573-578 (2002)), Pseudomonas aeruginosa (Scharfman, A., Delmotte, P., Beau, J., Lamblin, G., et al. Glycoconj J 17, 735-740 (2000)), and some strains of noroviruses (Rydell, G. E., Nilsson, J., Rodriguez-Diaz, J., Ruvoën-Clouet, N., et al. Glycobiology 19, 309-320 (2009)).
The nucleotide sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a key metabolic intermediate in bacteria, where it is involved in the synthesis and maintenance of the cell envelope. In all known bacterial classes, UDP-GlcNAc is used to make peptidoglycan (murein); a polymer comprising the bacterial cell wall whose structural integrity is absolutely essential for growth and survival. In addition, gram-negative bacteria use UDP-GlcNAc for the synthesis of lipid A, an important component of the outer cell membrane. Thus, for bacteria, the ability to maintain an adequate intracellular pool of UDP-GlcNAc is critical.
Biosynthesis of certain human milk oligosaccharides (hMOS) has been achieved in engineered strains of the bacterium, Escherichia coli K12. As described herein, simple fucosylated hMOS, e.g. 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), and lactodifucotetraose (LDFT), are produced efficiently by live E. coli through artificially enhancing existing intracellular pools of GDP-fucose (the nucleotide sugar donor) and lactose (the accepting sugar), and by then using these enhanced pools as substrates for heterologous recombinant fucosyltransferases (FIG. 1). Since neither the lactose nor GDP-fucose pools are essential for E. coli survival, biosynthesis of simple fucosylated hMOS is achieved at good yields without negative consequences on the host bacterium's growth or viability. However, to synthesize more complex hMOS in E. coli, use of the critical bacterial UDP-GlcNAc pool is required, with consequent potential impacts on cell viability.
The UDP-GlcNAc pool in E. coli is produced through the combined action of three glm genes, glmS (L-glutamine:D-fructose-6-phosphate aminotransferase), glmM (phosphoglucosamine mutase), and the bifunctional glmU (fused N-acetyl glucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyl transferase) (FIG. 2). These three genes direct a steady flow of carbon to UDP-GlcNAc, a flow that originates with fructose-6-phosphate (an abundant molecule of central energy metabolism). Expression of the glm genes is under positive control by the transcriptional activator protein, NagC.
When E. coli encounters glucosamine or N-acetyl-glucosamine in its environment, these molecules are each transported into the cell via specific membrane transport proteins and are used either to supplement the flow of carbon to the UDP-GlcNAc pool, or alternatively they are consumed to generate energy, under the action of nag operon gene products (i.e. nagA [N-acetylglucosamine-6-phosphate deacetylase] and nagB [glucosamine-6-phosphate deaminase]). In contrast to the glm genes, expression of nagA and nagB are under negative transcriptional control, but by the same regulatory protein as the glm genes, i.e. NagC. NagC is thus bi-functional, able to activate UDP-GlcNAc synthesis, while at the same time repressing the degradation of glucosamine-6-phosphate and N-acetylglucosamine-6-phosphate.
The binding of NagC to specific regulatory DNA sequences (operators), whether such binding results in gene activation or repression, is sensitive to fluctuations in the cytoplasmic level of the small-molecule inducer and metabolite, GlcNAc-6-phosphate. Intracellular concentrations of GlcNAc-6-phosphate increase when N-acetylglucosamine is available as a carbon source in the environment, and thus under these conditions the expression of the glm genes (essential to maintain the vital UDP-GlcNAc pool) would decrease, unless a compensatory mechanism is brought into play. E. coli maintains a baseline level of UDP-GlcNAc synthesis through continuous expression of nagC directed by two constitutive promoters, located within the upstream nagA gene. This constitutive level of nagC expression is supplemented approximately threefold under conditions where the degradative nag operon is induced, and by this means E. coli ensures an adequate level of glm gene expression under all conditions, even when N-acetylglucosamine is being utilized as a carbon source.
Many hMOS incorporate GlcNAc into their structures directly, and many also incorporate sialic acid, a sugar whose synthesis involves consumption of UDP-GlcNAc (FIG. 3, FIG. 13). Thus, synthesis of many types of hMOS in engineered E. coli carries the significant risk of reduced product yield and compromised cell viability resulting from depletion of the bacterium's UDP-GlcNAc pool. One way to address this problem during engineered synthesis of GlcNAc- or sialic acid-containing hMOS is to boost the UDP-GlcNAc pool through simultaneous over-expression of nagC, or preferably by simultaneous over-expression of both nagC and glmS.
While studies suggest that human milk glycans could be used as prebiotics and as antimicrobial anti-adhesion agents, the difficulty and expense of producing adequate quantities of these agents of a quality suitable for human consumption has limited their full-scale testing and perceived utility. What has been needed is a suitable method for producing the appropriate glycans in sufficient quantities at reasonable cost. Prior to the invention described herein, there were attempts to use several distinct synthetic approaches for glycan synthesis. Novel chemical approaches can synthesize oligosaccharides (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003)), but reactants for these methods are expensive and potentially toxic (Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). Enzymes expressed from engineered organisms (Albermann, C., Piepersberg, W. & Wehmeier, U. F. Carbohydr Res 334, 97-103 (2001); Bettler, E., Samain, E., Chazalet, V., Bosso, C., et al. Glycoconj J 16, 205-212 (1999); Johnson, K. F. Glycoconj J 16, 141-146 (1999); Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999); Wymer, N. & Toone, E. J. Curr Opin Chem Biol 4, 110-119 (2000)) provide a precise and efficient synthesis (Palcic, M. M. Curr Opin Biotechnol 10, 616-624 (1999)); Crout, D. H. & Vic, G. Curr Opin Chem Biol 2, 98-111 (1998)), but the high cost of the reactants, especially the sugar nucleotides, limits their utility for low-cost, large-scale production. Microbes have been genetically engineered to express the glycosyltransferases needed to synthesize oligosaccharides from the bacteria's innate pool of nucleotide sugars (Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 330, 439-443 (2001); Endo, T., Koizumi, S., Tabata, K. & Ozaki, A. Appl Microbiol Biotechnol 53, 257-261 (2000); Endo, T. & Koizumi, S. Curr Opin Struct Biol 10, 536-541 (2000); Endo, T., Koizumi, S., Tabata, K., Kakita, S. & Ozaki, A. Carbohydr Res 316, 179-183 (1999); Koizumi, S., Endo, T., Tabata, K. & Ozaki, A. Nat Biotechnol 16, 847-850 (1998)). However, low overall product yields and high process complexity have limited the commercial utility of these approaches.
Prior to the invention described herein, which enables the inexpensive production of large quantities of neutral and acidic hMOS, it had not been possible to fully investigate the ability of this class of molecule to inhibit pathogen binding, or indeed to explore their full range of potential additional functions.
Prior to the invention described herein, chemical syntheses of hMOS were possible, but were limited by stereo-specificity issues, precursor availability, product impurities, and high overall cost (Flowers, H. M. Methods Enzymol 50, 93-121 (1978); Seeberger, P. H. Chem Commun (Camb) 1115-1121 (2003); Koeller, K. M. & Wong, C. H. Chem Rev 100, 4465-4494 (2000)). Also, prior to the invention described herein, in vitro enzymatic syntheses were also possible, but were limited by a requirement for expensive nucleotide-sugar precursors. The invention overcomes the shortcomings of these previous attempts by providing new strategies to inexpensively manufacture large quantities of human milk oligosaccharides for use as dietary supplements. The invention described herein makes use of an engineered bacterium E. coli (or other bacteria) engineered to produce sialylated oligosaccharides in commercially viable levels, for example the methods described herein enable the production of 3′-SL at >50 g/L in bioreactors.
Variants and Functional Fragments
The present invention features introducing exogenous genes into bacterium to manipulate the pathways to increase UDP-GlcNAc pools, to produce sialylated oligosaccharides and to produce N-acetylglucosamine-containing oligosaccharides. In any of the methods described herein, the genes or gene products may be variants or functional fragments thereof.
A variant of any of genes or gene products disclosed herein may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid or amino acid sequences described herein. The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof.
Variants as disclosed herein also include homolog, orthologs, or paralogs of the genes or gene products described herein that retain the same biological function as the genes or gene products specified herein. These variants can be used interchangeably with the genes recited in these methods. Such variants may demonstrate a percentage of homology or identity, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, preferably in a functional domain, e.g. catalytic domain.
For sequence comparison, 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 BLAST and PSI-BLAST (Altschul et al., 1990, J Mol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17, 3389-402). For the PSI-BLAST 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”.
Changes can be introduced by mutation into the nucleic acid sequence or amino acid sequence of any of the genes or gene products described herein, leading to changes in the amino acid sequence of the encoded protein or enzyme, without altering the functional ability of the protein or enzyme. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of any of sequences expressly disclosed herein. A “non-essential” amino acid residue is a residue at a position in the sequence that can be altered from the wild-type sequence of the polypeptide without altering the biological activity, whereas an “essential” amino acid residue is a residue at a position that is required for biological activity. For example, amino acid residues that are conserved among members of a family of proteins are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are poorly conserved among members of the protein family) may not be as essential for activity and thus are more likely to be amenable to alteration. Thus, another aspect of the invention pertains to nucleic acid molecules encoding the proteins or enzymes disclosed herein that contain changes in amino acid residues relative to the amino acid sequences disclosed herein that are not essential for activity.
An isolated nucleic acid molecule encoding a protein homologous to any of the genes described herein can be created by introducing one or more nucleotide substitutions, additions or deletions into the corresponding nucleotide sequence, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into a nucleic acid sequence such that the encoded amino acid sequence is altered by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for mediating oligossacharide modification, synthesis, or degradation (via detection of the products).
The present invention also provides for functional fragments of the genes or gene products described herein. A fragment, in the case of these sequences and all others provided herein, is defined as a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above.
For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 350 amino acids, 350 to 400 amino acids, 400 to 450 amino acids, or 450 to 500 amino acids. The fragments encompassed in the present invention comprise fragments that retain functional fragments. As such, the fragments preferably retain the catalytic domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. The biological function of said fragment can be measured by measuring ability to synthesize or modify a substrate oligosaccharide, or conversely, to catabolize an oligosaccharide substrate.
EXAMPLE 1
Engineering of E. coli to Generate Host Strains for the Production of N-acetylglucosamine-Containing Human Milk Oligosaccharides
The E. coli K12 prototroph, W3110, was chosen as the parent background for hMOS biosynthesis. This strain had previously been modified at the ampC locus by the introduction of a tryptophan-inducible PtrpB-cI+ repressor construct (McCoy, J. & Lavallie, E. Current protocols in molecular biology/edited by Frederick M. Ausubel et al., (2001)), enabling economical production of recombinant proteins from the phage λ PL promoter (Sanger, F., Coulson, A. R., Hong, G. F., Hill, D. F. & Petersen, G. B. J Mol Biol 162, 729-773 (1982)) through induction with millimolar concentrations of tryptophan (Mieschendahl, M., Petri, T. & Hänggi, U. Nature Biotechnology 4, 802-808 (1986)). The strain GI724, an E. coli W3110 derivative containing the tryptophan-inducible PtrpB-cI+ repressor construct in ampC, was used at the basis for further E. coli strain manipulations
Biosynthesis of hMOS requires the generation of an enhanced cellular pool of lactose. This enhancement was achieved in strain GI724 through several manipulations of the chromosome using λ, Red recombineering (Court, D. L., Sawitzke, J. A. & Thomason, L. C. Annu Rev Genet 36, 361-388 (2002)) and generalized P1 phage transduction (Thomason, L. C., Costantino, N. & Court, D. L. Mol Biol Chapter 1, Unit 1.17 (2007)). The ability of the E. coli host strain to accumulate intracellular lactose was first engineered by simultaneous deletion of the endogenous β-galactosidase gene (lacZ) and the lactose operon repressor gene (lacI). During construction of this deletion, the lacIq 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 (β-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.
An additional modification useful for increasing the cytoplasmic pool of free lactose (and hence the final yield of hMOS) is the incorporation of a lacA mutation. LacA is a lactose acetyltransferase that is only active when high levels of lactose accumulate in the E. 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, A. Bioessays 31, 769-773 (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.
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 (SEQ ID NO: 10) (ΔthyA::(2.8RBS lacZ+,kanr). λ Red recombineering was used to perform the construction. FIG. 4 illustrates the new configuration of genes thus engineered at the thyA locus. The complete DNA sequence of the region, with annotations in GenBank format is disclosed herein. Genomic DNA sequence surrounding the lacZ+ insertion into the thyA region is set forth in SEQ ID NO: 1.
The thyA defect can be complemented in trans by supplying a wild-type thyA gene on a multicopy plasmid (Belfort, M., Maley, G. F. & Maley, F. Proceedings of the National Academy of Sciences 80, 1858 (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).
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 N-acetylglucosamine-containing oligosaccharides.
F′402 proA+B+, PlacIq-lacY, Δ(lacI-lacZ) 158, ΔlacA398/araC, Δgpt-mhpC, ΔthyA::(2.8RBS lacZ+, KAN), rpoS+, rph+, ampC::(Ptrp T7g10 RBS-λcI+, CAT)
E796 is a strain similar to E680 and carries a thyA (thymidylate synthase) mutation, introduced by almost entirely deleting the thyA gene and replacing it by an inserted functional, wild-type, but promoter-less E. coli lacZ+ gene but carrying the 0.8 ribosome binding site (SEQ ID NO: 11) [ΔthyA::(0.8RBS lacZ+, KAN)]. The genotype of strain E796 is given below. E796 incorporates all the changes discussed above and is a host strain suitable for the production of N-acetylglucosamine-containing oligosaccharides.
F′402 proA+B+, PlacIq-lacY, Δ(lacI-lacZ) 158, ΔlacA398/araC, Δgpt-mhpC, ΔthyA::(2.8RBS lacZ+, KAN), rpoS+, rph+, ampC::(Ptrp T7g10 RBS-λcI+, CAT)
E866 is a strain similar to E796 and is useful for dual plasmid selection. E866 also carries a thyA (thymidylate synthase) mutation, introduced by almost entirely deleting the thyA gene and replacing it by an inserted functional, wild-type, but promoter-less E. coli lacZ+ gene and carrying the 0.8 ribosome binding site (SEQ ID NO: 11) [ΔthyA::(0.8RBS lacZ+)]. In addition to the thyA deletion E866 also carries a deletion of the purA gene. The genotype of strain E866 is given below. E866 incorporates all the changes discussed above and is a host strain suitable for the production of N-acetylglucosamine-containing oligosaccharides.
F′402 proA+B+, PlacIq-lacY, Δ(lacI-lacZ) 158, ΔlacA398/araC, Δgpt-mhpC, ΔthyA::(0.8RBS lacZ+), rpoS+, rph+, ampC::(Ptrp T7g10 RBS-λcI+, CAT), ΔpurA727::KAN
EXAMPLE 2
Production of N-acetylglucosamine-containing Human Milk Oligosaccharides in E. coli: Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT)
The first step in the synthesis (from a lactose precursor) of both Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT) is the addition of a β(1,3)N-acetylglucosamine residue to lactose, utilizing a heterologous β(1,3)-N-acetylglucosaminyltransferase to form Lacto-N-triose 2 (LNT2). The plasmid pG292 (ColE1, thyA+, bla+, PL-lgtA) (SEQ ID NO: 2, FIG. 5) carries the lgtA β(1,3)-N-acetylglucosaminyltransferase gene of N. meningitidis and can direct the production of LNT2 in E. coli strain E680 under appropriate culture conditions. pG221 (ColE1, thyA+, bla+, PL-lgtA-wbgO) (SEQ ID NO: 3, FIG. 6) is a derivative of pG292 that carries (arranged as an operon) both the lgtA β(1,3)-N-acetylglucosaminyltransferase gene of N. meningitidis and the wbgO β(1,3)-galactosyltransferase gene of E. coli O55:H7. pG221 directs the production of LNT in E. coli strain E680 under appropriate culture conditions. pG222 (ColE1, thyA+, bla+, PL—IgtA-4GalT) (SEQ ID NO: 4, FIG. 7) is a derivative of pG292 that carries (arranged as an operon) both the lgtA β(1,3)-N-acetylglucosaminyltransferase gene of N. meningitidis and the 4GalT (jhp0765) β(1,4)-galactosyltransferase gene of H. pylori. pG222 directs the production of LNnT in E. coli strain E680 under appropriate culture conditions.
The addition of tryptophan to the lactose-containing growth medium of cultures of any one of the E680-derivative strains transformed with plasmids pG292, pG221 or pG222 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 cI levels, which results in a de-repression of PL, expression of lgtA, lgtA+wbgO, or IgtA+4GalT respectively, and production of LNT2, LNT, or LNnT respectively.
For LNT2, LNT, or LNnT production in small scale laboratory cultures (<100 ml), strains were grown at 30° C. in a selective medium lacking both thymidine and tryptophan to early exponential phase (e.g., M9 salts, 0.5% glucose, 0.4% casaminoacids). Lactose was then added to a final concentration of 0.5 or 1%, along with tryptophan (200 μM 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. 8 illustrates schematically the enzymatic reactions necessary to produce from lactose, via the intermediate trisaccharide lacto-N-triose 2 (LNT2), the two human milk oligosaccharides; Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT). A thin layer chromatogram (on left) is presented of culture medium samples taken from small scale E. coli cultures and demonstrating synthesis of LNT2, LNT, and LNnT (utilizing induced, lactose-containing cultures of E680 transformed with pG292, pG221 or pG222 respectively). A second thin layer chromatogram (on right) is presented of culture medium samples taken from an E. coli E680/pG222 15 L bioreactor culture and demonstrating synthesis of LNnT (as well as the higher molecular weight hMOS, Lacto-N-neohexaose, LNnH).
Although the above results clearly demonstrate how it is possible to synthesize GlcNAc-containing oligosaccharides (i.e. LNT2, LNT and LNnT) in engineered E. coli, FIG. 14 illustrates a serious problem faced when attempting to use the E. coli UDP-GlcNAc pool during such syntheses. In FIG. 14 four separate cultures of E680, transformed with pG292, were grown in the presence and absence of lactose, and with LgtA expression both induced and uninduced by tryptophan addition. It can clearly be seen that massive cell lysis occurs in the cultures where lactose is present—i.e. in those cultures where LgtA draws down the cellular UDP-GlcNAc pool by adding GlcNAc to lactose (and making LNT2). In so doing, UDP-GlcNAc is diverted from cell wall biosynthesis towards hMOS biosynthesis, and cell lysis results. This lysis can be monitored readily not only by the precipitous drop in culture density as seen in the figure, but also by the appearance of DNA in the culture medium.
EXAMPLE 3
Boosting the Cellular UDP-GlcNAc Pool Prevents Cell Lysis During the Biosynthesis of LNnT in Engineered E. coli 
To examine the impact of enhancing the E. coli cellular UDP-GlcNAc pool during synthesis of N-acetylglucosamine-containing hMOS the p15A replicon plasmid pG356 was constructed (FIG. 19 and SEQ ID NO:12). pG356 carries a p15A replicon (compatible with ColE1 replicons), purA and ampC selectable markers, and a synthetic operon (under control of the pL promoter) carrying the E. coli glmS (encoding L-glutamine:D-fructose-6-phosphate aminotransferase) and nagC (encoding the bi-functional transcriptional activator/repressor of glm and nag operons) genes. When pL is active in strains carrying the plasmid pG356, the UDP-GlcNAc pool increases. Strain E796 (see example 1) was transformed with pG222 (FIG. 7), and strain E866 (see example 1) was transformed with both pG222 (FIG. 7) and pG356 (FIG. 19). (Strains E796 and E866 are isogenic save for the purA mutation found in E866 that is used for pG356 plasmid retention). Identical 1.5 L fermentation runs were performed on each of the transformed strains. Optical density of the cultures and LNnT biosynthesis was followed, along with standard fermentation parameters. As can be seen in FIG. 18, the E796/pG222 culture produced LNnT, but lysed when the cell density reached 75 OD600, and achieved a final cell density at end-of-fermentation of only 50 OD600. In contrast (FIG. 19) with the E866/pG222+pG356 culture (where expression of the glmS and bagC genes enhance the intracellular UDP-GlcNAc pool) LNnT was also produced, but with no cell lysis observed. In this culture end-of-fermentation cell density reached 108 OD600—more than twice the density achieved for E796/pG222.
EXAMPLE 4
Production of 6′-sialyllactose (6′-SL) by Engineered E. coli (ΔnanRATEK)
For the production of 6′ sialyllactose, Escherichia coli GI724 (ATCC55151) was engineered with a set of mutations that cause cytoplasmic accumulation of non-acetylated lactose precursor and prevent the degradation of N-acetyl-5-neuraminic acid (FIG. 3). In particular, the lacZ (β-galactosidase) and lacA (lactose acetyl transferase) genes from the lac operon were deleted, leaving the LacIq repressor and the LacY permease fully functional. The LacY permease can be driven by weak (e.g. lac8) or strong (e.g. Ptac) promoters. The entire nan operon (nanRATEK; structural and regulatory genes involved in neuraminic acid degradation) was deleted in this example. E. coli genome manipulations were achieved using a combination of standard molecular genetics techniques, specifically lambda-Red recombineering, allele exchanges with positive selection suicide vectors, and P1 transductions (FIG. 3). The host genotype of strain E781, suitable for production of sialylated hMOS, is presented below:

    ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanRATEK471, ΔlacZ690, ΔlacA 745


To produce 6′-sialyllactose, the cellular UDP-GlcNAc pool must be converted into the sugar-nucleotide activated precursor, CMP-NeuAc, which in turn can function as a donor molecule for a sugar acceptor (i.e. lactose) in a sialyltransferase-catalyzed reaction (FIG. 3). To this purpose, three genes from Campylobacter jejuni ATCC43438, encoding i) UDP-N-acetylglucosamine 2-epimerase (NeuC), ii) N-acetylneuraminate synthase (NeuB), and iii) N-Acetylneuraminate cytidylyltransferase (NeuA), were constitutively co-expressed in the engineered E. coli strain described above, along with a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224 (SEQ ID NO:21 Genbank protein Accession BAF92026, incorporated herein by reference). The neu genes were expressed from a low copy number plasmid vector (pG317, FIG. 9, SEQ ID NO: 5) carrying a constitutive lac promoter (pBBR1 ori, cat+, Plac), while the α(2,6)sialyltransferase gene was expressed from a high copy number plasmid vector (pG315, FIG. 10, SEQ ID NO: 6) carrying a constitutive lac promoter (ColE1 ori, bla+, Plac). To prevent the synthesis of side-products, the relative expression for the α(2,6)sialyltransferase gene compared to the neu genes is modulated by engineering differing ribosomal binding sites (RBS) providing various degrees of translational efficiency upstream of the α(2,6)sialyltransferase gene. Engineered strains were grown to high density in pilot scale fermentors using a batch to fed-batch strategy. FIG. 11 is a TLC analysis of culture supernatants from two such fermentations, with samples to the left of the figure being taken from a fermentation of a strain containing pG315 (and thus carrying the RBS presented in SEQ ID NO: 7 in front of the α(2,6)sialyltransferase gene in the vector). Samples on the right of the figure are taken from a fermentation of a strain containing a close variant of pG315 (pG345, FIG. 12, SEQ ID NO:9, carrying the weaker RBS presented in SEQ ID NO: 8 in front of the α(2,6)sialyltransferase gene and replacing the RBS presented in SEQ ID NO: 7). In both cases, the lactose precursor was added at a cell density of 50 OD600 and efficient conversion to final products was achieved within 48 hours from the lactose addition. The final yield of 6′SL was increased when utilizing the plasmid with the weaker RBS upstream of the α(2,6)sialyltransferase gene, and moreover the level of KDO-lactose side product is very significantly decreased using this weaker RBS. The identity of the 6′-SL purified using activated carbon column chromatography was confirmed by ESI mass spectrometry and NMR.
EXAMPLE 5
Production of 6′-sialyllactose (6′-SL) by engineered E. coli. (ΔnanA, ΔnanATE)
For the production of 6′ sialyllactose, Escherichia coli GI724 (ATCC55151) was engineered with a set of mutations that cause cytoplasmic accumulation of non-acetylated lactose precursor and prevent the degradation of N-acetyl-5-neuraminic acid (FIG. 13). In particular, the lacZ (β-galactosidase) and lacA (lactose acetyl transferase) genes from the lac operon were deleted, leaving the Laclq repressor and the LacY permease fully functional. The LacY permease can be driven by weak (e.g. lac8) or strong (e.g. Ptac) promoters. While the entire nan operon (nanRATEK; structural and regulatory genes involved in neuraminic acid degradation) can be deleted to abolish neuraminic acid catabolism as in Example 4, lesser deletions encompassing just the nanA, or nanA, nanT and nanE, or nanA and nanE genes, are also suitable. In all the instances where the nanE gene was mutated, the last 104 bp of the nanE gene were left intact to allow for undisturbed transcription/translation of downstream nanK, although other lengths of residual nanE sequence are possible. E. coli genome manipulations were achieved using a combination of standard molecular genetics techniques, specifically lambda-Red recombineering, allele exchanges with positive selection suicide vectors, and P1 transductions (FIG. 13). The host genotypes of strains E971, E1017 and E1018, suitable for production of sialylated hMOS with various yield and purity, are presented below:

    ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanA:: kanR, ΔlacZ690, ΔlacA::scar,
    ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanATE::kanR::nanK+, ΔlacZ690, ΔlacA:: scar and
    ampC::(Ptrp-λcI+), lacIq lacPL8, ΔnanATE::scar::nanK+, ΔlacZ690, ΔlacA:: scar respectively


To produce 6′-sialyllactose, the cellular UDP-GlcNAc pool must be converted into the sugar-nucleotide activated precursor, CMP-NeuAc, which in turn can function as a donor molecule for a sugar acceptor (i.e. lactose) in a sialyltransferase-catalyzed reaction (FIG. 13). To this purpose, three genes from Campylobacter jejuni ATCC43438, encoding i) UDP-N-acetylglucosamine 2-epimerase (NeuC), ii) N-acetylneuraminate synthase (NeuB), and iii) N-Acetylneuraminate cytidylyltransferase (NeuA), were constitutively co-expressed in the engineered E. coli strain described above, along with a gene encoding an α(2,6) sialyltransferase from Photobacterium spp JT-ISH-224. The neu genes were expressed from a low copy number plasmid vector (pG317, FIG. 9, SEQ ID NO: 5) carrying a constitutive lac promoter (pBBR1 ori, cat+, Plac), while the α(2,6)sialyltransferase gene was expressed from the weak RBS of SEQ ID NO: 8 in a high copy number plasmid vector (pG345, FIG. 12, SEQ ID NO: 9) carrying a constitutive lac promoter (ColE1 ori, bla+, Plac). Engineered strains were grown to high density in pilot scale fermentors using a batch to fed-batch strategy. FIG. 14 is a TLC analysis of culture pellets or supernatants from three such fermentations. Panel A shows production and accumulation of 6′SL in the cells of three genetic backgrounds (only the relevant nan mutations are shown for strains E971, E1017 and E1018), Panel B and C show production and accumulation of 6′SL in the extracellular milieu (supernatants) in strains E971, E1017 and E1018 (only the relevant nan mutations are shown) with estimated maximum volumetric yields of 15 g per liter of supernatant. In all cases, the lactose precursor was added at a cell density of 40 OD600 and steady state conversion to final products was achieved within approximately 90 hours from the lactose addition (EFT is elapsed fermentation time).
The various sequences presented herein are recited below.








SEQ ID NO: 1



>E680_thyA::2.8RBS_lacZ Escherichia coli str.



GCAGCGGAACTCACAAGGCACCATAACGTCCCCTCCCTGATAACGCTGATACTGTGGTCG


 


CGGTTATGCCAGTTGGCATCTTCACGTAAATAGAGCAAATAGTCCCGCGCCTGGCTGGCG


 


GTTTGCCATAGCCGTTGCGACTGCTGCCAGTATTGCCAGCCATAGAGTCCACTTGCGCTT


 


AGCATGACCAAAATCAGCATCGCGACCAGCGTTTCAATCAGCGTATAACCACGTTGTGTT


 


TTCATGCCGGCAGTATGGAGCGAGGAGAAAAAAAGACGAGGGCCAGTTTCTATTTCTTCG


 


GCGCATCTTCCGGACTATTTACGCCGTTGCAGGACGTTGCAAAATTTCGGGAAGGCGTCT


 


CGAAGAATTTAACGGAGGGTAAAAAAACCGACGCACACTGGCGTCGGCTCTGGCAGGATG


 


TTTCGTAATTAGATAGCCACCGGCGCTTTattaaacctactATGACCATGATTACGGATT


 


CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC


 


GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATC


 


GCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCAC


 


CAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCG


 


TCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATC


 


CCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCA


 


CATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCG


 


TTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTC


 


GTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGG


 


TGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGA


 


GCGGCATTTTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCC


 


ATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGA


 


TGTGCGGCGAGTTGCGTGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGC


 


AGGTCGCCAGCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATG


 


CCGATCGCGTCACACTACGTCTGAACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCC


 


CGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGACGGCACGCTGATTGAAGCAG


 


AAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGTCTGCTGCTGCTGAACG


 


GCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCATCCTCTGCATGGTCAGG


 


TCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAACTTTAACG


 


CCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGCTACG


 


GCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTC


 


TGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGCAGC


 


GCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCG


 


CTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGT


 


ATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCG


 


TGGATGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGC


 


TACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTC


 


TTGGCGGTTTCGCTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCT


 


TCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGT


 


CGGCTTACGGCGGTGATTTTGGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTC


 


TGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGT


 


TTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGACCAGCGAATACCTGTTCCGTC


 


ATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGTAAGCCGCTGGCAAGCG


 


GTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATTGAACTGCCTGAACTAC


 


CGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACCGAACGCGA


 


CCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAAAACC


 


TCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCACCAGCGAAATGG


 


ATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTT


 


CACAGATGTGGATTGGCGATAAAAAACAACTGtTGACGCCGCTGCGCGATCAGTTCACCC


 


GTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCT


 


GGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCA


 


CGGCAGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATCAGG


 


GGAAAACCTTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGA


 


TTACCGTTGATGTTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGAACT


 


GCCAGCTGGCGCAGGTAGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAGAAAACT


 


ATCCCGACCGCCTTACTGCCGCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGT


 


ATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATT


 


ATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACATCAGCCGCTACAGTCAACAGC


 


AACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAAGGCACATGGCTGAATA


 


TCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGTCAGTATCGGCGG


 


AATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAAAAATAAGCGG


 


CCGCtTTATGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTT


 


CGGAATAGGAACTTCAAGATCCCCTTATTAGAAGAACTCGTCAAGAAGGCGATAGAAGGC


 


GATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTC


 


GCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGC


 


CACACCCAGCCGGCCACAGTCGATGAATCCtGAAAAGCGGCCATTTTCCACCATGATATT


 


CGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTT


 


GAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTG


 


ATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTG


 


GTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGAT


 


GGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCC


 


CAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAAC


 


GCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACC


 


GGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGC


 


GGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCA


 


AGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCC


 


TGTCTCTTGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCAT


 


CCAGTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGG


 


TTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGC


 


TACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATT


 


CATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGTTCCGCTTCCTTTAGCA


 


GCCCTTGCGCCCTGAGTGCTTGCGGCAGCGTGAGCTTCAAAAGCGCTCTGAAGTTCCTAT


 


ACTTTCTAGAGAATAGGAACTTCGAACTGCAGGTCGACGGATCCCCGGAATCATGGTTCC


 


TCAGGAAACGTGTTGCTGTGGGCTGCGACGATATGCCCAGACCATCATGATCACACCCGC


 


GACAATCATCGGGATGGAAAGAATTTGCCCCATGCTGATGTACTGCACCCAGGCACCGGT


 


AAACTGCGCGTCGGGCTGGCGGAAAAACTCAACAATGATGCGAAACGCGCCGTAACCAAT


 


CAGGAACAAACCTGAGACAGCTCCCATTGGGCGTGGTTTACGAATATACAGGTTGAGGAT


 


AATAAACAGCACCACACCTTCCAGCAGCAGCTCGTAAAGCTGTGATGGGTGGCGCGGCAG


 


CACACCGTAAGTGTCGAAAATGGATTGCCACTGCGGGTTGGTTTGCAGCAGCAAAATATC


 


TTCTGTACGGGAGCCAGGGAACAGCATGGCAAACGGGAAGTTCGGGTCAACGCGGCCCCA


 


CAATTCACCGTTAATAAAGTTGCCCAGACGCCCGGCACCAAGACCAAACGGAATGAGTGG


 


TGCGATAAAATCAGAGACCTGGAAGAAGGAACGTTTAGTACGGCGGGCGAAGATAATCAT


 


CACCACGATAACGCCAATCAGGCCGCCGTGGAAAGACATGCCGCCGTCCCAGACACGGAA


 


CAGATACAGCGGATCGGCCATAAACTGCGGGAAATTGTAGAACAGAACATAACCAATACG


 


TCCCCCGAGGAAGACGCCGAGGAAGCCCGCATAGAGTAAGTTTTCAACTTCATTTTTGGT


 


CCAGCCGCTGCCCGGACGATTCGCCCGTCGTGTTGCCAGCCACATTGCAAAAATGAAACC


 


CACCAGATACATCAGGCCGTACCAGTGAAGCGCCACGGGTCCTATTGAGAAAATGACCGG


 


ATCAAACTCCGGAAAATGCAGATAGCTACTGGTCATCTGTCACCACAAGTTCTTGTTATT


 


TCGCTGAAAGAGAACAGCGATTGAAATGCGCGCCGCAGGTTTCAGGCGCTCCAAAGGTGC


 


GAATAATAGCACAAGGGGACCTGGCTGGTTGCCGGATACCGTTAAAAGATATGTATA


 


SEQ ID NO: 2



>pG292, complete sequence.



TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA


 


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


 


GCCTTGCGCGATTTCGTGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCGTGAAG


 


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: 3



>pG221, complete sequence.



TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA


 


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: 4



>pG222, complete sequence.



TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA


 


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


 


TGCATGctcgagTTATACAAACTGCCAATATTTCAAATATTTAAAATGGAGTTCTCTCAT


 


TAAGGCGATTTTAGGGCTATAAGGTTCTTCTTTTCGTGCTATCGTAGAGATTTGCTCATC


 


ATCAGCGATCACAAAAGGTTGTAACACCAGATTTTTCACGCCATGGATAAAAGTAGCGTC


 


CATTATCGTATCCACAGGAACAACCCATTTTCGGCTGCATTTCAAAAAAACTTTGGCAAT


 


CTTAGGCGTGATCACATAGCCTTGAGTCCCCACCCCTTCGCTATAAGCTTTAATGATCCC


 


CACACGCTCTTGTATCTCGTGGTTTTTATGGCTCAATGGCTCACTTTTTACACTGGCATC


 


ATACAATAAATGCATCAAGCGGATATAGCCTAACTCTTGGATGTGTTTTTCTAAAAAATC


 


CAAGCCCTCTTTAAAATCCTCTTTCAAGGTTATATCGTCTTCTAAAATACAGATCGCTTC


 


ATTGAGTTCTATGCATTTTTCCCACAAGGAATAATGACTCGCATAGCACCCAAGCTCCCC


 


CAAGCTCATAAACTTCGCATGGTATTTTAAAGCGTAATAAAACTTAGAAACCTCACTGAT


 


GAGATTGGTTGTAATCCCCATGTCTTTGATGTTTTGCGTGATGAAATAAGGGTGTAAATG


 


CTTTTTCACTAAGGGGTGCAACCCGCCTTCAAAAGTTTTAGAATAAATCGCATCAAAAAT


 


TTGCGCTTGGTGGTGGGTGGCATTGATGCTATTGAGTAAAGTTGTGGTGTCTCTAAAAAC


 


TAAACCAAATGTATCGCACACTTTTTGATTTAAAGAAATGGCAAAAACACGCAtATGtat


 


atctccttcttCTCGAGTCAACGGTTTTTCAGCAATCGGTGCAAAATGCCGAAGTATTGC


 


CTCAAGGTAAACAGCCGCCGCATCCTGCCGTCTGCCGCAAAATCCAGCCACGCGCCGGCG


 


GGCAGCGTGTCCGTCCGTTTGAAGCATTGGTACAAAAACCGGCGGGCGCGTTCAAAATCT


 


TCTTCCGGCAAATGTTTCTCCAGCAATTCATACGCTACTGCTTTTATTTGGCGGTATTCA


 


AGGCTGTCGAACCGGGTTTTAAAACCCATAGACTGCAAAAAATCGTTTCTGGCGGTTTTT


 


TGGATGCCTTGCGCGATTTCGTGTTGGCGGATGCTGTATTTGGATGAAACCTGATTGGCG


 


TGAAGGCGGTATTTGACCAAGGCTTCGGGATAATAAGCCAGCCTGCCCAATTTGCTGACA


 


TCGTACCAAAATTGGTAATCTTCCGCCCAATCCCGCTCGGTGTTGTAACGCAAACCGCCG


 


TCAATGACGCTGCGCCTCATAATCATCGTGTTGTTGTGTATGGGGTTGCCGAAAGGGAAA


 


AAGTCGGCAATGTCTTCGTGTCGGGTCGGTTTTTTCCAAATTTTGCCGTGTTCGTGGTGC


 


CGCGCCAGCCGGTTGCCGTCCTTTTCTTCCGACAAAACTTCCAGCCACGCACCCATCGCG


 


ATGATGCTGCGGTCTTTTTCCATCTCACCCACGATTTTCTCAATCCAGTCGGGGGCGGCA


 


ATATCGTCTGCATCGGTGCGCGCAATATATTCCCCCCCCCCCCCCGACTTTGCCAATTCA


 


TCCAGCCCGATGTTTAAAGAGGGAATCAGACCGGAATTGCGCGGCTGCGCGAGGATGCGG


 


ATGCGGCCGTCCTGTTCTTGGAAACGCTGGGCAATGGCAAGCGTACCGTCCGTCGAGCCG


 


TCATCGACAATCAAAATATCCAAGTTGCGCCAAGTTTGATTCACGACGGCGGCTAATGAT


 


TGGGCGAAATATTTTTCTACGTTGTAGGCGCAAATCAATACGCTGACTAAAGGCTGCAAT


 


TTATTCTCCCGATAGGCACGATGCCGTCTGAAGGCTTCAGACGGCATATGtatatctcct


 


tcttgaaTTCTAACAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGT


 


TTAATTTGATGCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTG


 


TTTTTTTGTTACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTA


 


TAGTTAAAAAAATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCT


 


GCTTTCCATTGAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATC


 


TGGATTCTCCTGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACC


 


CCCCGCGATTGGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTT


 


CGTATCACACACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTT


 


TAAGAGCGTCACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCA


 


GTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGT


 


TTTTTATATGAATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCT


 


GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC


 


TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA


 


CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG


 


AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA


 


TAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA


 


CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC


 


TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC


 


GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCT


 


GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG


 


TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG


 


GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA


 


CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG


 


AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT


 


TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT


 


TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG


 


ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT


 


CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC


 


TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGAT


 


AACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCC


 


ACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAG


 


AAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG


 


AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGT


 


GGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG


 


AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT


 


TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTC


 


TCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC


 


ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAA


 


TACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCG


 


AAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC


 


CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG


 


GCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTT


 


CCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATT


 


TGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCC


 


ACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCAC


 


GAGGCCCTTTCGTC


 


SEQ ID NO: 5



>pG317, complete sequence.



GTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCA


 


TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA


 


AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG


 


CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC


 


CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCATGCATAAAAACTGTTGTAATTCA


 


TTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGC


 


GGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAG


 


AAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCT


 


GAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAA


 


CACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTC


 


CAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTA


 


TCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAATTCCGGATGAGCATTCATC


 


AGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTC


 


TTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGAC


 


TGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCA


 


GTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAAT


 


ACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCA


 


ACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGA


 


TTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGAAGACGAAAGGGCCT


 


CGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGG


 


TGGCACTTTTCGGGGAAATGTGCGCGCCCGCGTTCCTGCTGGCGCTGGGCCTGTTTCTGG


 


CGCTGGACTTCCCGCTGTTCCGTCAGCAGCTTTTCGCCCACGGCCTTGATGATCGCGGCG


 


GCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGC


 


TCCCGAAGGTCTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCT


 


CCTGCGGCGGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCG


 


GCCACGGCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGC


 


GGTGCATAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGC


 


CGCTCCAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATG


 


CCCCGTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATC


 


TGCGCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACC


 


ACGAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGA


 


TCCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGT


 


TCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAAC


 


ACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCG


 


GTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTC


 


TCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTC


 


TTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGGC


 


TCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATACTCA


 


CTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACGGGCTTG


 


CTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATATGTGGA


 


CGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAACCCTGCTG


 


GACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGATTGCCCACCGG


 


CTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCC


 


ATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCT


 


TGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCT


 


TGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAG


 


CCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGGGTTCCAAGGGGGCAGCG


 


CCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAACAAGCCCCGGAGGGGCCACTTT


 


TTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGGAACCCCGCAGGGGTGCCCTTCTTTG


 


GGCACCAAAGAACTAGATATAGGGCGAAATGCGAAAGACTTAAAAATCAACAACTTAAAA


 


AAGGGGGGTACGCAACAGCTCATTGCGGCACCCCCCGCAATAGCTCATTGCGTAGGTTAA


 


AGAAAATCTGTAATTGACTGCCACTTTTACGCAACGCATAATTGTTGTCGCGCTGCCGAA


 


AAGTTGCAGCTGATTGCGCATGGTGCCGCAACCGTGCGGCACCCTACCGCATGGAGATAA


 


GCATGGCCACGCAGTCCAGAGAAATCGGCATTCAAGCCAAGAACAAGCCCGGTCACTGGG


 


TGCAAACGGAACGCAAAGCGCATGAGGCGTGGGCCGGGCTTATTGCGAGGAAACCCACGG


 


CGGCAATGCTGCTGCATCACCTCGTGGCGCAGATGGGCCACCAGAACGCCGTGGTGGTCA


 


GCCAGAAGACACTTTCCAAGCTCATCGGACGTTCTTTGCGGACGGTCCAATACGCAGTCA


 


AGGACTTGGTGGCCGAGCGCTGGATCTCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGT


 


CGGCCTACGTGGTCAATGACCGCGTGGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGT


 


CGGTGTTCAGTGCCGCCGTGGTGGTTGATCACGACGACCAGGACGAATCGCTGTTGGGGC


 


ATGGCGACCTGCGCCGCATCCCGACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCC


 


CCGGCGAGGAGCCGCCCAGCCAGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCCT


 


TGACCGAAACGGAGGAATGGGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGATGAGC


 


CGTGTTTTCTGGACGATGGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTGCCGCGCC


 


GGTAGCACTTGGGTTGCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGC


 


CGCCTCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGG


 


GCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCGTTTTTATCA


 


GGCTCTGGGAGGCAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCT


 


GAGCAAACTGGCCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATA


 


AACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGA


 


CCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGC


 


GTAGCACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGC


 


CACTCATCGCAGTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCG


 


AATTTTAACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTT


 


GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTG


 


CTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA


 


CGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTG


 


GCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCG


 


ATACCGTCGACCTCGAGTTAAGTCTCTAATCGATTGTTTTCCAATGGAATGGTTATAAAA


 


TCTTTGGTTTTTAGTCTTGAAAATCTTCTAGGATTTTCTATGTAAGTTTTTGTATAAATA


 


TTATATTGCTTTAATAAATTTAATATATTTTTATTGCATTTTAAGGTTATTTTTTCCATA


 


TCTGTTCAACCTTTTTTAAATCCTCCAAACAGTCAATATCTAAACTTGAGCTTTCGTCCA


 


TTAAAAAATGCTTGGTTTTGCTTTGTAAAAAGCTAGGATTGTTTAAAAATTCTTTTATCT


 


TTAAAATATAAATTGCACCATTGCTCATATAAGTTTTAGGCAATTTTTGCCTTGGCATAA


 


AAGGATATTCATCATTACAAATCCCTGCTAAATCGCCACAATCATTACAAACAAAGGCTT


 


TTAGAATTTTATTATCACATTCGCTTACGCTAATTAGGGCATTTGCATTGCTATTTTTAT


 


AAAGATTAAAAGCTTCATTAATATGAATATTTGTTCTTAGCGGTGAAGTGGGTTGTAAAA


 


AAACTACATCTTCATAATCTTTATAAAATTTTAGAGCATGTAACAGCACTTTATCGCTTG


 


TGGTATCATCTTGTGCAAGGCTAATTGGGCGTTTTAAAATATCAACATTTTGACTTTTTG


 


CATAATTTAAAATTTCATCACTATCACTGCTTACAACAACTTTACTAATGCTTTTAGCAT


 


TTAGTGCAGCTTTGATCGTGTAGTAAATTAAAGGTTTATTGTTTAATAAAACCAAATTTT


 


TATTTTTAATACCCTTTGAGCCACCACGAGCAGGGATTATTGCTAAGCTCATTTTATATC


 


CTTAAAAACTTTTTGTGTGCTGAGTTTAAAAAAATCTCCGCTTTGTAAATATTCAAAAAA


 


TAATTTTGAGCTATCTAAAATCTCTAACTTAGCGCTAAATAAATCTTGTTTTTTATGAAT


 


AGTGTTAATAGCTTTTAGTATTTCATCACTATTTGCATTAACTTTTAGTGTATTTTCATT


 


GCCAAGTCTTCCATTTTGTCTTGAGCCAACTAAAATCCCTGCTGTTTTTAAGTATAAGGC


 


CTCTTTTAAAATACAACTTGAATTACCTATTATAAAATCAGCATTTTTTAACAAAGTTAT


 


AAAATACTCAAATCTAAGCGATGGAAAAAGCTTAAATCTAGGGTTATTTTTAAACTCTTC


 


ATAGCTTTGCAAGATTAATTCAAAACCTAAATCATTATTTGGATAAATAACAATATAATT


 


TTTATTACTTTGTATCAGTGCTTTTACTAAATTGTCTGCTTGATTTTTAATGCTAGTAAT


 


TTCAGTTGTAACAGGATGAAACATAAGCAAAGCGTAGTTTTCATAATTTATATCATAATA


 


TTTTTTTGCTTCGCTAAGTGAAATTTTATTATCGTTTAAAAGTTCTAAATCAGGCGAACC


 


TATGATAAAAATAGATTTTTCATCTTCTCCAAGCTGCATTAAACGCCTTTTTGCAAACTC


 


ATCATTTACTAAATGAATATGAGCTAGTTTTGATATAGCGTGGCGTAAGCTATCGTCAAT


 


AGTTCCTGAAATCTCTCCGCCTTCAATATGCGCTACTAAGATATTATTTAATGCTCCAAC


 


AATAGCTGCTGCTAAAGGCTCAATTCTATCTCCATGTACTACGATTAAATCAGGTTTTAG


 


CTCATTTGCATACCTTGAAAATCCATCAATTGTAGTAGCTAAAGCCTTATCAGTTTGATA


 


ATATTTATCATAATTTATAAATTCATAAATATTTTTAAAGCCATTTTTATAAAGTTCTTT


 


AACTGTATAGCCAAAATTTTTACTTAAGTGCATTCCTGTTGCAAAGATGTAAAGTTCAAA


 


TTCGCTTGAGTTTTGCACCCTGTACATTAAAGATTTAATCTTAGAATAATCAGCCCTAGA


 


GCCTGTTATAAAAAGGATTTTTTTCACGCAAAATCCTCATAGCTTAACTGAGCATCATTT


 


TCTATATCTCTTAATGCTTTTTTGCCTAAAATATTTTCAAATTCAGCCGCACTAATTCCA


 


CCAAGTCCAGGTCTTTTAACCCAAATATTATCCATAGATAAAACTTCGCCTTTTTTAATA


 


TCTTTAATGCTAACTACACTTGCAAAGGCAAAATCAATTGTAACTTGTTCTTGTTTAGCC


 


GCTTTTTTACTTTCATTATTTCCTCTTATTATAGCCATTTGCTCACTTTGTATAATTAGC


 


TCTTTTAAAGCCTTTGTATCCATAGAACAAACTATATCAGGGCCACTTCTATGCATACTA


 


TCAGTAAAATGTCTTTCAAGCACACAAGCTCCAAGTACAACTGCACCTAAACACGCAAGA


 


TTATCTGTTGTGTGGTCGCTTAAGCCTACCATACAAGAAAATTCTTTTTTTAACTCAAGC


 


ATAGCGTTTAATCTTACAAGATTATGCGGGGTTGGGTAAAGATTGGTCGTGTGCATTAAA


 


ACAAAAGGAATTTCATTGTCTAATAAGATTTTTACAGTTGGTTTTATACTTTCAATACTA


 


TTCATTCCTGTGCTAACTATCATAGGCTTTTTAAAGGCTGCTATGTGTTTAATAAGCGGA


 


TAATTATTACACTCACCTGAACCAATCTTAAAAGCACTAACTCCCATATCTTCTAAGCGG


 


TTCGCACCTGCACGAGAAAAAGGTGTGCTAAGATAAACAAGACCTAATTTTTCTGTGTAT


 


TCTTTAAGTGCTAGCTCATCTTTATAATCCAAAGCACATTTTTGCATAATCTCATAAATG


 


CTTATTTTTGCATTACCAGGAATTACTTTTTTAGCGGCCTTACTCATCTCATCTTCAACA


 


ATATGAGTTTGATGCTTTATAATCTTAGCACCTGCGCTAAAGGCTGCATCTACCATAATT


 


TTAGCTAGTTCTAAACTGCCATTATGATTAATGCCTATTTCAGGTACGACTAAGGGTGCT


 


TTTTCTTCACTTATGATTATATTTTGTATTTTTATTTCTTTCATTTATTTTCCTCCTTAG


 


SEQ ID NO: 6



>pG315, complete sequence



CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTC


 


ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGA


 


GATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC


 


CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC


 


CTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG


 


CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA


 


AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC


 


CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCG


 


CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG


 


GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG


 


TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCA


 


CCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCTAGACTGCAATACAAACACCTGTTT


 


CACAATTTGGCAGATCAGCCCAAAAAAGTACATTCTCTTCTTTTACAATACCTAGTTTTA


 


TCATTACTTGAACTAAAGGACTTCTCAAAGCAGTTTCACGATCAGTTATAGTTTCTGTCG


 


ATGTAAAAACTATAAATTTAATTTTTTCAGCTGGTATCGTGAAATATAAAGAGCTCGCTA


 


TACCAGCAACTGCATCAGGAAGCATATCTGTCATCATCAAAACTTCAAATGATATTTTTG


 


ATGGAATATCAACCATTGAAGGATAGTTTTGCATTATTAATGTATTAATGATACCGCCAC


 


CAGGGTGACCTTTGAAGAACAAATCATAACTATTGCCTAAATAATGTGGGCTCGATTCAT


 


TAATTGCATTATTAATGACATTAATTTGTTGTTTCGCATAATACTCTCTTTCATGGTTAC


 


CAGCCCATACAGTCGTACCTGTAAACACAAAGTTTGGTAAATTAGATGAATTATATTCAT


 


TTTGTAATTTTTGTTTGTCAAAATTAACAATCGATAAGAATAATTCTTGTTGTTTGCTAT


 


TGAATTTTTTGAAACCATCCCATTGCATTTGCTTTAAACTATCACCAATATAGTCTCGTA


 


ACTCATGTAATGATGGTTCTAAAGTTAAATAATCTTTTCTTAAAAAATGGTAGTTAGCTG


 


GATATAGTTTTTGCCAGTTATAAACAGATGATGTTCCTGTATTTGAAGTGTCTTCATTGA


 


TACCATTAATGACATCCTCAAGATAATCTTTACCAATTTTTAAATTATCTGTTTTATTTA


 


ATGTATCTCTCCAGTTATATAAATTTACATATTCTGCTGAACCATCATCATATAAATCTA


 


TATTTGTTACCGTAACGTTATTAAACGAATTTAATTCTTTTAGTATTGGCACTAAATTAT


 


CAAATGAATGAGCAGTGTTAGAGCTAAGTTTAACATTCAATCTATGCTTTGTTTGTGCTT


 


GCTTAACAATTTCTTGTACTAAGTCAGCTGGTGTATGGTTATTTATCAATGCAAACGATG


 


TAATATTTAACTCTTTCATTTGCTCATCAGTCGGAACTATTCTCCCCCAAGCTATATATC


 


TTTGTGCTGTAGGATTTTCTTCTTCCGATTTAATAATATCCATTAGCTGCTGAAGAGTTG


 


GAAGAGATGCATGATCAACATAAACCTCTAAAGATGGAGCCACTACGTTTAATGTTACTT


 


TTGTTATATATTTTTCACCTTTATTACTAACACCATTAAAATCAAAGCAGTACTTTTCAT


 


CGTCATCTAATCGTGGCGCCACTACAGATAATGATATTGACTCTTTATTTTGTTCTGTTA


 


ATAGTTGTTGCGTACCACAAGTTTGTACCCAAGAGTGTTTTGTAAAAGAGATGTTTGATT


 


GATTAATTGGCTCTAAATTAACATACTCCTCATCAATAATAGTTTTATTAATATCATTTT


 


TAATAATAGATTGTGTATTTTCTTCTGACATggtctgtttcctcCTCGAGGGGGGGCCCG


 


GTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCA


 


TAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA


 


AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG


 


CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC


 


CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGAC


 


TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA


 


CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA


 


AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCT


 


GACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA


 


AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG


 


CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA


 


CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA


 


CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG


 


GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG


 


TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG


 


ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC


 


TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG


 


ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC


 


GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC


 


TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG


 


TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGT


 


CTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG


 


GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA


 


GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACT


 


TTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCA


 


GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG


 


TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC


 


ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG


 


GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA


 


TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT


 


ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC


 


AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATC


 


TTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA


 


TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA


 


AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT


 


TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAA


 


AATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC


 


SEQ ID NO: 7



CTCGAGgaggaaacagaccATG



 


SEQ ID NO: 8



CTCGAGgaaagaggggacaaactagATG



 


SEQ ID NO: 9



>pG345, complete sequence



CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTC


 


ATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGA


 


GATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTC


 


CAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACC


 


CTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG


 


CCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAA


 


AGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCAC


 


CACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCG


 


CAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG


 


GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTG


 


TAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCA


 


CCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCTAGACTGCAATACAAACACCTGTTT


 


CACAATTTGGCAGATCAGCCCAAAAAAGTACATTCTCTTCTTTTACAATACCTAGTTTTA


 


TCATTACTTGAACTAAAGGACTTCTCAAAGCAGTTTCACGATCAGTTATAGTTTCTGTCG


 


ATGTAAAAACTATAAATTTAATTTTTTCAGCTGGTATCGTGAAATATAAAGAGCTCGCTA


 


TACCAGCAACTGCATCAGGAAGCATATCTGTCATCATCAAAACTTCAAATGATATTTTTG


 


ATGGAATATCAACCATTGAAGGATAGTTTTGCATTATTAATGTATTAATGATACCGCCAC


 


CAGGGTGACCTTTGAAGAACAAATCATAACTATTGCCTAAATAATGTGGGCTCGATTCAT


 


TAATTGCATTATTAATGACATTAATTTGTTGTTTCGCATAATACTCTCTTTCATGGTTAC


 


CAGCCCATACAGTCGTACCTGTAAACACAAAGTTTGGTAAATTAGATGAATTATATTCAT


 


TTTGTAATTTTTGTTTGTCAAAATTAACAATCGATAAGAATAATTCTTGTTGTTTGCTAT


 


TGAATTTTTTGAAACCATCCCATTGCATTTGCTTTAAACTATCACCAATATAGTCTCGTA


 


ACTCATGTAATGATGGTTCTAAAGTTAAATAATCTTTTCTTAAAAAATGGTAGTTAGCTG


 


GATATAGTTTTTGCCAGTTATAAACAGATGATGTTCCTGTATTTGAAGTGTCTTCATTGA


 


TACCATTAATGACATCCTCAAGATAATCTTTACCAATTTTTAAATTATCTGTTTTATTTA


 


ATGTATCTCTCCAGTTATATAAATTTACATATTCTGCTGAACCATCATCATATAAATCTA


 


TATTTGTTACCGTAACGTTATTAAACGAATTTAATTCTTTTAGTATTGGCACTAAATTAT


 


CAAATGAATGAGCAGTGTTAGAGCTAAGTTTAACATTCAATCTATGCTTTGTTTGTGCTT


 


GCTTAACAATTTCTTGTACTAAGTCAGCTGGTGTATGGTTATTTATCAATGCAAACGATG


 


TAATATTTAACTCTTTCATTTGCTCATCAGTCGGAACTATTCTCCCCCAAGCTATATATC


 


TTTGTGCTGTAGGATTTTCTTCTTCCGATTTAATAATATCCATTAGCTGCTGAAGAGTTG


 


GAAGAGATGCATGATCAACATAAACCTCTAAAGATGGAGCCACTACGTTTAATGTTACTT


 


TTGTTATATATTTTTCACCTTTATTACTAACACCATTAAAATCAAAGCAGTACTTTTCAT


 


CGTCATCTAATCGTGGCGCCACTACAGATAATGATATTGACTCTTTATTTTGTTCTGTTA


 


ATAGTTGTTGCGTACCACAAGTTTGTACCCAAGAGTGTTTTGTAAAAGAGATGTTTGATT


 


GATTAATTGGCTCTAAATTAACATACTCCTCATCAATAATAGTTTTATTAATATCATTTT


 


TAATAATAGATTGTGTATTTTCTTCTGACATctagtttgtcccctctttcCTCGAGGGGG


 


GGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCA


 


TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA


 


GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATT


 


GCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA


 


ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTC


 


ACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCG


 


GTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC


 


CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC


 


CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA


 


CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC


 


CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAT


 


AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG


 


CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC


 


AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA


 


GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACT


 


AGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTT


 


GGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG


 


CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGG


 


TCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAA


 


AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA


 


TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG


 


ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA


 


CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCG


 


GCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCT


 


GCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGT


 


TCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGC


 


TCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGA


 


TCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT


 


AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTC


 


ATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA


 


TAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCA


 


CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA


 


AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCT


 


TCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCC


 


GCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA


 


TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATT


 


TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC


 


SEQ ID NO: 10



CTTTattaaacctactATG



 


SEQ ID NO: 11



CTTTcttcaacctactATG



 


SEQ ID NO: 12



>pEC3′-(T7)GlmS-(T7)NagC-purA_(pG356)



TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA


 


CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTG


 


TTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGC


 


ACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCG


 


CCactagtGTTGAGGAAAACGATTGGCTGAACAAAAAACAGACTGATCGAGGTCATTTTT


 


GAGTGCAAAAAGTGCTGTAACTCTGAAAAAGCGATGGTAGAATCCATTTTTAAGCAAACG


 


GTGATTTTGAAAAATGGGTAACAACGTCGTCGTACTGGGCACCCAATGGGGTGACGAAGG


 


TAAAGGTAAGATCGTCGATCTTCTGACTGAACGGGCTAAATATGTTGTACGCTACCAGGG


 


CGGTCACAACGCAGGCCATACTCTCGTAATCAACGGTGAAAAAACCGTTCTCCATCTTAT


 


TCCATCAGGTATTCTCCGCGAGAATGTAACCAGCATCATCGGTAACGGTGTTGTGCTGTC


 


TCCGGCCGCGCTGATGAAAGAGATGAAAGAACTGGAAGACCGTGGCATCCCCGTTCGTGA


 


GCGTCTGCTGCTGTCTGAAGCATGTCCGCTGATCCTTGATTATCACGTTGCGCTGGATAA


 


CGCGCGTGAGAAAGCGCGTGGCGCGAAAGCGATCGGCACCACCGGTCGTGGTATCGGGCC


 


TGCTTATGAAGATAAAGTAGCACGTCGCGGTCTGCGTGTTGGCGACCTTTTCGACAAAGA


 


AACCTTCGCTGAAAAACTGAAAGAAGTGATGGAATATCACAACTTCCAGTTGGTTAACTA


 


CTACAAAGCTGAAGCGGTTGATTACCAGAAAGTTCTGGATGATACGATGGCTGTTGCCGA


 


CATCCTGACTTCTATGGTGGTTGACGTTTCTGACCTGCTCGACCAGGCGCGTCAGCGTGG


 


CGATTTCGTCATGTTTGAAGGTGCGCAGGGTACGCTGCTGGATATCGACCACGGTACTTA


 


TCCGTACGTAACTTCTTCCAACACCACTGCTGGTGGCGTGGCGACCGGTTCCGGCCTGGG


 


CCCGCGTTATGTTGATTACGTTCTGGGTATCCTCAAAGCTTACTCCACTCGTGTAGGTGC


 


AGGTCCGTTCCCGACCGAACTGTTTGATGAAACTGGCGAGTTCCTCTGCAAGCAGGGTAA


 


CGAATTCGGCGCAACTACGGGGCGTCGTCGTCGTACCGGCTGGCTGGACACCGTTGCCGT


 


TCGTCGTGCGGTACAGCTGAACTCCCTGTCTGGCTTCTGCCTGACTAAACTGGACGTTCT


 


GGATGGCCTGAAAGAGGTTAAACTCTGCGTGGCTTACCGTATGCCGGATGGTCGCGAAGT


 


GACTACCACTCCGCTGGCAGCTGACGACTGGAAAGGTGTAGAGCCGATTTACGAAACCAT


 


GCCGGGCTGGTCTGAATCCACCTTCGGCGTGAAAGATCGTAGCGGCCTGCCGCAGGCGGC


 


GCTGAACTATATCAAGCGTATTGAAGAGCTGACTGGTGTGCCGATCGATATCATCTCTAC


 


CGGTCCGGATCGTACTGAAACCATGATTCTGCGCGACCCGTTCGACGCGTAATTCTGGTA


 


CGCCTGGCAGATATTTTGCCTGCCGGGCGAACAGTGTGATACATTGCTGTGTCGGGTAAG


 


CCATTACGCTATCCGACACAGTGTTAAATCCTCGCTTTTTTCCTTCCCCagatctGGCGC


 


CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTA


 


TTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG


 


TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTACTGCTCACAAGAAA


 


AAAGGCACGTCATCTGACGTGCCTTTTTTATTTGTACTACCCTGTACGATTACTGCAGGT


 


CGACTTAATTTTCCAGCAAATGCTGGAGCAAAATACCGTTGAGCATGGCGCGTTTTACCA


 


GCGCAAAAGCGCCGATTGCCGAGCGGTGATCCAGCTCAGAACGTACCACCGGCAGATTAG


 


TGCGAAACGCCTTCAGCGCCTGGGTATTAATGCAGCTTTCAATAGCAGGGAGCAGCACTT


 


TATCGGCTTCGGTGATTTCACCGGCAATAACAATTTTTTGCGGATTAAATAAGTTGATAG


 


CAATGGCGATGGTTTTACCCAGATGACGACCGACATACTCAATTACTTCCGACGCCAGAC


 


TATCGCCTTTGTTCGCGGCTTTGCAGATAGTTTTGATGGTGCAGTCGTCCAGCGGCACGC


 


GGCTCTGGTAGCCCTGCTTTAACAGATTCAACACCCGTTGTTCAATGGCAGCGTTGGCAG


 


CGATAGTTTCCAGGCAGCCAAAGTTGCCGCAGTGGCAGCGTTCACCCAGCGGTTCGACCT


 


GAATATGGCCAATTTCACCGACGTTGCCGTTGCGGCCAATAAAAATGCGCCCGTTAGAGA


 


TAATCCCGGCCCCGGTTCCGCGATGGACACGCACCAGAATGGAGTCTTCGCAATCCTGAC


 


TTGCACCGAAGTAGTGCTCCGCCAGCGCCAGACTACGGATATCGTGACCAACGAAACAGG


 


TCACTTTAAAACGTTCTTCCAGAGCTTCTACCAGCCCCCAGTTTTCTACCTGAATATGCG


 


GCATGTAATGAATTTTGCCGCTGTCCGGGTCAACAAGCCCTGGCAGGATCACCGAAATCG


 


CGATCAGCTCGCGCAGTTTGCGCTGGTAGCTATCAATAAACTGAGCAATGGCATTCAACA


 


GGGCATGTTCCAGCGTTTGCTGGGTACGTTCCGGCAGCGGGTAATGTTCTTCTGCCAGCA


 


CTTTGCTGCTGAGATCAAACAGAGTGATGGTGGCGTCATGACGACCAAGCCGTACGCCGA


 


TTGCGTGGAAATTGCGGGTTTCGGTGACGATGGAGATAGCGCGGCGGCCCCCGGTGGAGG


 


CCTGCTGATCAACTTCTTTGATCAGCCCGCGTTCGATAAGCTGACGCGTAATTTTGGTTA


 


CGCTGGCGGGGGCAAGCTGGCTTTGCTCGGCAATCTGAATCCGCGAGATTGGCCCGTACT


 


GGTCAATCAGGCGATAAACCGCCGCGCTGTTAAGCTGTTTTACGAGATCAACATTACCTA


 


TCTGAGCTTGTCCGCCTGGTGTCATATGTATATCTCCTTCTTgtcgacTCTAGATGCATG


 


CTCGAGATTACTCAACCGTAACCGATTTTGCCAGGTTACGCGGCTGGTCAACGTCGGTGC


 


CTTTGATCAGCGCGACATGGTAAGCCAGCAGCTGCAGCGGAACGGTGTAGAAGATCGGTG


 


CAATCACCTCTTCCACATGCGGCATCTCGATGATGTGCATGTTATCGCTACTTACAAAAC


 


CCGCATCCTGATCGGCGAAGACATACAACTGACCGCCACGCGCGCGAACTTCTTCAATGT


 


TGGATTTCAGTTTTTCCAGCAATTCGTTGTTCGGTGCAACAACAATAACCGGCATATCGG


 


CATCAATTAGCGCCAGCGGACCGTGTTTCAGTTCGCCAGCAGCGTAGGCTTCAGCGTGAA


 


TGTAAGAGATCTCTTTCAACTTCAATGCGCCTTCCAGCGCGATTGGGTACTGATCGCCAC


 


GGCCCAGGAACAGCGCGTGATGTTTGTCAGAGAAATCTTCTGCCAGCGCTTCAATGCGTT


 


TGTCCTGAGACAGCATCTGCTCAATACGGCTCGGCAGCGCCTGCAGACCATGCACGATGT


 


CATGTTCAATGGAGGCATCCAGACCTTTCAGGCGAGACAGCTTCGCCACCAGCATCAACA


 


GCACAGTTAACTGAGTGGTGAATGCTTTAGTGGATGCCACGCCGATTTCTGTACCCGCGT


 


TGGTCATTAGCGCCAGATCGGATTCGCGCACCAGAGAAGAACCCGGAACGTTACAGATTG


 


CCAGTGAACCAAGGTAACCCAGCTCTTTCGACAGACGCAGGCCAGCCAGGGTATCCGCGG


 


TTTCGCCAGACTGTGACAAGGTGATCATCAGGCTGTTACGACGCACGGCAGATTTGCGAT


 


AGCGGAATTCAGAGGCGATTTCGACGTCGCACGGAATACCTGCTAGCGATTCAAACCAGT


 


AGCGGGAAACCATACCGGAGTTATAAGAAGTACCACAGGCGAGGATCTGAATATGCTCAA


 


CCTTCGACAGCAGTTCGTCGGCGTTCGGTCCCAGCTCGCTTAAATCAACCTGACCGTGGC


 


TGATGCGTCCGGTAAGGGTGTTTTTGATCGCGTTCGGCTGTTCGTAGATCTCTTTCTGCA


 


TGTAGTGACGGTAAATGCCTTTATCGCCCGCGTCATATTGCAGATTGGATTCGATATCCT


 


GACGTTTTACTTCCGCGCCAGTTTTATCGAAGATGTTTACCGAACGGCGAGTGATTTCCG


 


CAATATCGCCCTCTTCAAGGAAGATAAAGCGACGGGTCACCGGCAACAGCGCCAGCTGGT


 


CAGAAGCGATAAAGTTTTCGCCCATCCCCAGGCCAATCACCAGCGGACTACCAGAACGTG


 


CCGCCAGCAGGGTATCCGGGTGACGGGAGTCCATGATCACTGTACCGTACGCACCACGCA


 


GCTGCGGGATAGCACGCAGAACGGCCTCACGCAGAGTCCCGCCTTGTTTCAGCTCCCAGT


 


TCACCAGATGGGCAATCACTTCGGTGTCGGTTTCAGAAACGAAGGTATAGCCACGCGCTT


 


TTAGCTCTTCACGCAGCGGTTCATGGTTTTCGATGATGCCGTTATGCACCACCACAATGT


 


GTTCAGAAACATGCGGATGCGCATTCACTTCTGAAGGTTCACCGTGGGTCGCCCAGCGAG


 


TGTGAGCAATACCAGTGCCGCCATGCAGAGGATGTTCTTCCGCTGCCTGTGCCAGCATCT


 


GGACTTTACCGAGGCGACGCAGGCGGGTCATATGACCTTCTGCATCAACAACGGCCAGAC


 


CGGCAGAGTCATATCCGCGGTATTCCAGACGACGTAAACCTTCAAGAAGGATTTCTGCTA


 


CATCACGTTGCGCGATCGCGCCAACAATTCCACACATATGtatatctccttcttgaaTTC


 


TAACAATTGATTGAATGTATGCAAATAAATGCATACACCATAGGTGTGGTTTAATTTGAT


 


GCCCTTTTTCAGGGCTGGAATGTGTAAGAGCGGGGTTATTTATGCTGTTGTTTTTTTGTT


 


ACTCGGGAAGGGCTTTACCTCTTCCGCATAAACGCTTCCATCAGCGTTTATAGTTAAAAA


 


AATCTTTCGGAACTGGTTTTGCGCTTACCCCAACCAACAGGGGATTTGCTGCTTTCCATT


 


GAGCCTGTTTCTCTGCGCGACGTTCGCGGCGGCGTGTTTGTGCATCCATCTGGATTCTCC


 


TGTCAGTTAGCTTTGGTGGTGTGTGGCAGTTGTAGTCCTGAACGAAAACCCCCCGCGATT


 


GGCACATTGGCAGCTAATCCGGAATCGCACTTACGGCCAATGCTTCGTTTCGTATCACAC


 


ACCCCAAAGCCTTCTGCTTTGAATGCTGCCCTTCTTCAGGGCTTAATTTTTAAGAGCGTC


 


ACCTTCATGGTGGTCAGTGCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTA


 


TGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGTATGTTTTTTATATG


 


AATTTATTTTTTGCAGGGGGGCATTGTTTGGTAGGTGAGAGATCAATTCTGCATTAATGA


 


ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTGCTAGCGGA


 


GTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGC


 


AGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCT


 


TCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTAC


 


GAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGG


 


CCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGAC


 


GCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG


 


GCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGT


 


TATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCC


 


AAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAAC


 


TATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGT


 


AATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGAC


 


AAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCA


 


GAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTAC


 


GCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGGC


 


ggccgcGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTT


 


CACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA


 


AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT


 


ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGG


 


CTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGA


 


TTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTT


 


ATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT


 


TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTT


 


TGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT


 


GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGC


 


CGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC


 


CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTAT


 


GCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAG


 


AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT


 


ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC


 


TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAA


 


GGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTG


 


AAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAA


 


TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAAC


 


CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC


 


SEQ ID NO: 13



>neuC_N-acetylglucosamine-6-phosphate-2-



epimerase_GI_15193223_in_pG317


MKKILFITGSRADYSKIKSLMYRVQNSSEFELYIFATGMHLSKNFGYTVKELYKNGFKNI


 


YEFINYDKYYQTDKALATTIDGFSRYANELKPDLIVVHGDRIEPLAAAIVGALNNILVAH


 


IEGGEISGTIDDSLRHAISKLAHIHLVNDEFAKRRLMQLGEDEKSIFIIGSPDLELLNDN


 


KISLSEAKKYYDINYENYALLMFHPVTTEITSIKNQADNLVKALIQSNKNYIVIYPNNDL


 


GFELILQSYEEFKNNPRFKLFPSLRFEYFITLLKNADFIIGNSSCILKEALYLKTAGILV


 


GSRQNGRLGNENTLKVNANSDEILKAINTIHKKQDLFSAKLEILDSSKLFFEYLQSGDFF


 


KLSTQKVFKDIK


 


SEQ ID NO: 14



>neuB_sialic_acid_synthase_GI_15193222_in_pG317



MKEIKIQNIIISEEKAPLVVPEIGINHNGSLELAKIMVDAAFSAGAKIIKHQTHIVEDEM


 


SKAAKKVIPGNAKISIYEIMQKCALDYKDELALKEYTEKLGLVYLSTPFSRAGANRLEDM


 


GVSAFKIGSGECNNYPLIKHIAAFKKPMIVSTGMNSIESIKPTVKILLDNEIPFVLMHTT


 


NLYPTPHNLVRLNAMLELKKEFSCMVGLSDHTTDNLACLGAVVLGACVLERHFTDSMHRS


 


GPDIVCSMDTKALKELIIQSEQMAIIRGNNESKKAAKQEQVTIDFAFASVVSIKDIKKGE


 


VLSMDNIWVKRPGLGGISAAEFENILGKKALRDIENDAQLSYEDFA


 


SEQ ID NO: 15



>neuA_CMP-Neu5Ac_synthase_GI_15193224_in_pG317



MSLAIIPARGGSKGIKNKNLVLLNNKPLIYYTIKAALNAKSISKVVVSSDSDEILNYAKS


 


QNVDILKRPISLAQDDTTSDKVLLHALKFYKDYEDVVFLQPTSPLRTNIHINEAFNLYKN


 


SNANALISVSECDNKILKAFVCNDCGDLAGICNDEYPFMPRQKLPKTYMSNGAIYILKIK


 


EFLNNPSFLQSKTKHFLMDESSSLDIDCLEDLKKVEQIWKK


 


SEQ ID NO: 16



>AAF42258 lacto-N-neotetraose biosynthesis glycosyl



transferase LgtA [Neisseria meningitidis MC58].


MPSEAFRRHRAYRENKLQPLVSVLICAYNVEKYFAQSLAAVVNQTWRNLDILIVDDGSTD


 


GTLAIAQRFQEQDGRIRILAQPRNSGLIPSLNIGLDELAKSGGGGEYIARTDADDIAAPD


 


WIEKIVGEMEKDRSIIAMGAWLEVLSEEKDGNRLARHHEHGKIWKKPTRHEDIADFFPFG


 


NPIHNNTMIMRRSVIDGGLRYNTERDWAEDYQFWYDVSKLGRLAYYPEALVKYRLHANQV


 


SSKYSIRQHEIAQGIQKTARNDFLQSMGFKTRFDSLEYRQIKAVAYELLEKHLPEEDFER


 


ARRFLYQCFKRTDTLPAGAWLDFAADGRMRRLFTLRQYFGILHRLLKNR


 


SEQ ID NO: 17



>NP_207619 lipooligosaccharide 5G8 epitope



biosynthesis-associated protein Lex2B


[Helicobacter pylori 26695].


MRVFAISLNQKVCDTFGLVFRDTTTLLNSINATHHQAQIFDAIYSKTFEGGLHPLVKKHL


 


HPYFITQNIKDMGITTNLISEVSKFYYALKYHAKFMSLGELGCYASHYSLWEKCIELNEA


 


ICILEDDITLKEDFKEGLDFLEKHIQELGYIRLMHLLYDASVKSEPLSHKNHEIQERVGI


 


IKAYSEGVGTQGYVITPKIAKVFLKCSRKWVVPVDTIMDATFIHGVKNLVLQPFVIADDE


 


QISTIARKEEPYSPKIALMRELHFKYLKYWQFV


 


SEQ ID NO: 18



[E.coli_WbgO_YP_003500090 putative glycosyl



transferase WbgO [Escherichia coli O55:H7


str. CB9615].


MIIDEAESAESTHPVVSVILPVNKKNPFLDEAINSILSQTFSSFEIIIVANCCTDDFYNE


 


LKHKVNDKIKLIRTNIAYLPYSLNKAIDLSNGEFIARMDSDDISHPDRFTKQVDFLKNNP


 


YVDVVGTNAIFIDDKGREINKTKLPEENLDIVKNLPYKCCIVHPSVMFRKKVIASIGGYM


 


FSNYSEDYELWNRLSLAKIKFQNLPEYLFYYRLHEGQSTAKKNLYMVMVNDLVIKMKCFF


 


LTGNINYLFGGIRTIASFIYCKYIK


 


SEQ ID NO: 19



>BAA35319 DNA-binding transcriptional dual regulator



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


MTPGGQAQIGNVDLVKQLNSAAVYRLIDQYGPISRIQIAEQSQLAPASVTKITRQLIERG


 


LIKEVDQQASTGGRRAISIVTETRNFHAIGVRLGRHDATITLFDLSSKVLAEEHYPLPER


 


TQQTLEHALLNAIAQFIDSYQRKLRELIAISVILPGLVDPDSGKIHYMPHIQVENWGLVE


 


ALEERFKVTCFVGHDIRSLALAEHYFGASQDCEDSILVRVHRGTGAGIISNGRIFIGRNG


 


NVGEIGHIQVEPLGERCHCGNFGCLETIAANAAIEQRVLNLLKQGYQSRVPLDDCTIKTI


 


CKAANKGDSLASEVIEYVGRHLGKTIAIAINLFNPQKIVIAGEITEADKVLLPAIESCIN


 


TQALKAFRTNLPVVRSELDHRSAIGAFALVKRAMLNGILLQHLLEN


 


SEQ ID NO: 20



>NP_418185 L-glutamine:D-fructose-6-phosphate



aminotransferase glmS [Escherichia coli str.


K-12 substr. MG1655].


MCGIVGAIAQRDVAEILLEGLRRLEYRGYDSAGLAVVDAEGHMTRLRRLGKVQMLAQAAE


 


EHPLHGGTGIAHTRWATHGEPSEVNAHPHVSEHIVVVHNGIIENHEPLREELKARGYTFV


 


SETDTEVIAHLVNWELKQGGTLREAVLRAIPQLRGAYGTVIMDSRHPDTLLAARSGSPLV


 


IGLGMGENFIASDQLALLPVTRRFIFLEEGDIAEITRRSVNIFDKTGAEVKRQDIESNLQ


 


YDAGDKGIYRHYMQKEIYEQPNAIKNTLTGRISHGQVDLSELGPNADELLSKVEHIQILA


 


CGTSYNSGMVSRYWFESLAGIPCDVEIASEFRYRKSAVRRNSLMITLSQSGETADTLAGL


 


RLSKELGYLGSLAICNVPGSSLVRESDLALMTNAGTEIGVASTKAFTTQLTVLLMLVAKL


 


SRLKGLDASIEHDIVHGLQALPSRIEQMLSQDKRIEALAEDFSDKHHALFLGRGDQYPIA


 


LEGALKLKEISYIHAEAYAAGELKHGPLALIDADMPVIVVAPNNELLEKLKSNIEEVRAR


 


GGQLYVFADQDAGFVSSDNMHIIEMPHVEEVIAPIFYTVPLQLLAYHVALIKGTDVDQPR


 


NLAKSVTVE


 


SEQ ID NO: 21



>BAF92026 beta-galactoside alpha-2,6-sialyltransferase



[Photobacterium sp. JT-ISH-224].


MKNFLLLTLILLTACNNSEENTQSIIKNDINKTIIDEEYVNLEPINQSNISFTKHSWVQT


 


CGTQQLLTEQNKESISLSVVAPRLDDDEKYCFDFNGVSNKGEKYITKVTLNVVAPSLEVY


 


VDHASLPTLQQLMDIIKSEEENPTAQRYIAWGRIVPTDEQMKELNITSFALINNHTPADL


 


VQEIVKQAQTKHRLNVKLSSNTAHSFDNLVPILKELNSFNNVTVTNIDLYDDGSAEYVNL


 


YNWRDTLNKTDNLKIGKDYLEDVINGINEDTSNTGTSSVYNWQKLYPANYHFLRKDYLTL


 


EPSLHELRDYIGDSLKQMQWDGFKKFNSKQQELFLSIVNFDKQKLQNEYNSSNLPNFVFT


 


GTTVWAGNHEREYYAKQQINVINNAINESSPHYLGNSYDLFFKGHPGGGIINTLIMQNYP


 


SMVDIPSKISFEVLMMTDMLPDAVAGIASSLYFTIPAEKIKFIVFTSTETITDRETALRS


 


PLVQVMIKLGIVKEENVLFWADLPNCETGVCIAV






Provided below is the DNA sequence in Genbank format of the new configuration of genes engineered at the Escherichia coli thyA locus in strains used to produce N-acetylglucosamine-containing oligosaccharides.










LOCUS
E680_thyA::2.8RBS_lacZ 5877 bp DNA linear BCT




04 MAR. 2013


 


DEFINITION
Escherichia coli str. K-12 substr. MG1655, complete genome.


 


ACCESSION
NC_000913


 


VERSION
NC_000913.2 GI:49175990


 


KEYWORDS
.


 


SOURCE
Escherichia coli str. K-12 substr. MG1655 (unknown)


 


ORGANISM
Escherichia coli str. K-12 substr. MG1655



Bacteria; Proteobacteria; Gammaproteobacteria;



Enterobacteriales;



Enterobacteriaceae; Escherichia.


 


REFERENCE
1 (bases 1 to 4639675)


 


AUTHORS
Riley,M., Abe,T., Arnaud,M.B., Berlyn,M.K., Blattner,F.R.,



Chaudhuri,R.R., Glasner,J.D., Horiuchi,T., Keseler,I.M.,



Kosuge,T.,



Mori,H., Perna,N.T., Plunkett,G. III, Rudd,K.E., Serres,M.H.,



Thomas,G.H., Thomson,N.R., Wishart,D. and Wanner,B.L.


 


TITLE
Escherichia coli K-12: a cooperatively developed annotation



snapshot--2005


 


JOURNAL
Nucleic Acids Res. 34 (1), 1-9 (2006)


 


PUBMED
16397293


 


REMARK
Publication Status: Online-Only


 


REFERENCE
2 (bases 1 to 4639675)


 


AUTHORS
Blattner,F.R., Plunkett,G. III, Bloch,C.A., Perna,N.T.,



Burland,V.,



Riley,M., Collado-Vides,J., Glasner,J.D., Rode,C.K.,



Mayhew,G.F.,



Gregor,J., Davis,N.W., Kirkpatrick,H.A., Goeden,M.A.,



Rose,D.J.,



Mau,B. and Shao,Y.


 


TITLE
The complete genome sequence of Escherichia coli K-12


 


JOURNAL
Science 277 (5331), 1453-1474 (1997)


 


PUBMED
9278503


 


REFERENCE
3 (bases 1 to 4639675)


 


AUTHORS
Arnaud,M., Berlyn,M.K.B., Blattner,F.R., Galperin,M.Y.,



Glasner,J.D., Horiuchi,T., Kosuge,T., Mori,H., Perna,N.T.,



Plunkett,G. III, Riley,M., Rudd,K.E., Serres,M.H., Thomas,G.H.



and



Wanner,B.L.


 


TITLE
Workshop on Annotation of Escherichia coli K-12


 


JOURNAL
Unpublished


 


REMARK
Woods Hole, Mass., on 14-18 Nov. 2003 (sequence



corrections)


 


REFERENCE
4 (bases 1 to 4639675)


 


AUTHORS
Glasner,J.D., Perna,N.T., Plunkett,G. III, Anderson,B.D.,



Bockhorst,J., Hu,J.C., Riley,M., Rudd,K.E. and Serres,M.H.


 


TITLE
ASAP: Escherichia coli K-12 strain MG1655 version m56


 


JOURNAL
Unpublished


 


REMARK
ASAP download 10 June 2004 (annotation updates)


 


REFERENCE
5 (bases 1 to 4639675)


 


AUTHORS
Hayashi,K., Morooka,N., Mori,H. and Horiuchi,T.


 


TITLE
A more accurate sequence comparison between genomes of



Escherichia



coli K12 W3110 and MG1655 strains


 


JOURNAL
Unpublished


 


REMARK
GenBank accessions AG613214 to AG613378 (sequence corrections)


 


REFERENCE
6 (bases 1 to 4639675)


 


AUTHORS
Perna, N. T.


 


TITLE
Escherichia coli K-12 MG1655 yqiK-rfaE intergenic region,



genomic



sequence correction


 


JOURNAL
Unpublished


 


REMARK
GenBank accession AY605712 (sequence corrections)


 


REFERENCE
7 (bases 1 to 4639675)


 


AUTHORS
Rudd,K.E.


 


TITLE
A manual approach to accurate translation start site



annotation: an



E. coli K-12 case study


 


JOURNAL
Unpublished


 


REFERENCE
8 (bases 1 to 4639675)


 


CONSRTM
NCBI Genome Project


 


TITLE
Direct Submission


 


JOURNAL
Submitted (04-MAR-2013) National Center for Biotechnology



Information, NIH, Bethesda, MD 20894, USA


 


REFERENCE
9 (bases 1 to 4639675)


 


AUTHORS
Rudd,K.E.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (06-FEB-2013) Department of Biochemistry and



Molecular



Biology, University of Miami Miller School of Medicine, 118



GautierBldg., Miami, FL 33136, USA


 


REMARK
Sequence update by submitter


 


REFERENCE
10 (bases 1 to 4639675)


 


AUTHORS
Rudd,K.E.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (24-APR-2007) Department of Biochemistry and



Molecular



Biology, University of Miami Miller School of Medicine, 118



Gautier Bldg., Miami, FL 33136, USA


 


REMARK
Annotation update from ecogene.org as a multi-database



collaboration


 


REFERENCE
11 (bases 1 to 4639675)


 


AUTHORS
Plunkett,G. III.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (07-FEB-2006) Laboratory of Genetics, University of



Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA


 


REMARK
Protein updates by submitter


 


REFERENCE
12 (bases 1 to 4639675)


 


AUTHORS
Plunkett,G. III.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (10-JUN-2004) Laboratory of Genetics, University of



Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA


 


REMARK
Sequence update by submitter


 


REFERENCE
13 (bases 1 to 4639675)


 


AUTHORS
Plunkett,G. III.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (13-OCT-1998) Laboratory of Genetics, University of



Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA


 


REFERENCE
14 (bases 1 to 4639675)


 


AUTHORS
Blattner,F.R. and Plunkett,G. III.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (02-SEP-1997) Laboratory of Genetics, University of



Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA


 


REFERENCE
15 (bases 1 to 4639675)


 


AUTHORS
Blattner,F.R. and Plunkett,G. III.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (16-JAN-1997) Laboratory of Genetics, University of



Wisconsin, 425G Henry Mall, Madison, WI 53706-1580, USA


 


COMMENT
PROVISIONAL REFSEQ: This record has not yet been subject to



final



NCBI review. The reference sequence is identical to U00096.



On Jun 24, 2004 this sequence version replaced gi:16127994.



Current U00096 annotation updates are derived from EcoGene



ecogene.org. Suggestions for updates can be sent to Dr.



Kenneth Rudd (krudd@miami.edu). These updates are being



generated



from a collaboration that also includes ASAP/ERIC,the Coli



Genetic



Stock Center, EcoliHub, EcoCyc, RegulonDB and UniProtKB/Swiss-



Prot.


 


COMPLETENESS:
full length.


 


FEATURES
Location/Qualifiers


 


gene
complement(<1..245)



/gene=′′ppdA′′



/locus_tag=′′b2826′′



/gene_synonym=′′ECK2822; JW2794′′



/db_xref=′′EcoGene:EG12081′′



/db_xref=′′GeneID:945393′′


 


CDS
complement(<1..245)



/gene=′′ppdA′′



/locus_tag=′′b2826′′



/gene_synonym=′′ECK2822; JW2794′′



/function=′′putative enzyme; Not classified′′



/GO_component=′′GO:0009289 - pilus′′



/GO_process=′′GO:0009101 - glycoprotein biosynthetic



process′′



/note=′′prepilin peptidase dependent protein A′′



/codon_start=1



/transl_table=11



/product=′′hypothetical protein′′



/protein_id=′′NP 417303.1′′



/db_xref=′′GI:16130730′′



/db_xref=′′ASAP:ABE-0009266′′



/db_xref=′′UniProtKB/Swiss-Prot:P33554′′



/db_xref=′′EcoGene:EG12081′′



/db_xref=′′GeneID:945393′′


 








(SEQ ID NO: 22)



/translation=′′MKTQRGYTLIETLVAMLILVMLSASGLYGWQYWQQSQRLWQTAS



 


QARDYLLYLREDANWHNRDHSISVIREGTLWCLVSSAAGANTCHGSSPLVFVPRWPEV


 


EMSDLTPSLAFFGLRNTAWAGHIRFKNSTGEWWLVVSPWGRLRLCQQGETEGCL′′ 


 









source
join(<1..449,4852..+225877)




/organism=′′Escherichia coli str.K-12 substr. MG1655′′



/mol_type=′′genomic DNA′′



/strain=′′K-12′′



/sub_strain=′′MG1655′′



/db_xref=′′taxon:511145′′


 


primer
346..366



/note=cagtcagtcaggcgccTTCGGGAAGGCGTCTCGAAGA (SEQ ID



NO: 23)



/label=0268-THYA-R


 


misc_feature
complement(388..394)



/feature_type=′′Hairpin loop′′



/label=Terminator


 


primer
400..449



/note=GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTag



GaaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 24)



/label=′′50bp thyA 3 prime homology′′


 


primer
400..483



/note=GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTat



taaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 25)



/label=1389-thyAKANlacZ-R-2-8


 


primer
400..483



/note=GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTCt



tCaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 26)



/label=1516-thyAKANlacZ-R-0-8


 


primer
400..483



/note=GGCGTCGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTag



GaaacctactATGACCATGATTACGGATTCAC (SEQ ID NO: 27)



/label=′′1041-thyAKANlacZ-R (4-8)′′


 


misc_feature
complement(401..407)



/feature_type=′′Hairpin loop′′



/label=Terminator


 


primer
405..472



/note=CGGCTCTGGCAGGATGTTTCGTAATTAGATAGCCACCGGCGCTTTaTTaaac 



ctactATGACCATGAT (SEQ ID NO: 28)



/label=1394-2/8-F


 


gene
complement(join(429..449,4852..4854))



/gene=′′thyA′′


 


CDS
complement(join(429..449,4852..4854))



/gene=′′thyA′′



/note=′′ECK2823:JW2795:b2827′′



/codon_start=1



/transl_table=11



/product=′′thymidylate synthetase′′



/protein_id=′′BAE76896.1′′



/db_xref=′′GI:85675643′′


 








(SEQ ID NO: 43)



/translation=′′MKQYLELMQKVLDEGTQKNDRTGTGTLSIFGHQMRFNLQDGFPL



 


VTTKRCHLRSIIHELLWFLQGDTNIAYLHENNVTIWDEWADENGDLGPVYGKQWRAWP


 


TPDGRHIDQITTVLNQLKNDPDSRRIIVSAWNVGELDKMALAPCHAFFQFYVADGKLS


 


CQLYQRSCDVFLGLPFNIASYALLVHMMAQQCDLEVGDFVWTGGDTHLYSNHMDQTHL


 


QLSREPRPLPKLIIKRKPESIFDYRFEDFEIEGYDPHPGIKAPVAI′′ 


 









RBS
450..461




/label=′′2.8 RBS′′


 


source
450..3536



/organism=′′Escherichia coli W3110′′



/mol_type=′′genomic DNA′′



/strain=′′K-12′′



/sub_strain=′′W3110′′



/db_xref=′′taxon:316407′′



/note=′′synonym: Escherichia coli str. K12 substr.



W3110′′


 


misc_feature
450..4851



/feature_type=Insertion



/note=′′originates from KanR-lacZRBS (E403)′′



/label=Insert


 


misc_feature
449′′450



/feature type=′′RBS variation site′′



/label=′′C in 0/8′′


 


misc_feature
450..453



/feature_type=′′RBS variation site′′



/label=′′CTTC in 0/8′′


 


misc_feature
451..452



/feature_type=′′RBS variation site′′



/label=′′GG in 4/8′′


 


misc_feature
451..452



/feature_type=′′RBS variation site′′



/label=′′TT in 2/8′′


 


CDS
462..3536



/gene=′′lacZ′′



/note=′′ECK0341:JW0335:b0344′′



/codon_start=1



/transl_table=11



/product=′′beta-D-galactosidase′′



/protein_id=′′BAE76126.1′′



/db_xref=′′GI:85674486′′


 








(SEQ ID NO: 29)



/translation=′′MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR



 


TDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYT


 


NVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWV


 


GYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHK


 


PTTQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFG


 


GEIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFR


 


EVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSH


 


YPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRN


 


HPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVD


 


EDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGG


 


FVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQ


 


QQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIE


 


LPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPH


 


LTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSE


 


ATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKT


 


YRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAA


 


CFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETS


 


HRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK′′


 










/label=′′wild-type lacZ+CDS′′



primer
complement(1325..1345)



/note=TTCAGACGTAGTGTGACGCGA



/label=1042-thyAlacZcheck


 


primer
2754..2776



/note=TTTCTTTCACAGATGTGGATTGG



/label=′′1395-mid lacZ-F′′


 


primer
complement(2779..2801)



/note=CGGCGTCAGCAGTTGTTTTTTAT



/label=′′1396-mid lacZ-R′′


 


mutation
2793



/label=′′C in MG1655 lacZ (silent change)′′


 


scar
complement(3549..3567)



/label=′′KD13 downstream scar sequence′′


 


source
3549..4851



/organism=′′Template plasmid pKD13′′



/mol_type=′′genomic DNA′′



/db_xref=′′taxon:170493′′


 


primer
3549..3568



/label=′′0339 Plw-P2b′′


 


repeat unit
3568..3579



/label=′′FLP site′′


 


misc_feature
complement(3568..3601)



/feature type=′′FRT site′′



/label=′′34bp FRT site′′


 


note
complement(3568..4789)



/label=′′excised region upon pCP20 introduction′′


 


repeat unit
complement(3590..3601)



/label=′′Flp site′′


 


misc_feature
complement(3602..3615)



/feature type=′′FRT site′′



/note=′′natural FRT site′′



/label=′′upstream FRT site′′


 


repeat_unit
complement(3604..3615)



/label=′′Flp site′′


 


misc_feature
complement(3628..4422)



/feature type=′′CDS (KAN resistance)′′



/note=′′kanamycin resistance′′



/codon_start=1



/transl_table=11



/product=′′Tn5 neomycin phosphotransferase′′



/protein_id=′′AAL02037.1′′



/db_xref=′′GI:15554336′′


 








(SEQ ID NO: 30)



/translation=′′MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR



 


PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL


 


LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE


 


EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY


 


QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF′′ 


 









primer
complement(3677..3696)




/label=′′0389 KD13 K4′′


 


primer_bind
3791..3810



/label=′′common priming site kt′′


 


primer
3791..3810



/label=′′0344 Wanner Kt primer′′


 


mutation
3811



/label=′′A in wt (silent change)′′


 


primer
complement(4242..4261)



/label=′′0343 Wanner K2 primer′′


 


primer_bind
4261..4280



/label=′′common priming site k2′′


 


primer_bind
4352..4371



/label=′′common priming site k1′′


 


primer
4352..4371



/label=′′0342 Wanner K1 primer′′


 


repeat_unit
4790..4801



/label=′′FLP site′′


 


scar
complement(4790..4851)



/label=′′KD13 upstream scar′′


 


misc_feature
complement(4790..4823)



/feature type=′′FRT site′′



/label=′′34bp FRT site′′


 


repeat_unit
complement(4812..4823)



/label=′′Flp site′′


 


primer
complement(4832..4851)



/label=′′0338 P4w-P1b′′


 


primer
complement(4832..4901)



/note=TCTGGGCATATCGTCGCAGCCCACAGCAACACGTTTCCTGAGGAACCATGAT



TCCGGGGATCCGTCGACC (SEQ ID NO: 31)



/label=1040-thyAKANlacZ-F


 


Site
complement(4858..4863)



/site_type=′′binding site′′



/label=′′thyA RBS′′


 


gene
complement(4861..5736)



/gene=′′lgt′′


 


CDS
complement(4861..5736)



/gene=′′lgt′′



/note=′′ECK2824:JW2796:b2828′′



/codon_start=1



/transl_table=11



/product=′′phosphatidylglycerol-prolipoprotein



diacylglyceryl transferase′′



/protein_id=′′BAE76897.1′′



/db_xref=′′GI:85675644′′


 








(SEQ ID NO: 32)



/translation=′′MTSSYLHFPEFDPVIFSIGPVALHWYGLMYLVGFIFAMWLATRR



 


ANRPGSGWTKNEVENLLYAGFLGVFLGGRIGYVLFYNFPQFMADPLYLFRVWDGGMSF


 


HGGLIGVIVVMIIFARRTKRSFFQVSDFIAPLIPFGLGAGRLGNFINGELWGRVDPNF


 


PFAMLFPGSRTEDILLLQTNPQWQSIFDTYGVLPRHPSQLYELLLEGVVLFIILNLYI


 


RKPRPMGAVSGLFLIGYGAFRIIVEFFRQPDAQFTGAWVQYISMGQILSIPMIVAGVI


 


MMVWAYRRSPQQHVS′′ 


 









promoter
complement(4957..4962)




/label=′′thyA WEAK -10′′


 


promoter
complement(4978..4983)



/label=′′thyA -35′′


 


primer
complement(5076..5099)



/note=cagtcagtcaggcgccTCCTCAACCTGTATATTCGTAAAC 



(SEQ ID NO: 33)



/label=0267-THYA-F


 


Site
complement(5739..5744)



/site type=′′binding site′′



/label=′′Igt RBS′′


 


promoter
complement(5823..5828)



/label=′′Igt -10 (strong)′′


 








ORIGIN



(SEQ ID NO: 34)










   1
GCAGCGGAAC TCACAAGGCA CCATAACGTC CCCTCCCTGA TAACGCTGAT ACTGTGGTCG



 


  61
CGGTTATGCC AGTTGGCATC TTCACGTAAA TAGAGCAAAT AGTCCCGCGC CTGGCTGGCG


 


 121
GTTTGCCATA GCCGTTGCGA CTGCTGCCAG TATTGCCAGC CATAGAGTCC ACTTGCGCTT


 


 181
AGCATGACCA AAATCAGCAT CGCGACCAGC GTTTCAATCA GCGTATAACC ACGTTGTGTT


 


 241
TTCATGCCGG CAGTATGGAG CGAGGAGAAA AAAAGACGAG GGCCAGTTTC TATTTCTTCG


 


 301
GCGCATCTTC CGGACTATTT ACGCCGTTGC AGGACGTTGC AAAATTTCGG GAAGGCGTCT


 


 361
CGAAGAATTT AACGGAGGGT AAAAAAACCG ACGCACACTG GCGTCGGCTC TGGCAGGATG


 


 421
TTTCGTAATT AGATAGCCAC CGGCGCTTTa ttaaacctac tATGACCATG ATTACGGATT


 


 481
CACTGGCCGT CGTTTTACAA CGTCGTGACT GGGAAAACCC TGGCGTTACC CAACTTAATC


 


 541
GCCTTGCAGC ACATCCCCCT TTCGCCAGCT GGCGTAATAG CGAAGAGGCC CGCACCGATC


 


 601
GCCCTTCCCA ACAGTTGCGC AGCCTGAATG GCGAATGGCG CTTTGCCTGG TTTCCGGCAC


 


 661
CAGAAGCGGT GCCGGAAAGC TGGCTGGAGT GCGATCTTCC TGAGGCCGAT ACTGTCGTCG


 


 721
TCCCCTCAAA CTGGCAGATG CACGGTTACG ATGCGCCCAT CTACACCAAC GTGACCTATC


 


 781
CCATTACGGT CAATCCGCCG TTTGTTCCCA CGGAGAATCC GACGGGTTGT TACTCGCTCA


 


 841
CATTTAATGT TGATGAAAGC TGGCTACAGG AAGGCCAGAC GCGAATTATT TTTGATGGCG


 


 901
TTAACTCGGC GTTTCATCTG TGGTGCAACG GGCGCTGGGT CGGTTACGGC CAGGACAGTC


 


 961
GTTTGCCGTC TGAATTTGAC CTGAGCGCAT TTTTACGCGC CGGAGAAAAC CGCCTCGCGG


 


1021
TGATGGTGCT GCGCTGGAGT GACGGCAGTT ATCTGGAAGA TCAGGATATG TGGCGGATGA


 


1081
GCGGCATTTT CCGTGACGTC TCGTTGCTGC ATAAACCGAC TACACAAATC AGCGATTTCC


 


1141
ATGTTGCCAC TCGCTTTAAT GATGATTTCA GCCGCGCTGT ACTGGAGGCT GAAGTTCAGA


 


1201
TGTGCGGCGA GTTGCGTGAC TACCTACGGG TAACAGTTTC TTTATGGCAG GGTGAAACGC


 


1261
AGGTCGCCAG CGGCACCGCG CCTTTCGGCG GTGAAATTAT CGATGAGCGT GGTGGTTATG


 


1321
CCGATCGCGT CACACTACGT CTGAACGTCG AAAACCCGAA ACTGTGGAGC GCCGAAATCC


 


1381
CGAATCTCTA TCGTGCGGTG GTTGAACTGC ACACCGCCGA CGGCACGCTG ATTGAAGCAG


 


1441
AAGCCTGCGA TGTCGGTTTC CGCGAGGTGC GGATTGAAAA TGGTCTGCTG CTGCTGAACG


 


1501
GCAAGCCGTT GCTGATTCGA GGCGTTAACC GTCACGAGCA TCATCCTCTG CATGGTCAGG


 


1561
TCATGGATGA GCAGACGATG GTGCAGGATA TCCTGCTGAT GAAGCAGAAC AACTTTAACG


 


1621
CCGTGCGCTG TTCGCATTAT CCGAACCATC CGCTGTGGTA CACGCTGTGC GACCGCTACG


 


1681
GCCTGTATGT GGTGGATGAA GCCAATATTG AAACCCACGG CATGGTGCCA ATGAATCGTC


 


1741
TGACCGATGA TCCGCGCTGG CTACCGGCGA TGAGCGAACG CGTAACGCGA ATGGTGCAGC


 


1801
GCGATCGTAA TCACCCGAGT GTGATCATCT GGTCGCTGGG GAATGAATCA GGCCACGGCG


 


1861
CTAATCACGA CGCGCTGTAT CGCTGGATCA AATCTGTCGA TCCTTCCCGC CCGGTGCAGT


 


1921
ATGAAGGCGG CGGAGCCGAC ACCACGGCCA CCGATATTAT TTGCCCGATG TACGCGCGCG


 


1981
TGGATGAAGA CCAGCCCTTC CCGGCTGTGC CGAAATGGTC CATCAAAAAA TGGCTTTCGC


 


2041
TACCTGGAGA GACGCGCCCG CTGATCCTTT GCGAATACGC CCACGCGATG GGTAACAGTC


 


2101
TTGGCGGTTT CGCTAAATAC TGGCAGGCGT TTCGTCAGTA TCCCCGTTTA CAGGGCGGCT


 


2161
TCGTCTGGGA CTGGGTGGAT CAGTCGCTGA TTAAATATGA TGAAAACGGC AACCCGTGGT


 


2221
CGGCTTACGG CGGTGATTTT GGCGATACGC CGAACGATCG CCAGTTCTGT ATGAACGGTC


 


2281
TGGTCTTTGC CGACCGCACG CCGCATCCAG CGCTGACGGA AGCAAAACAC CAGCAGCAGT


 


2341
TTTTCCAGTT CCGTTTATCC GGGCAAACCA TCGAAGTGAC CAGCGAATAC CTGTTCCGTC


 


2401
ATAGCGATAA CGAGCTCCTG CACTGGATGG TGGCGCTGGA TGGTAAGCCG CTGGCAAGCG


 


2461
GTGAAGTGCC TCTGGATGTC GCTCCACAAG GTAAACAGTT GATTGAACTG CCTGAACTAC


 


2521
CGCAGCCGGA GAGCGCCGGG CAACTCTGGC TCACAGTACG CGTAGTGCAA CCGAACGCGA


 


2581
CCGCATGGTC AGAAGCCGGG CACATCAGCG CCTGGCAGCA GTGGCGTCTG GCGGAAAACC


 


2641
TCAGTGTGAC GCTCCCCGCC GCGTCCCACG CCATCCCGCA TCTGACCACC AGCGAAATGG


 


2701
ATTTTTGCAT CGAGCTGGGT AATAAGCGTT GGCAATTTAA CCGCCAGTCA GGCTTTCTTT


 


2761
CACAGATGTG GATTGGCGAT AAAAAACAAC TGtTGACGCC GCTGCGCGAT CAGTTCACCC


 


2821
GTGCACCGCT GGATAACGAC ATTGGCGTAA GTGAAGCGAC CCGCATTGAC CCTAACGCCT


 


2881
GGGTCGAACG CTGGAAGGCG GCGGGCCATT ACCAGGCCGA AGCAGCGTTG TTGCAGTGCA


 


2941
CGGCAGATAC ACTTGCTGAT GCGGTGCTGA TTACGACCGC TCACGCGTGG CAGCATCAGG


 


3001
GGAAAACCTT ATTTATCAGC CGGAAAACCT ACCGGATTGA TGGTAGTGGT CAAATGGCGA


 


3061
TTACCGTTGA TGTTGAAGTG GCGAGCGATA CACCGCATCC GGCGCGGATT GGCCTGAACT


 


3121
GCCAGCTGGC GCAGGTAGCA GAGCGGGTAA ACTGGCTCGG ATTAGGGCCG CAAGAAAACT


 


3181
ATCCCGACCG CCTTACTGCC GCCTGTTTTG ACCGCTGGGA TCTGCCATTG TCAGACATGT


 


3241
ATACCCCGTA CGTCTTCCCG AGCGAAAACG GTCTGCGCTG CGGGACGCGC GAATTGAATT


 


3301
ATGGCCCACA CCAGTGGCGC GGCGACTTCC AGTTCAACAT CAGCCGCTAC AGTCAACAGC


 


3361
AACTGATGGA AACCAGCCAT CGCCATCTGC TGCACGCGGA AGAAGGCACA TGGCTGAATA


 


3421
TCGACGGTTT CCATATGGGG ATTGGTGGCG ACGACTCCTG GAGCCCGTCA GTATCGGCGG


 


3481
AATTCCAGCT GAGCGCCGGT CGCTACCATT ACCAGTTGGT CTGGTGTCAA AAATAAGCGG


 


3541
CCGCtTTATG TAGGCTGGAG CTGCTTCGAA GTTCCTATAC TTTCTAGAGA ATAGGAACTT


 


3601
CGGAATAGGA ACTTCAAGAT CCCCTTATTA GAAGAACTCG TCAAGAAGGC GATAGAAGGC


 


3661
GATGCGCTGC GAATCGGGAG CGGCGATACC GTAAAGCACG AGGAAGCGGT CAGCCCATTC


 


3721
GCCGCCAAGC TCTTCAGCAA TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC


 


3781
CACACCCAGC CGGCCACAGT CGATGAATCC tGAAAAGCGG CCATTTTCCA CCATGATATT


 


3841
CGGCAAGCAG GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA TGCGCGCCTT


 


3901
GAGCCTGGCG AACAGTTCGG CTGGCGCGAG CCCCTGATGC TCTTCGTCCA GATCATCCTG


 


3961
ATCGACAAGA CCGGCTTCCA TCCGAGTACG TGCTCGCTCG ATGCGATGTT TCGCTTGGTG


 


4021
GTCGAATGGG CAGGTAGCCG GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT


 


4081
GGATACTTTC TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC


 


4141
CAATAGCAGC CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG CGCAAGGAAC


 


4201
GCCCGTCGTG GCCAGCCACG ATAGCCGCGC TGCCTCGTCC TGCAGTTCAT TCAGGGCACC


 


4261
GGACAGGTCG GTCTTGACAA AAAGAACCGG GCGCCCCTGC GCTGACAGCC GGAACACGGC


 


4321
GGCATCAGAG CAGCCGATTG TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA


 


4381
AGCGGCCGGA GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC


 


4441
TGTCTCTTGA TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA AGAAAGCCAT


 


4501 
CCAGTTTACT TTGCAGGGCT TCCCAACCTT ACCAGAGGGC GCCCCAGCTG GCAATTCCGG


 


4561
TTCGCTTGCT GTCCATAAAA CCGCCCAGTC TAGCTATCGC CATGTAAGCC CACTGCAAGC


 


4621
TACCTGCTTT CTCTTTGCGC TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT


 


4681
CATCCGGGGT CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA


 


4741
GCCCTTGCGC CCTGAGTGCT TGCGGCAGCG TGAGCTTCAA AAGCGCTCTG AAGTTCCTAT


 


4801
ACTTTCTAGA GAATAGGAAC TTCGAACTGC AGGTCGACGG ATCCCCGGAA TCATGGTTCC


 


4861
TCAGGAAACG TGTTGCTGTG GGCTGCGACG ATATGCCCAG ACCATCATGA TCACACCCGC


 


4921
GACAATCATC GGGATGGAAA GAATTTGCCC CATGCTGATG TACTGCACCC AGGCACCGGT


 


4981
AAACTGCGCG TCGGGCTGGC GGAAAAACTC AACAATGATG CGAAACGCGC CGTAACCAAT


 


5041
CAGGAACAAA CCTGAGACAG CTCCCATTGG GCGTGGTTTA CGAATATACA GGTTGAGGAT


 


5101
AATAAACAGC ACCACACCTT CCAGCAGCAG CTCGTAAAGC TGTGATGGGT GGCGCGGCAG


 


5161
CACACCGTAA GTGTCGAAAA TGGATTGCCA CTGCGGGTTG GTTTGCAGCA GCAAAATATC


 


5221
TTCTGTACGG GAGCCAGGGA ACAGCATGGC AAACGGGAAG TTCGGGTCAA CGCGGCCCCA


 


5281
CAATTCACCG TTAATAAAGT TGCCCAGACG CCCGGCACCA AGACCAAACG GAATGAGTGG


 


5341
TGCGATAAAA TCAGAGACCT GGAAGAAGGA ACGTTTAGTA CGGCGGGCGA AGATAATCAT


 


5401
CACCACGATA ACGCCAATCA GGCCGCCGTG GAAAGACATG CCGCCGTCCC AGACACGGAA


 


5461
CAGATACAGC GGATCGGCCA TAAACTGCGG GAAATTGTAG AACAGAACAT AACCAATACG


 


5521
TCCCCCGAGG AAGACGCCGA GGAAGCCCGC ATAGAGTAAG TTTTCAACTT CATTTTTGGT


 


5581
CCAGCCGCTG CCCGGACGAT TCGCCCGTCG TGTTGCCAGC CACATTGCAA AAATGAAACC


 


5641 
CACCAGATAC ATCAGGCCGT ACCAGTGAAG CGCCACGGGT CCTATTGAGA AAATGACCGG


 


5701 
ATCAAACTCC GGAAAATGCA GATAGCTACT GGTCATCTGT CACCACAAGT TCTTGTTATT


 


5761 
TCGCTGAAAG AGAACAGCGA TTGAAATGCG CGCCGCAGGT TTCAGGCGCT CCAAAGGTGC


 


5821 
GAATAATAGC ACAAGGGGAC CTGGCTGGTT GCCGGATACC GTTAAAAGAT ATGTATA


 








//







Provided below is the DNA sequence in Genbank format of the configuration of genes at the Escherichia coli nan locus, and the details of the deletion endpoints found in engineered strains E1017 and E1018.










LOCUS
W3110_nanRATEKyhcH_region 5861 bp DNA linear BCT 




19FEB. 2009


 


DEFINITION
Escherichia coli str. K-12 substr. W3110 strain K-12.


 


ACCESSION
AC_000091


 


VERSION
AC_000091.1 GI:89106884


 


KEYWORDS
.


 


SOURCE
Escherichia coli str. K-12 substr. W3110 (unknown)


 


ORGANISM
Escherichia coli str. K-12 substr. W3110



Bacteria; Proteobacteria; Gammaproteobacteria;



Enterobacteriales;



Enterobacteriaceae; Escherichia.


 


REFERENCE
1


 


AUTHORS
Riley,M., Abe,T., Arnaud,M.B., Berlyn,M.K., Blattner,F.R.,



Chaudhuri,R.R., Glasner,J.D., Horiuchi,T., Keseler,I.M.,



Kosuge,T.,



Mori,H., Perna,N.T., Plunkett,G. III, Rudd,K.E., Serres,M.H.,



Thomas,G.H., Thomson,N.R., Wishart,D. and Wanner,B.L.


 


TITLE
Escherichia coli K-12: a cooperatively developed annotation



snapshot--2005


 


JOURNAL
Nucleic Acids Res. 34 (1), 1-9 (2006)


 


PUBMED
16397293


 


REMARK
Publication Status: Online-Only


 


REFERENCE
2 (bases 1 to 4646332)


 


AUTHORS
Hayashi,K., Morooka,N., Yamamoto,Y., Fujita,K., Isono,K.,



Choi, S., Ohtsubo,E., Baba,T., Wanner,B.L., Mori,H. and 



Horiuchi,T.


 


TITLE
Highly accurate genome sequences of Escherichia coli K-12



strains



MG1655 and W3110


 


JOURNAL
Mol. Syst. Biol. 2, 2006 (2006)


 


PUBMED
16738553


 


REFERENCE
3


 


AUTHORS
Yamamoto,Y., Aiba,H., Baba,T., Hayashi,K., Inada,T., Isono,K.,



Itoh,T., Kimura,S., Kitagawa,M., Makino,K., Miki,T.,



Mitsuhashi,N., Mizobuchi,K., Mori,H., Nakade,S., Nakamura,Y., 



Nashimoto,H.,



Oshima,T., Oyama,S., Saito,N., Sampei,G., Satoh,Y.,



Sivasundaram,S., Tagami,H., Takahashi,H., Takeda,J.,



Takemoto,K.,



Uehara,K., Wada,C., Yamagata,S. and Horiuchi,T.


 


TITLE
Construction of a contiguous 874-kb sequence of the



Escherichia



coli-K12 genome corresponding to 50.0-68.8 min on the linkage



map



and analysis of its sequence features


 


JOURNAL
DNA Res. 4 (2), 91-113 (1997)


 


PUBMED
9205837


 


REFERENCE
4


 


AUTHORS
Itoh,T., Aiba,H., Baba,T., Hayashi,K., Inada,T., Isono,K.,



Kasai,H., Kimura,S., Kitakawa,M., Kitagawa,M., Makino,K.,



Miki,T., 



Mizobuchi,K., Mori,H., Mori,T., Motomura,K., Nakade,S.,



Nakamura,Y., Nashimoto,H., Nishio,Y., Oshima,T., Saito,N.,



Sampei,G., Seki,Y., Sivasundaram,S., Tagami,H., Takeda,J.,



Takemoto,K., Wada,C., Yamamoto,Y. and Horiuchi,T.


 


TITLE
A 460-kb DNA sequence of the Escherichia coli K-12 genome



corresponding to the 40.1-50.0 min region on the linkage map


 


JOURNAL
DNA Res. 3 (6), 379-392 (1996)


 


PUBMED
9097040


 


REFERENCE
5


 


AUTHORS
Aiba,H., Baba,T., Hayashi,K., Inada,T., Isono,K., Itoh,T.,



Kasai,H., Kashimoto,K., Kimura,S., Kitakawa,M., Kitagawa,M.,



Makino,K., Miki,T., Mizobuchi,K., Mori,H., Mori,T.,



Motomura,K., 



Nakade,S., Nakamura,Y., Nashimoto,H., Nishio,Y., Oshima,T.,



Saito,N., Sampei,G., Seki,Y., Sivasundaram,S., Tagami,H.,



Takeda,J., Takemoto,K., Takeuchi,Y., Wada,C., Yamamoto,Y. and



Horiuchi,T. 


 


TITLE
A 570-kb DNA sequence of the Escherichia coli K-12 genome



corresponding to the 28.0-40.1 min region on the linkage map


 


JOURNAL
DNA Res. 3 (6), 363-377 (1996)


 


PUBMED
9097039


 


REFERENCE
6


 


AUTHORS
Arn,E.A. and Abelson,J.N.


 


TITLE
The 2′-5′ RNA ligase of Escherichia coli Purification,



cloning,



and genomic disruption


 


JOURNAL
J. Biol. Chem. 271 (49), 31145-31153 (1996)


 


PUBMED
8940112


 


REFERENCE
7


 


AUTHORS
Oshima,T., Aiba,H., Baba,T., Fujita,K., Hayashi,K., Honjo,A.,



Ikemoto,K., Inada,T., Itoh,T., Kajihara,M., Kanai,K.,



Kashimoto,K., 



Kimura,S., Kitagawa,M., Makino,K., Masuda,S., Miki,T.,



Mizobuchi,K., Mori,H., Motomura,K., Nakamura,Y., Nashimoto,H.,



Nishio,Y., Saito,N., Sampei,G., Seki,Y., Tagami,H.,



Takemoto,K.,



Wada,C., Yamamoto,Y., Yano,M. and Horiuchi,T.


 


TITLE
A 718-kb DNA sequence of the Escherichia coli K-12 genome



corresponding to the 12.7-28.0 min region on the linkage map


 


JOURNAL
DNA Res. 3 (3), 137-155 (1996)


 


PUBMED
8905232


 


REFERENCE
8


 


AUTHORS
Fujita,N., Mori,H., Yura,T. and Ishihama,A.


 


TITLE
Systematic sequencing of the Escherichia coli genome: analysis



of



the 2.4-4.1 min (110,917-193,643 bp) region


 


JOURNAL
Nucleic Acids Res. 22 (9), 1637-1639 (1994)


 


PUBMED
8202364


 


REFERENCE
9


 


AUTHORS
Janosi,L., Shimizu,I. and Kaji,A.


 


TITLE
Ribosome recycling factor (ribosome releasing factor) is



essential



for bacterial growth


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 91 (10), 4249-4253 (1994)


 


PUBMED
8183897


 


REFERENCE
10


 


AUTHORS
Allikmets,R., Gerrard,B., Court,D. and Dean,M.


 


TITLE
Cloning and organization of the abc and mdl genes of



Escherichia



coli: relationship to eukaryotic multidrug resistance


 


JOURNAL
Gene 136 (1-2), 231-236 (1993)


 


PUBMED
7904973


 


REFERENCE
11


 


AUTHORS
van Heeswijk,W.C., Rabenberg,M., Westerhoff,H.V. and Kahn,D.


 


TITLE
The genes of the glutamine synthetase adenylylation cascade



are not



regulated by nitrogen in Escherichia coli


 


JOURNAL
Mol. Microbiol. 9 (3), 443-457 (1993)


 


PUBMED
8412694


 


REFERENCE
12


 


AUTHORS
Zhao,S., Sandt,C.H., Feulner,G., Vlazny,D.A., Gray,J.A. and



Hill,C.W.


 


TITLE
Rhs elements of Escherichia coli K-12: complex composites of



shared



and unique components that have different evolutionary



histories


 


JOURNAL
J. Bacteriol. 175 (10), 2799-2808 (1993)


 


PUBMED
8387990


 


REFERENCE
13


 


AUTHORS
Yamada,M., Asaoka,S., Saier,M.H. Jr. and Yamada,Y.


 


TITLE
Characterization of the gcd gene from Escherichia coli K-12



W3110



and regulation of its expression


 


JOURNAL
J. Bacteriol. 175 (2), 568-571 (1993)


 


PUBMED
8419307


 


REFERENCE
14


 


AUTHORS
Cormack,R.S. and Mackie,G.A.


 


TITLE
Structural requirements for the processing of Escherichia coli



ribosomal RNA by RNase E in vitro


 


JOURNAL
J. Mol. Biol. 228 (4), 1078-1090 (1992)


 


PUBMED
1474579


 


REFERENCE
15


 


AUTHORS
Gervais,F.G. and Drapeau,G.R.


 


TITLE
Identification, cloning, and characterization of rcsF, a new



regulator gene for exopolysaccharide synthesis that suppresses



the



division mutation ftsZ84 in Escherichia coli K-12


 


JOURNAL
J. Bacteriol. 174 (24), 8016-8022 (1992)


 


PUBMED
1459951


 


REFERENCE
16


 


AUTHORS
Yamanaka,K., Ogura,T., Niki,H. and Hiraga,S.


 


TITLE
Identification and characterization of the smbA gene, a



suppressor



of the mukB null mutant of Escherichia coli


 


JOURNAL
J. Bacteriol. 174 (23), 7517-7526 (1992)


 


PUBMED
1447125


 


REFERENCE
17


 


AUTHORS
Condon,C., Philips,J., Fu,Z.Y., Squires,C. and Squires,C.L.


 


TITLE
Comparison of the expression of the seven ribosomal RNA



operons in



Escherichia coli


 


JOURNAL
EMBO J. 11 (11), 4175-4185 (1992)


 


PUBMED
1396599


 


REFERENCE
18


 


AUTHORS
Arnqvist,A., Olsen,A., Pfeifer,J., Russell,D.G. and Normark,S.


 


TITLE
The Crl protein activates cryptic genes for curli formation



and



fibronectin binding in Escherichia coli HB101


 


JOURNAL
Mol. Microbiol. 6 (17), 2443-2452 (1992)


 


PUBMED
1357528


 


REFERENCE
19


 


AUTHORS
Talarico,T.L., Ray,P.H., Dev,I.K., Merrill,B.M. and



Dallas,W.S.


 


TITLE
Cloning, sequence analysis, and overexpression of Escherichia



coli



folK, the gene coding for



7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase


 


JOURNAL
J. Bacteriol. 174 (18), 5971-5977 (1992)


 


PUBMED
1325970


 


REFERENCE
20


 


AUTHORS
Li,S.J. and Cronan,J.E. Jr.


 


TITLE
The genes encoding the two carboxyltransferase subunits of



Escherichia coli acetyl-CoA carboxylase


 


JOURNAL
J. Biol. Chem. 267 (24), 16841-16847 (1992)


 


PUBMED
1355089


 


REFERENCE
21


 


AUTHORS
Yura,T., Mori,H., Nagai,H., Nagata,T., Ishihama,A., Fujita,N.,



Isono,K., Mizobuchi,K. and Nakata,A.


 


TITLE
Systematic sequencing of the Escherichia coli genome: analysis



of



the 0-2.4 min region


 


JOURNAL
Nucleic Acids Res. 20 (13), 3305-3308 (1992)


 


PUBMED
1630901


 


REFERENCE
22


 


AUTHORS
Ghosh,S.K., Biswas,S.K., Paul,K. and Das,J.


 


TITLE
Nucleotide and deduced amino acid sequence of the recA gene of



Vibrio cholerae


 


JOURNAL
Nucleic Acids Res. 20 (2), 372 (1992)


 


PUBMED
1741267


 


REFERENCE
23


 


AUTHORS
Smallshaw,J.E. and Kelln,R.A.


 


TITLE
Cloning, nucleotide sequence and expression of the Escherichia



coli



K-12 pyrH gene encoding UMP kinase


 


JOURNAL
Genetics (Life Sci. Adv.) 11, 59-65 (1992)


 


REFERENCE
24


 


AUTHORS
O′Neill,G.P., Grygorczyk,R., Adam,M. and Ford-Hutchinson,A.W.


 


TITLE
The nucleotide sequence of a voltage-gated chloride channel



from



the electric organ of Torpedo californica


 


JOURNAL
Biochim. Biophys. Acta 1129 (1), 131-134 (1991)


 


PUBMED
1721838


 


REFERENCE
25


 


AUTHORS
Kajie,S., Ideta,R., Yamato,I. and Anraku,Y.


 


TITLE
Molecular cloning and DNA sequence of dniR, a gene affecting



anaerobic expression of the Escherichia coli hexaheme nitrite



reductase


 


JOURNAL
FEMS Microbiol. Lett. 67 (2), 205-211 (1991)


 


PUBMED
1663890


 


REFERENCE
26


 


AUTHORS
Hershfield,M.S., Chaffee,S., Koro-Johnson,L., Mary,A.,



Smith,A.A.



and Short,S.A.


 


TITLE
Use of site-directed mutagenesis to enhance the epitope-



shielding



effect of covalent modification of proteins with polyethylene



glycol


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 88 (16), 7185-7189 (1991)


 


PUBMED
1714590


 


REFERENCE
27


 


AUTHORS
Shimizu,I. and Kaji,A.


 


TITLE
Identification of the promoter region of the ribosome-



releasing



factor cistron (frr)


 


JOURNAL
J. Bacteriol. 173 (16), 5181-5187 (1991)


 


PUBMED
1860827


 


REFERENCE
28


 


AUTHORS
Poulsen,L.K., Refn,A., Molin,S. and Andersson,P.


 


TITLE
The gef gene from Escherichia coli is regulated at the level



of



translation


 


JOURNAL
Mol. Microbiol. 5 (7), 1639-1648 (1991)


 


PUBMED
1943701


 


REFERENCE
29


 


AUTHORS
Poulsen,L.K., Refn,A., Molin,S. and Andersson,P.


 


TITLE
Topographic analysis of the toxic Gef protein from Escherichia



coli


 


JOURNAL
Mol. Microbiol. 5 (7), 1627-1637 (1991)


 


PUBMED
1943700


 


REFERENCE
30


 


AUTHORS
Kawamukai,M., Utsumi,R., Takeda,K., Higashi,A., Matsuda,H.,



Choi,Y.L. and Komano,T.


 


TITLE
Nucleotide sequence and characterization of the sfsl gene:



sfsl is



Involved in CRP*-dependent mal gene expression in Escherichia



coli


 


JOURNAL
J. Bacteriol. 173 (8), 2644-2648 (1991)


 


PUBMED
2013578


 


REFERENCE
31


 


AUTHORS
Hulton,C.S., Higgins,C.F. and Sharp,P.M.


 


TITLE
ERIC sequences: a novel family of repetitive elements in the



genomes of Escherichia coli, Salmonella typhimurium and other



enterobacteria


 


JOURNAL
Mol. Microbiol. 5 (4), 825-834 (1991)


 


PUBMED
1713281


 


REFERENCE
32


 


AUTHORS
Munro,A.W., Ritchie,G.Y., Lamb,A.J., Douglas,R.M. and



Booth, I.R.


 


TITLE
The cloning and DNA sequence of the gene for the



glutathione-regulated potassium-efflux system KefC of



Escherichia



coli


 


JOURNAL
Mol. Microbiol. 5 (3), 607-616 (1991)


 


PUBMED
2046548


 


REFERENCE
33


 


AUTHORS
Arigoni,F., Kaminski,P.A., Hennecke,H. and Elmerich,C.


 


TITLE
Nucleotide sequence of the fixABC region of Azorhizobium



caulinodans ORS571: similarity of the fixB product with



eukaryotic



flavoproteins, characterization of fixX, and identification of



nifW


 


JOURNAL
Mol. Gen. Genet. 225 (3), 514-520 (1991)


 


PUBMED
1850088


 


REFERENCE
34


 


AUTHORS
Mattick,J.S., Anderson,B.J., Cox,P.T., Dalrymple,B.P.,



Bills,M.M.,



Hobbs,M. and Egerton,J.R.


 


TITLE
Gene sequences and comparison of the fimbrial subunits



representative of Bacteroides nodosus serotypes A to I: class



I and



class II strains


 


JOURNAL
Mol. Microbiol. 5 (3), 561-573 (1991)


 


PUBMED
1675419


 


REFERENCE
35


 


AUTHORS
Company,M., Arenas,J. and Abelson,J.


 


TITLE
Requirement of the RNA helicase-like protein PRP22 for release



of



messenger RNA from spliceosomes


 


JOURNAL
Nature 349 (6309), 487-493 (1991)


 


PUBMED
1992352


 


REFERENCE
36


 


AUTHORS
Umeda,M. and Ohtsubo,E.


 


TITLE
Four types of IS1 with differences in nucleotide sequence



reside in



the Escherichia coli K-12 chromosome


 


JOURNAL
Gene 98 (1), 1-5 (1991)


 


PUBMED
1849492


 


REFERENCE
37


 


AUTHORS
Hirvas,L., Koski,P. and Vaara,M.


 


TITLE
The ompH gene of Yersinia enterocolitica: cloning, sequencing,



expression, and comparison with known enterobacterial ompH



sequences


 


JOURNAL
J. Bacteriol. 173 (3), 1223-1229 (1991)


 


PUBMED
1991717


 


REFERENCE
38


 


AUTHORS
Bouvier,J. and Stragier,P.


 


TITLE
Nucleotide sequence of the lsp-dapB interval in Escherichia



coli


 


JOURNAL
Nucleic Acids Res. 19 (1), 180 (1991)


 


PUBMED
2011499


 


REFERENCE
39


 


AUTHORS
Dicker,I.B. and Seetharam,S.


 


TITLE
Cloning and nucleotide sequence of the firA gene and the



firA200(Ts) allele from Escherichia coli


 


JOURNAL
J. Bacteriol. 173 (1), 334-344 (1991)


 


PUBMED
1987124


 


REFERENCE
40


 


AUTHORS
Grimm,B., Bull,A. and Breu,V.


 


TITLE
Structural genes of glutamate 1-semialdehyde aminotransferase



for



porphyrin synthesis in a cyanobacterium and Escherichia coli


 


JOURNAL
Mol. Gen. Genet. 225 (1), 1-10 (1991)


 


PUBMED
1900346


 


REFERENCE
41


 


AUTHORS
Allen,B.L., Gerlach,G.F. and Clegg,S.


 


TITLE
Nucleotide sequence and functions of mrk determinants



necessary for



expression of type 3 fimbriae in Klebsiella pneumoniae


 


JOURNAL
J. Bacteriol. 173 (2), 916-920 (1991)


 


PUBMED
1670938


 


REFERENCE
42


 


AUTHORS
Chen,H., Lawrence,C.B., Bryan,S.K. and Moses,R.E.


 


TITLE
Aphidicolin inhibits DNA polymerase II of Escherichia coli, an



alpha-like DNA polymerase


 


JOURNAL
Nucleic Acids Res. 18 (23), 7185-7186 (1990)


 


PUBMED
2124684


 


REFERENCE
43


 


AUTHORS
Mallonee,D.H., White,W.B. and Hylemon,P.B.


 


TITLE
Cloning and sequencing of a bile acid-inducible operon from



Eubacterium sp. strain VPI 12708


 


JOURNAL
J. Bacteriol. 172 (12), 7011-7019 (1990)


 


PUBMED
2254270


 


REFERENCE
44


 


AUTHORS
Young,C., Collins-Emerson,J.M., Terzaghi,E.A. and Scott,D.B.


 


TITLE
Nucleotide sequence of Rhizobium loti nodl


 


JOURNAL
Nucleic Acids Res. 18 (22), 6691 (1990)


 


PUBMED
2251131


 


REFERENCE
45


 


AUTHORS
Chen,H., Sun,Y., Stark,T., Beattie,W. and Moses,R.E.


 


TITLE
Nucleotide sequence and deletion analysis of the polB gene of



Escherichia coli


 


JOURNAL
DNA Cell Biol. 9 (9), 631-635 (1990)


 


PUBMED
2261080


 


REFERENCE
46


 


AUTHORS
Eriani,G., Delarue,M., Poch,O., Gangloff,J. and Moras,D.


 


TITLE
Partition of tRNA synthetases into two classes based on



mutually



exclusive sets of sequence motifs


 


JOURNAL
Nature 347 (6289), 203-206 (1990)


 


PUBMED
2203971


 


REFERENCE
47


 


AUTHORS
Showalter,R.E. and Silverman,M.R.


 


TITLE
Nucleotide sequence of a gene, hpt, for hypoxanthine



phosphoribosyltransferase from Vibrio harveyi


 


JOURNAL
Nucleic Acids Res. 18 (15), 4621 (1990)


 


PUBMED
2388850


 


REFERENCE
48


 


AUTHORS
Martin-Verstraete,I., Debarbouille,M., Klier,A. and



Rapoport, G.


 


TITLE
Levanase operon of Bacillus subtilis includes a fructose-



specific



phosphotransferase system regulating the expression of the



operon


 


JOURNAL
J. Mol. Biol. 214 (3), 657-671 (1990)


 


PUBMED
2117666


 


REFERENCE
49


 


AUTHORS
Henrich,B., Monnerjahn,U. and Plapp,R.


 


TITLE
Peptidase D gene (pepD) of Escherichia coli K-12: nucleotide



sequence, transcript mapping, and comparison with other



peptidase



genes


 


JOURNAL
J. Bacteriol. 172 (8), 4641-4651 (1990)


 


PUBMED
1695895


 


REFERENCE
50


 


AUTHORS
Nunn,D., Bergman,S. and Lory,S.


 


TITLE
Products of three accessory genes, pilB, pilC, and pilD, are



required for biogenesis of Pseudomonas aeruginosa pili


 


JOURNAL
J. Bacteriol. 172 (6), 2911-2919 (1990)


 


PUBMED
1971619


 


REFERENCE
51


 


AUTHORS
Rosenthal,E.R. and Calvo,J.M.


 


TITLE
The nucleotide sequence of leuC from Salmonella typhimurium


 


JOURNAL
Nucleic Acids Res. 18 (10), 3072 (1990)


 


PUBMED
2190189


 


REFERENCE
52


 


AUTHORS
Kang,P.J. and Craig,E.A.


 


TITLE
Identification and characterization of a new Escherichia coli



gene



that is a dosage-dependent suppressor of a dnaK deletion



mutation


 


JOURNAL
J. Bacteriol. 172 (4), 2055-2064 (1990)


 


PUBMED
2180916


 


REFERENCE
53


 


AUTHORS
Wurgler,S.M. and Richardson,C.C.


 


TITLE
Structure and regulation of the gene for dGTP



triphosphohydrolase



from Escherichia coli


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 87 (7), 2740-2744 (1990)


 


PUBMED
2157212


 


REFERENCE
54


 


AUTHORS
Schaaff,I., Hohmann,S. and Zimmermann,F.K.


 


TITLE
Molecular analysis of the structural gene for yeast



transaldolase


 


JOURNAL
Eur. J. Biochem. 188 (3), 597-603 (1990)


 


PUBMED
2185015


 


REFERENCE
55


 


AUTHORS
Ricca,E. and Calvo,J.M.


 


TITLE
The nucleotide sequence of leuA from Salmonella typhimurium


 


JOURNAL
Nucleic Acids Res. 18 (5), 1290 (1990)


 


PUBMED
2181403


 


REFERENCE
56


 


AUTHORS
Honore,N. and Cole,S.T.


 


TITLE
Nucleotide sequence of the aroP gene encoding the general



aromatic



amino acid transport protein of Escherichia coli K-12:



homology



with yeast transport proteins


 


JOURNAL
Nucleic Acids Res. 18 (3), 653 (1990)


 


PUBMED
2408019


 


REFERENCE
57


 


AUTHORS
Angerer,A., Gaisser,S. and Braun,V.


 


TITLE
Nucleotide sequences of the sfuA, sfuB, and sfuC genes of



Serratia



marcescens suggest a periplasmic-binding-protein-dependent



iron



transport mechanism


 


JOURNAL
J. Bacteriol. 172 (2), 572-578 (1990)


 


PUBMED
2404942


 


REFERENCE
58


 


AUTHORS
Surin,B.P., Watson,J.M., Hamilton,W.D., Economou,A. and



Downie, J.A.


 


TITLE
Molecular characterization of the nodulation gene, nodT, from



two



biovars of Rhizobium leguminosarum


 


JOURNAL
Mol. Microbiol. 4 (2), 245-252 (1990)


 


PUBMED
2338917


 


REFERENCE
59


 


AUTHORS
Zhou,Z. and Syvanen,M.


 


TITLE
Identification and sequence of the drpA gene from Escherichia



coli


 


JOURNAL
J. Bacteriol. 172 (1), 281-286 (1990)


 


PUBMED
1688424


 


REFERENCE
60


 


AUTHORS
Roncero,M.I., Jepsen,L.P., Stroman,P. and van Heeswijck,R.


 


TITLE
Characterization of a leuA gene and an ARS element from Mucor



circinelloides


 


JOURNAL
Gene 84 (2), 335-343 (1989)


 


PUBMED
2693214


 


REFERENCE
61


 


AUTHORS
Ichikawa,S. and Kaji,A.


 


TITLE
Molecular cloning and expression of ribosome releasing factor


 


JOURNAL
J. Biol. Chem. 264 (33), 20054-20059 (1989)


 


PUBMED
2684966


 


REFERENCE
62


 


AUTHORS
Minami-Ishii,N., Taketani,S., Osumi,T. and Hashimoto,T.


 


TITLE
Molecular cloning and sequence analysis of the cDNA for rat



mitochondrial enoyl-CoA hydratase. Structural and evolutionary



relationships linked to the bifunctional enzyme of the



peroxisomal



beta-oxidation system


 


JOURNAL
Eur. J. Biochem. 185 (1), 73-78 (1989)


 


PUBMED
2806264


 


REFERENCE
63


 


AUTHORS
Matsubara,Y., Indo,Y., Naito,E., Ozasa,H., Glassberg,R.,



Vockley,J., Ikeda,Y., Kraus,J. and Tanaka,K.


 


TITLE
Molecular cloning and nucleotide sequence of cDNAs encoding



the



precursors of rat long chain acyl-coenzyme A, short chain



acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases.



Sequence



homology of four enzymes of the acyl-CoA dehydrogenase family


 


JOURNAL
J. Biol. Chem. 264 (27), 16321-16331 (1989)


 


PUBMED
2777793


 


REFERENCE
64


 


AUTHORS
Roa,B.B., Connolly,D.M. and Winkler,M.E.


 


TITLE
Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-



apaH



operon of Escherichia coli K-12


 


JOURNAL
J. Bacteriol. 171 (9), 4767-4777 (1989)


 


PUBMED
2670894


 


REFERENCE
65


 


AUTHORS
Lindquist,S., Galleni,M., Lindberg,F. and Normark,S.


 


TITLE
Signalling proteins in enterobacterial AmpC beta-lactamase



regulation


 


JOURNAL
Mol. Microbiol. 3 (8), 1091-1102 (1989)


 


PUBMED
2691840


 


REFERENCE
66


 


AUTHORS
Xie,Q.W., Tabor,C.W. and Tabor,H.


 


TITLE
Spermidine biosynthesis in Escherichia coli: promoter and



termination regions of the speED operon


 


JOURNAL
J. Bacteriol. 171 (8), 4457-4465 (1989)


 


PUBMED
2666401


 


REFERENCE
67


 


AUTHORS
Sato,S., Nakada,Y. and Shiratsuchi,A.


 


TITLE
IS421, a new insertion sequence in Escherichia coli


 


JOURNAL
FEBS Lett. 249 (1), 21-26 (1989)


 


PUBMED
2542093


 


REFERENCE
68


 


AUTHORS
Liu,J.D. and Parkinson,J.S.


 


TITLE
Genetics and sequence analysis of the pcnB locus, an



Escherichia



coli gene involved in plasmid copy number control


 


JOURNAL
J. Bacteriol. 171 (3), 1254-1261 (1989)


 


PUBMED
2537812


 


REFERENCE
69


 


AUTHORS
Henrich,B., Schroeder,U., Frank,R.W. and Plapp,R.


 


TITLE
Accurate mapping of the Escherichia coli pepD gene by sequence



analysis of its 5′ flanking region


 


JOURNAL
Mol. Gen. Genet. 215 (3), 369-373 (1989)


 


PUBMED
2651887


 


REFERENCE
70


 


AUTHORS
Lipinska,B., Sharma,S. and Georgopoulos,C.


 


TITLE
Sequence analysis and regulation of the htrA gene of



Escherichia



coli: a sigma 32-independent mechanism of heat-inducible



transcription


 


JOURNAL
Nucleic Acids Res. 16 (21), 10053-10067 (1988)


 


PUBMED
3057437


 


REFERENCE
71


 


AUTHORS
Sung,Y.C. and Fuchs,J.A.


 


TITLE
Characterization of the cyn operon in Escherichia coli K12


 


JOURNAL
J. Biol. Chem. 263 (29), 14769-14775 (1988)


 


PUBMED
3049588


 


REFERENCE
72


 


AUTHORS
Lozoya,E., Hoffmann,H., Douglas,C., Schulz,W., Scheel,D. and



Hahlbrock,K.


 


TITLE
Primary structures and catalytic properties of isoenzymes



encoded



by the two 4-coumarate: CoA ligase genes in parsley


 


JOURNAL
Eur. J. Biochem. 176 (3), 661-667 (1988)


 


PUBMED
3169018


 


REFERENCE
73


 


AUTHORS
Andrews,S.C. and Guest,J.R.


 


TITLE
Nucleotide sequence of the gene encoding the GMP reductase of



Escherichia coli K12


 


JOURNAL
Biochem. J. 255 (1), 35-43 (1988)


 


PUBMED
2904262


 


REFERENCE
74


 


AUTHORS
Jaiswal,A.K., McBride,O.W., Adesnik,M. and Nebert,D.W.


 


TITLE
Human dioxin-inducible cytosolic NAD(P)H:menadione



oxidoreductase.



cDNA sequence and localization of gene to chromosome 16


 


JOURNAL
J. Biol. Chem. 263 (27), 13572-13578 (1988)


 


PUBMED
2843525


 


REFERENCE
75


 


AUTHORS
Karpel,R., Olami,Y., Taglicht,D., Schuldiner,S. and Padan,E.


 


TITLE
Sequencing of the gene ant which affects the Na+/H+ antiporter



activity in Escherichia coli


 


JOURNAL
J. Biol. Chem. 263 (21), 10408-10414 (1988)


 


PUBMED
2839489


 


REFERENCE
76


 


AUTHORS
Mellano,M.A. and Cooksey,D.A.


 


TITLE
Nucleotide sequence and organization of copper resistance



genes



from Pseudomonas syringae pv. tomato


 


JOURNAL
J. Bacteriol. 170 (6), 2879-2883 (1988)


 


PUBMED
3372485


 


REFERENCE
77


 


AUTHORS
Coleman,J. and Raetz,C.R.


 


TITLE
First committed step of lipid A biosynthesis in Escherichia



coli:



sequence of the 1pxA gene


 


JOURNAL
J. Bacteriol. 170 (3), 1268-1274 (1988)


 


PUBMED
3277952


 


REFERENCE
78


 


AUTHORS
Gebhard,W., Schreitmuller,T., Hochstrasser,K. and Wachter,E.


 


TITLE
Complementary DNA and derived amino acid sequence of the



precursor



of one of the three protein components of the inter-alpha-



trypsin



Inhibitor complex


 


JOURNAL
FEBS Lett. 229 (1), 63-67 (1988)


 


PUBMED
2450046


 


REFERENCE
79


 


AUTHORS
Tomasiewicz,H.G. and McHenry,C.S.


 


TITLE
Sequence analysis of the Escherichia coli dnaE gene


 


JOURNAL
J. Bacteriol. 169 (12), 5735-5744 (1987)


 


PUBMED
3316192


 


REFERENCE
80


 


AUTHORS
Crowell,D.N., Reznikoff,W.S. and Raetz,C.R.


 


TITLE
Nucleotide sequence of the Escherichia coli gene for lipid A



disaccharide synthase


 


JOURNAL
J. Bacteriol. 169 (12), 5727-5734 (1987)


 


PUBMED
2824445


 


REFERENCE
81


 


AUTHORS
Tabor,C.W. and Tabor,H.


 


TITLE
The speEspeD operon of Escherichia coli. Formation and



processing



of a proenzyme form of S-adenosylmethionine decarboxylase


 


JOURNAL
J. Biol. Chem. 262 (33), 16037-16040 (1987)


 


PUBMED
3316212


 


REFERENCE
82


 


AUTHORS
Nonet,M.L., Marvel,C.C. and Tolan,D.R.


 


TITLE
The hisT-purF region of the Escherichia coli K-12 chromosome.



Identification of additional genes of the hisT and purF



operons


 


JOURNAL
J. Biol. Chem. 262 (25), 12209-12217 (1987)


 


PUBMED
3040734


 


REFERENCE
83


 


AUTHORS
Coulton,J.W., Mason,P. and Allatt,D.D.


 


TITLE
fhuC and fhuD genes for iron (III)-ferrichrome transport into



Escherichia coli K-12


 


JOURNAL
J. Bacteriol. 169 (8), 3844-3849 (1987)


 


PUBMED
3301821


 


REFERENCE
84


 


AUTHORS
Horiuchi,T., Nagasawa,T., Takano,K. and Sekiguchi,M.


 


TITLE
A newly discovered tRNA(lAsp) gene (aspV) of Escherichia coli



K12


 


JOURNAL
Mol. Gen. Genet. 206 (2), 356-357 (1987)


 


PUBMED
3295485


 


REFERENCE
85


 


AUTHORS
Ben-Bassat,A., Bauer,K., Chang,S.Y., Myambo,K., Boosman,A. and



Chang, S.


 


TITLE
Processing of the initiation methionine from proteins:



properties



of the Escherichia coli methionine aminopeptidase and its gene



structure


 


JOURNAL
J. Bacteriol. 169 (2), 751-757 (1987)


 


PUBMED
3027045


 


REFERENCE
86


 


AUTHORS
Gronger,P., Manian,S.S., Reilander,H., O′Connell,M.,



Priefer,U.B.



and Puhler,A.


 


TITLE
Organization and partial sequence of a DNA region of the



Rhizobium



leguminosarum symbiotic plasmid pRL6JI containing the genes



fixABC,



nifA, nifB and a novel open reading frame


 


JOURNAL
Nucleic Acids Res. 15 (1), 31-49 (1987)


 


PUBMED
3029674


 


REFERENCE
87


 


AUTHORS
Richardson,K.K., Richardson,F.C., Crosby,R.M., Swenberg,J.A.



and



Skopek,T.R.


 


TITLE
DNA base changes and alkylation following in vivo exposure of



Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-



nitrosourea


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 84 (2), 344-348 (1987)


 


PUBMED
3540961


 


REFERENCE
88


 


AUTHORS
Chye,M.L. and Pittard,J.


 


TITLE
Transcription control of the aroP gene in Escherichia coli K-



12:



analysis of operator mutants


 


JOURNAL
J. Bacteriol. 169 (1), 386-393 (1987)


 


PUBMED
3025182


 


REFERENCE
89


 


AUTHORS
Blanchin-Roland, S., Blanquet,S., Schmitter,J.M. and Fayat,G.


 


TITLE
The gene for Escherichia coli diadenosine tetraphosphatase is



located immediately clockwise to folA and forms an operon with



ksgA


 


JOURNAL
Mol. Gen. Genet. 205 (3), 515-522 (1986)


 


PUBMED
3031429


 


REFERENCE
90


 


AUTHORS
Takano,K., Nakabeppu,Y., Maki,H., Horiuchi,T. and Sekiguchi,M.


 


TITLE
Structure and function of dnaQ and mutD mutators of



Escherichia



coli


 


JOURNAL
Mol. Gen. Genet. 205 (1), 9-13 (1986)


 


PUBMED
3540531


 


REFERENCE
91


 


AUTHORS
Mackie,G.A.


 


TITLE
Structure of the DNA distal to the gene for ribosomal protein



S20



in Escherichia coli K12: presence of a strong terminator and



an IS1



element


 


JOURNAL
Nucleic Acids Res. 14 (17), 6965-6981 (1986)


 


PUBMED
2429258


 


REFERENCE
92


 


AUTHORS
Koster,W. and Braun,V.


 


TITLE
Iron hydroxamate transport of Escherichia coli: nucleotide



sequence



of the fhuB gene and identification of the protein


 


JOURNAL
Mol. Gen. Genet. 204 (3), 435-442 (1986)


 


PUBMED
3020380


 


REFERENCE
93


 


AUTHORS
Breton,R., Sanfacon,H., Papayannopoulos,I., Biemann,K. and



Lapointe, J.


 


TITLE
Glutamyl-tRNA synthetase of Escherichia coli. Isolation and



primary



structure of the gltX gene and homology with other aminoacyl-



tRNA



synthetases


 


JOURNAL
J. Biol. Chem. 261 (23), 10610-10617 (1986)


 


PUBMED
3015933


 


REFERENCE
94


 


AUTHORS
Birnbaum,M.J., Haspel,H.C. and Rosen,O.M.


 


TITLE
Cloning and characterization of a cDNA encoding the rat brain



glucose-transporter protein


 


JOURNAL
Proc. Natl. Acad. Sol. U.S.A. 83 (16), 5784-5788 (1986)


 


PUBMED
3016720


 


REFERENCE
95


 


AUTHORS
Cox,E.C. and Horner,D.L.


 


TITLE
DNA sequence and coding properties of mutD(dnaQ) a dominant



Escherichia coli mutator gene


 


JOURNAL
J. Mol. Biol. 190 (1), 113-117 (1986)


 


PUBMED
3023634


 


REFERENCE
96


 


AUTHORS
Ohki,M., Tamura,F., Nishimura,S. and Uchida,H.


 


TITLE
Nucleotide sequence of the Escherichia coli dnaJ gene and



purification of the gene product


 


JOURNAL
J. Biol. Chem. 261 (4), 1778-1781 (1986)


 


PUBMED
3003084


 


REFERENCE
97


 


AUTHORS
Coulton,J.W., Mason,P., Cameron,D.R., Carmel,G., Jean,R. and



Rode, H.N.


 


TITLE
Protein fusions of beta-galactosidase to the ferrichrome-iron



receptor of Escherichia coli K-12


 


JOURNAL
J. Bacteriol. 165 (1), 181-192 (1986)


 


PUBMED
3079747


 


REFERENCE
98


 


AUTHORS
Lee,N., Gielow,W., Martin,R., Hamilton,E. and Fowler,A.


 


TITLE
The organization of the araBAD operon of Escherichia coli


 


JOURNAL
Gene 47 (2-3), 231-244 (1986)


 


PUBMED
3549454


 


REFERENCE
99


 


AUTHORS
Sekiguchi,T., Ortega-Cesena,J., Nosoh,Y., Ohashi,S., Tsuda,K.



and



Kanaya,S.


 


TITLE
DNA and amino-acid sequences of 3-isopropylmalate



dehydrogenase of



Bacillus coagulans. Comparison with the enzymes of



Saccharomycescerevisiae and Thermus thermophilus


 


JOURNAL
Biochim. Biophys. Acta 867, 36-44 (1986)


 


REFERENCE
100


 


AUTHORS
Chong,P., Hui,I., Loo,T. and Gillam,S.


 


TITLE
Structural analysis of a new GC-specific insertion element



IS186


 


JOURNAL
FEBS Lett. 192 (1), 47-52 (1985)


 


PUBMED
2996940


 


REFERENCE
101


 


AUTHORS
Icho,T., Sparrow,C.P. and Raetz,C.R.


 


TITLE
Molecular cloning and sequencing of the gene for CDP-



diglyceride



synthetase of Escherichia coli


 


JOURNAL
J. Biol. Chem. 260 (22), 12078-12083 (1985)


 


PUBMED
2995358


 


REFERENCE
102


 


AUTHORS
Nomura,T., Aiba,H. and Ishihama,A.


 


TITLE
Transcriptional organization of the convergent overlapping



dnaQ-rnh



genes of Escherichia coli


 


JOURNAL
J. Biol. Chem. 260 (11), 7122-7125 (1985)


 


PUBMED
2987244


 


REFERENCE
103


 


AUTHORS
Kamio,Y., Lin,C.K., Regue,M. and Wu,H.C.


 


TITLE
Characterization of the ileS-lsp operon in Escherichia coli.



Identification of an open reading frame upstream of the ileS



gene



and potential promoter(s) for the ileS-lsp operon


 


JOURNAL
J. Biol. Chem. 260 (9), 5616-5620 (1985)


 


PUBMED
2985604


 


REFERENCE
104


 


AUTHORS
Cowing,D.W., Bardwell,J.C., Craig,E.A., Woolford,C.,



Hendrix, R.W.



and Gross,C.A.


 


TITLE
Consensus sequence for Escherichia coli heat shock gene



promoters


 


JOURNAL
Proc. Natl. Acad. Sol. U.S.A. 82 (9), 2679-2683 (1985)


 


PUBMED
3887408


 


REFERENCE
105


 


AUTHORS
Broome-Smith,J.K., Edelman,A., Yousif,S. and Spratt,B.G.


 


TITLE
The nucleotide sequences of the ponA and ponB genes encoding



penicillin-binding protein 1A and 1B of Escherichia coli K12


 


JOURNAL
Eur. J. Biochem. 147 (2), 437-446 (1985)


 


PUBMED
3882429


 


REFERENCE
106


 


AUTHORS
Becerril,B., Valle,F., Merino,E., Riba,L. and Bolivar,F.


 


TITLE
Repetitive extragenic palindromic (REP) sequences in the



Escherichia coli gdhA gene


 


JOURNAL
Gene 37 (1-3), 53-62 (1985)


 


PUBMED
3902576


 


REFERENCE
107


 


AUTHORS
Friedberg,D., Rosenthal,E.R., Jones,J.W. and Calvo,J.M.


 


TITLE
Characterization of the 3′ end of the leucine operon of



Salmonella typhimurium


 


JOURNAL
Mol. Gen. Genet. 199 (3), 486-494 (1985)


 


PUBMED
2993799


 


REFERENCE
108


 


AUTHORS
Bouvier,J., Richaud,C., Richaud,F., Patte,J.C. and Stragier,P.


 


TITLE
Nucleotide sequence and expression of the Escherichia coli



dapB gene


 


JOURNAL
J. Biol. Chem. 259 (23), 14829-14834 (1984)


 


PUBMED
6094578


 


REFERENCE
109


 


AUTHORS
Richaud,C., Richaud,F., Martin,C., Haziza,C. and Patte,J.C.


 


TITLE
Regulation of expression and nucleotide sequence of the



Escherichia



coli dapD gene


 


JOURNAL
J. Biol. Chem. 259 (23), 14824-14828 (1984)


 


PUBMED
6094577


 


REFERENCE
110


 


AUTHORS
Nuesch,J. and Schumperli,D.


 


TITLE
Structural and functional organization of the gpt gene region



of



Escherichia coli


 


JOURNAL
Gene 32 (1-2), 243-249 (1984)


 


PUBMED
6397401


 


REFERENCE
111


 


AUTHORS
Jagadeeswaran,P., Ashman,C.R., Roberts,S. and Langenberg,J.


 


TITLE
Nucleotide sequence and analysis of deletion mutants of the



Escherichia coli gpt gene in plasmid pSV2 gpt


 


JOURNAL
Gene 31 (1-3), 309-313 (1984)


 


PUBMED
6396164


 


REFERENCE
112


 


AUTHORS
Deutch,A.H., Rushlow,K.E. and Smith,C.J.


 


TITLE
Analysis of the Escherichia coli proBA locus by DNA and



protein



sequencing


 


JOURNAL
Nucleic Acids Res. 12 (15), 6337-6355 (1984)


 


PUBMED
6089111


 


REFERENCE
113


 


AUTHORS
Bouvier,J., Patte,J.C. and Stragier,P.


 


TITLE
Multiple regulatory signals in the control region of the



Escherichia coli carAB operon


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 81 (13), 4139-4143 (1984)


 


PUBMED
6377309


 


REFERENCE
114


 


AUTHORS
Innis,M.A., Tokunaga,M., Williams,M.E., Loranger,J.M.,



Chang,S.Y.,



Chang,S. and Wu,H.C.


 


TITLE
Nucleotide sequence of the Escherichia coli prolipoprotein



signal



peptidase (lsp) gene


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 81 (12), 3708-3712 (1984)


 


PUBMED
6374664


 


REFERENCE
115


 


AUTHORS
Bardwell,J.C. and Craig,E.A.


 


TITLE
Major heat shock gene of Drosophila and the Escherichia coli



heat-inducible dnaK gene are homologous


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 81 (3), 848-852 (1984)


 


PUBMED
6322174


 


REFERENCE
116


 


AUTHORS
Pratt,D. and Subramani,S.


 


TITLE
Nucleotide sequence of the Escherichia coli xanthine-guanine



phosphoribosyl transferase gene


 


JOURNAL
Nucleic Acids Res. 11 (24), 8817-8823 (1983)


 


PUBMED
6324103


 


REFERENCE
117


 


AUTHORS
Richardson,K.K., Fostel,J. and Skopek,T.R.


 


TITLE
Nucleotide sequence of the xanthine guanine phosphoribosyl



transferase gene of E. coli


 


JOURNAL
Nucleic Acids Res. 11 (24), 8809-8816 (1983)


 


PUBMED
6324102


 


REFERENCE
118


 


AUTHORS
Parsot,C., Cossart,P., Saint-Girons,I. and Cohen,G.N.


 


TITLE
Nucleotide sequence of thrC and of the transcription



termination



region of the threonine operon in Escherichia coli K12


 


JOURNAL
Nucleic Acids Res. 11 (21), 7331-7345 (1983)


 


PUBMED
6316258


 


REFERENCE
119


 


AUTHORS
Stephens,P.E., Lewis,H.M., Darlison,M.G. and Guest,J.R.


 


TITLE
Nucleotide sequence of the lipoamide dehydrogenase gene of



Escherichia coli K12


 


JOURNAL
Eur. J. Biochem. 135 (3), 519-527 (1983)


 


PUBMED
6352260


 


REFERENCE
120


 


AUTHORS
Stephens,P.E., Darlison,M.G., Lewis,H.M. and Guest,J.R.


 


TITLE
The pyruvate dehydrogenase complex of Escherichia coli K12.



Nucleotide sequence encoding the dihydrolipoamide



acetyltransferase



component


 


JOURNAL
Eur. J. Biochem. 133 (3), 481-489 (1983)


 


PUBMED
6345153


 


REFERENCE
121


 


AUTHORS
Stephens,P.E., Darlison,M.G., Lewis,H.M. and Guest,J.R.


 


TITLE
The pyruvate dehydrogenase complex of Escherichia coli K12.



Nucleotide sequence encoding the pyruvate dehydrogenase



component


 


JOURNAL
Eur. J. Biochem. 133 (1), 155-162 (1983)


 


PUBMED
6343085


 


REFERENCE
122


 


AUTHORS
Kanaya,S. and Crouch,R.J.


 


TITLE
Low levels of RNase H activity in Escherichia coli FB2 rnh



result



from a single-base change in the structural gene of RNase H


 


JOURNAL
J. Bacteriol. 154 (2), 1021-1026 (1983)


 


PUBMED
6302075


 


REFERENCE
123


 


AUTHORS
Overbeeke,N., Bergmans,H., van Mansfeld,F. and Lugtenberg,B.


 


TITLE
Complete nucleotide sequence of phoE, the structural gene for



the



phosphate limitation inducible outer membrane pore protein of



Escherichia coli K12


 


JOURNAL
J. Mol. Biol. 163 (4), 513-532 (1983)


 


PUBMED
6341601


 


REFERENCE
124


 


AUTHORS
Gilson,E., Nikaido,H. and Hofnung,M.


 


TITLE
Sequence of the malK gene in E.coli K12


 


JOURNAL
Nucleic Acids Res. 10 (22), 7449-7458 (1982)


 


PUBMED
6296778


 


REFERENCE
125


 


AUTHORS
Stoner,C.M. and Schleif,R.


 


TITLE
Is the amino acid but not the nucleotide sequence of the



Escherichia coli araC gene conserved?


 


JOURNAL
J. Mol. Biol. 154 (4), 649-652 (1982)


 


PUBMED
6283093


 


REFERENCE
126


 


AUTHORS
An,G., Bendiak,D.S., Mamelak,L.A. and Friesen,J.D.


 


TITLE
Organization and nucleotide sequence of a new ribosomal operon



in



Escherichia coli containing the genes for ribosomal protein S2



and



elongation factor Ts


 


JOURNAL
Nucleic Acids Res. 9 (16), 4163-4172 (1981)


 


PUBMED
6272196


 


REFERENCE
127


 


AUTHORS
Mackie,G.A.


 


TITLE
Nucleotide sequence of the gene for ribosomal protein S20 and



its



flanking regions


 


JOURNAL
J. Biol. Chem. 256 (15), 8177-8182 (1981)


 


PUBMED
6267039


 


REFERENCE
128


 


AUTHORS
Little,J.W., Mount,D.W. and Yanisch-Perron,C.R.


 


TITLE
Purified lexA protein is a repressor of the recA and lexA



genes


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 78 (7), 4199-4203 (1981)


 


PUBMED
7027255


 


REFERENCE
129


 


AUTHORS
Mulligan,R.C. and Berg,P.


 


TITLE
Factors governing the expression of a bacterial gene in



mammalian



cells


 


JOURNAL
Mol. Cell. Biol. 1 (5), 449-459 (1981)


 


PUBMED
6100966


 


REFERENCE
130


 


AUTHORS
Lee,N.L., Gielow,W.O. and Wallace,R.G.


 


TITLE
Mechanism of araC autoregulation and the domains of two



overlapping



promoters, Pc and PBAD, in the L-arabinose regulatory region



of



Escherichia coli


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 78 (2), 752-756 (1981)


 


PUBMED
6262769


 


REFERENCE
131


 


AUTHORS
Cossart,P., Katinka,M. and Yaniv,M.


 


TITLE
Nucleotide sequence of the thrB gene of E. coli, and its two



adjacent regions; the thrAB and thrBC junctions


 


JOURNAL
Nucleic Acids Res. 9 (2), 339-347 (1981)


 


PUBMED
6259626


 


REFERENCE
132


 


AUTHORS
Miyada,C.G., Horwitz,A.H., Cass,L.G., Timko,J. and Wilcox,G.


 


TITLE
DNA sequence of the araC regulatory gene from Escherichia coli



B/r


 


JOURNAL
Nucleic Acids Res. 8 (22), 5267-5274 (1980)


 


PUBMED
7008027


 


REFERENCE
133


 


AUTHORS
Katinka,M., Cossart,P., Sibilli,L., Saint-Girons,I.,



Chalvignac,M.A., Le Bras,G., Cohen,G.N. and Yaniv,M.


 


TITLE
Nucleotide sequence of the thrA gene of Escherichia coli


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 77 (10), 5730-5733 (1980)


 


PUBMED
7003595


 


REFERENCE
134


 


AUTHORS
Ogden,S., Haggerty,D., Stoner,C.M., Kolodrubetz,D. and



Schleif,R.


 


TITLE
The Escherichia coli L-arabinose operon: binding sites of the



regulatory proteins and a mechanism of positive and negative



regulation


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 77 (6), 3346-3350 (1980)


 


PUBMED
6251457


 


REFERENCE
135


 


AUTHORS
Smith,D.R. and Calvo,J.M.


 


TITLE
Nucleotide sequence of the E coli gene coding for



dihydrofolate



reductase


 


JOURNAL
Nucleic Acids Res. 8 (10), 2255-2274 (1980)


 


PUBMED
6159575


 


REFERENCE
136


 


AUTHORS
Johnsrud,L.


 


TITLE
DNA sequence of the transposable element IS1


 


JOURNAL
Mol. Gen. Genet. 169 (2), 213-218 (1979)


 


PUBMED
375010


 


REFERENCE
137


 


AUTHORS
Smith,B.R. and Schleif,R.


 


TITLE
Nucleotide sequence of the L-arabinose regulatory region of



Escherichia coli K12


 


JOURNAL
J. Biol. Chem. 253 (19), 6931-6933 (1978)


 


PUBMED
357433


 


REFERENCE
138


 


AUTHORS
Greenfield,L., Boone,T. and Wilcox,G.


 


TITLE
DNA sequence of the araBAD promoter in Escherichia coli B/r


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 75 (10), 4724-4728 (1978)


 


PUBMED
368797


 


REFERENCE
139


 


AUTHORS
Young,R.A. and Steitz,J.A.


 


TITLE
Complementary sequences 1700 nucleotides apart form a



ribonuclease



III cleavage site in Escherichia coli ribosomal precursor RNA


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 75 (8), 3593-3597 (1978)


 


PUBMED
358189


 


REFERENCE
140


 


AUTHORS
Ohtsubo,H. and Ohtsubo,E.


 


TITLE
Nucleotide sequence of an insertion element, IS1


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 75 (2), 615-619 (1978)


 


PUBMED
273224


 


REFERENCE
141


 


AUTHORS
Musso,R., Di Lauro,R., Rosenberg,M. and de Crombrugghe,B.


 


TITLE
Nucleotide sequence of the operator-promoter region of the



galactose operon of Escherichia coli


 


JOURNAL
Proc. Natl. Acad. Sci. U.S.A. 74 (1), 106-110 (1977)


 


PUBMED
319453


 


REFERENCE
142 (bases 1 to 4646332)


 


CONSRTM
NCBI Genome Project


 


TITLE
Direct Submission


 


JOURNAL
Submitted (10-NOV-2005) National Center for Biotechnology



Information, NIH, Bethesda, MD 20894, USA


 


REFERENCE
143 (bases 1 to 4646332)


 


AUTHORS
Mori,H., Horiuchi,T. and Hirai,A.


 


TITLE
Direct Submission


 


JOURNAL
Submitted (22-AUG-2005) Hirotada Mori, Graduate School of



Biological Sciences, Nara Institute of Science and Technology;



8916-5 Takayama, Ikoma, Nara 630-0101, Japan



(E-mail:hmori@gtc.naist.jp, Tel:81-743-72-5660, Fax:81-743-72-



5669)


 


COMMENT
PROVISIONAL REFSEQ: This record has not yet been subject to



final



NCBI review. The reference sequence was derived from AP009048.


 


COMPLETENESS:
full length.


 


FEATURES
Location/Qualifiers


 


source
complement(<1..>5861)



/organism=′′Escherichia coli str. K-12 substr. W3110′′



/mol_type=′′genomic DNA′′



/strain=′′K-12′′



/sub_strain=′′W3110′′



/db_xref=′′taxon:316407′′


 


gene
complement(<1..6)



/gene=′′dcuD′′


 


CDS
complement(<1..6)



/gene=′′dcuD′′



/note=′′ECK3216:JW3196:b3227′′



/codon_start=1



/transl_table=11



/product=′′predicted transporter′′



/protein_id=′′AP 003769.1′′



/db_xref=′′GI:89109989′′


 








(SEQ ID NO: 35)



/translation=′′MFGIIISVIVLITMGYLILKNYKPQVVLAAAGIFLMMCGVWLGF



 


GGVLDPTKSSGYLIVDIYNEILRMLSNRIAGLGLSIMAVGGYARYMERIGASRAMVSL


 


LSRPLKLIRSPYIILSATYVIGQIMAQFITSASGLGMLLMVTLFPTLVSLGVSRLSAV


 


AVIATTMSIEWGILETNSIFAAQVAGMKIATYFFHYQLPVASCVIISVAISHFFVQRA


 


FDKKDKNINHEQAEQKALDNVPPLYYAILPVMPLILMLGSLFLAHVGLMQSELHLVVV


 


MLLSLTVTMFVEFFRKHNLRETMDDVQAFFDGMGTQFANVVTLVVAGEIFAKGLTTIG


 


TVDAVIRGAEHSGLGGIGVMIIMALVIAICAIVMGSGNAPFMSFASLIPNIAAGLHVP


 


AVVMIMPMHFATTLARAVSPITAVVVVTSGIAGVSPFAVVKRTAIPMAVGFVVNMIAT


 


ITLFY′′ 


 









primer
330..348




/label=′′ck nanR3 control primer′′


 


gene
386..1177



/gene=′′nanR′′


 


CDS
386..1177



/gene=′′nanR′′



/note=′′ECK3215:JW3195:b3226′′



/codon_start=1



/transl_table=11



/product=′′DNA-binding transcriptional dual regulator′′



/protein_id=′′AP 003768.1′′



/db_xref=′′GI:89109988′′


 








(SEQ ID NO: 36)



/translation=′′MGLMNAFDSQTEDSSPAIGRNLRSRPLARKKLSEMVEEELEQMI



 


RRREFGEGEQLPSERELMAFFNVGRPSVREALAALKRKGLVQINNGERARVSRPSADT


 


IIGELSGMAKDFLSHPGGIAHFEQLRLFFESSLVRYAAEHATDEQIDLLAKALEINSQ


 


SLDNNAAFIRSDVDFHRVLAEIPGNPIFMAIHVALLDWLIAARPTVTDQALHEHNNVS


 


YQQHIAIVDAIRRHDPDEADRALQSHLNSVSATWHAFGQTTNKKK′′ 


 









primer
1005..1025




/label=′′nanR ck2 control primer′′


 


primer
1126..1146



/label=′′nanAFck control primer′′


 


promoter
1178..1278



/label=′′nan operon promoter region′′


 


Site
1187..1191



/site_type=′′binding site′′



/label=′′CAP binding′′


 


Site
1198..1202



/site_type=′′binding site′′



/label=′′CAP binding′′


 


promoter
1241..1246



/label=-10


 


primer_bind
1252..1301



/note=′′for dnanA:: or dnanATE::scar deletions′′



/label=′′H1-dnanA lambda red primer′′



mRNA 1255



/label=+1


 


mRNA
1267



/label=+13


 


mRNA
1279



/label=+25


 


gene
1299..2192



/gene=′′nanA′′


 


CDS
1299..2192



/gene=′′nanA′′



/note=′′ECK3214:JW3194:b3225′′



/codon_start=1



/transl_table=11



/product=′′N-acetylneuraminate lyase′′



/protein_id=′′AP 003767.1′′



/db_xref=′′GI:89109987′′


 








(SEQ ID NO: 37)



/translation=′′MATNLRGVMAALLTPFDQQQALDKASLRRLVQFNIQQGIDGLYV



 


GGSTGEAFVQSLSEREQVLEIVAEEAKGKIKLIAHVGCVSTAESQQLAASAKRYGFDA


 


VSAVTPFYYPFSFEEHCDHYRAIIDSADGLPMVVYNIPALSGVKLTLDQINTLVTLPG


 


VGALKQTSGDLYQMEQIRREHPDLVLYNGYDEIFASGLLAGADGGIGSTYNIMGWRYQ


 


GIVKALKEGDIQTAQKLQTECNKVIDLLIKTGVFRGLKTVLHYMDVVSVPLCRKPFGP


 


VDEKYLPELKALAQQLMQERG′′ 


 









Region
1302..4424




/label=′′DELETION nanATE′′


 


primer_bind
complement(2175..2224)



/label=′′H2-dnanA lambda red primer′′


 


gene
2301..3791



/gene=′′nanT′′


 


CDS
2301..3791



/gene=′′nanT′′



/note=′′ECK3213:JW3193:b3224′′



/codon_start=1



/transl_table=11



/product=′′sialic acid transporter′′



/protein_id=′′AP 003766.1′′



/db_xref=′′GI:89109986′′


 








(SEQ ID NO: 38)



/translation=′′MSTTTQNIPWYRHLNRAQWRAFSAAWLGYLLDGFDFVLIALVLT



 


EVQGEFGLTTVQAASLISAAFISRWFGGLMLGAMGDRYGRRLAMVTSIVLFSAGTLAC


 


GFAPGYITMFIARLVIGMGMAGEYGSSATYVIESWPKHLRNKASGFLISGFSVGAVVA


 


AQVYSLVVPVWGWRALFFIGILPIIFALWLRKNIPEAEDWKEKHAGKAPVRTMVDILY


 


RGEHRIANIVMTLAAATALWFCFAGNLQNAAIVAVLGLLCAAIFISFMVQSAGKRWPT


 


GVMLMVVVLFAFLYSWPIQALLPTYLKTDLAYNPHTVANVLFFSGFGAAVGCCVGGFL


 


GDWLGTRKAYVCSLLASQLLIIPVFAIGGANVWVLGLLLFFQQMLGQGIAGILPKLIG


 


GYFDTDQRAAGLGFTYNVGALGGALAPIIGALIAQRLDLGTALASLSFSLTFVVILLI


 


GLDMPSRVQRWLRPEALRTHDAIDGKPFSGAVPFGSAKNDLVKTKS′′ 


 









primer
complement(2329..2350)




/label=′′nanARck control primer′′


 


primer_bind
3792..3841



/label=′′H1-dnanE lambda red primer′′


 


gene
3839..4528



/gene=′′nanE′′


 


CDS
3839..4528



/gene=′′nanE′′



/note=′′ECK3212:JW3192:b3223′′



/codon_start=1



/transl_table=11



/product=′′predicted N-acetylmannosamine-6-P



epimerase′′



/protein_id=′′AP 003765.1′′



/db_xref=′′GI:89109985′′


 








(SEQ ID NO: 39)



/translation=′′MSLLAQLDQKIAANGGLIVSCQPVPDSPLDKPEIVAAMALAAEQ



 


AGAVAIRIEGVANLQATRAVVSVPIIGIVKRDLEDSPVRITAYIEDVDALAQAGADII


 


AIDGTDRPRPVPVETLLARIHHHGLLAMTDCSTPEDGLACQKLGAEIIGTTLSGYTTP


 


ETPEEPDLALVKTLSDAGCRVIAEGRYNTPAQAADAMRHGAWAVTVGSAITRLEHICQ


 


WYNTAMKKAVL′′ 


 









primer_bind
complement(4425..4474)




/note=′′for dnanATE::scar deletion′′



/label=′′H2-dnanE lambda red primer′′


 


RBS
4425..4448



/label=′′C-terminal gibberish peptide fused to KD13



scar peptide′′


 


RBS
4449..4451



/label=′′NEW STOP gibberish peptide after resolution



of cassette′′


 


primer_bind
4486..4530



/label=′′nanK-H1 lambda red primer′′


 


RBS
4515..4520



/label=′′nanK RBS′′


 


gene
4525..5400



/gene=′′nanK′′


 


CDS
4525..5400



/gene=′′nanK′′



/note=′′ECK3211:JW5538:b3222′′



/codon_start=1



/transl_table=11



/product=′′predicted N-acetylmannosamine kinase′′



/protein_id=′′AP 003764.1′′



/db_xref=′′GI:89109984′′


 








(SEQ ID NO:40



/translation=′′MTTLAIDIGGTKLAAALIGADGQIRDRRELPTPASQTPEALRDA



 


LSALVSPLQAHAQRVAIASTGIIRDGSLLALNPHNLGGLLHFPLVKTLEQLTNLPTIA


 


INDAQAAAWAEFQALDGDITDMVFITVSTGVGGGVVSGCKLLTGPGGLAGHIGHTLAD


 


PHGPVCGCGRTGCVEAIASGRGIAAAAQGELAGADAKTIFTRAGQGDEQAQQLIHRSA


 


RTLARLIADIKATTDCQCVVVGGSVGLAEGYLALVETYLAQEPAAFHVDLLAAHYRHD


 


AGLLGAALLAQGEKL′′ 


 









RBS
4526..4528




/label=′′Native Stop for NanE′′


 


primer
complement(5065..5083)



/label=′′nanKckl control primer′′


 


primer_bind
complement(5380..5424)



/label=′′nanK-H2 lambda red primer′′


 


gene
5397..5861



/gene=′′yhcH′′


 


CDS
5397..5861



/gene=′′yhcH′′



/note=′′ECK3210:JW3190:b3221′′



/codon_start=1



/transl_table=11



/product=′′hypothetical protein′′



/protein_id=′′AP 003763.1′′



/db_xref=′′GI:89109983′′


 








(SEQ ID NO: 41)



/translation=′′MMMGEVQSLPSAGLHPALQDALTLALAARPQEKAPGRYELQGDN



 


IFMNVMTFNTQSPVEKKAELHEQYIDIQLLLNGEERILFGMAGTARQCEEFHHEDDYQ


 


LCSTIDNEQAIILKPGMFAVFMPGEPHKPGCVVGEPGEIKKVVVKVKADLMA′′


 


ORIGIN


(SEQ ID NO: 42)










   1
GAACATTGTT GAACTCCGTG TCAAAAGAAA ACGGTCAATC CCATAAACGG CAGATTGAAA



 


  61
ACAACGATGT TATATTTTTT GCAAGGCTAT TTATGGTGCG GATGTCGTGT TTTTAATTGT


 


 121
AGGTGAGGTG ATTTTTCATT AAAAAATATG CGCTTATGAT TATTTTGTAA GAACACATTC


 


 181
ATAATATTCA TAATGCTCGT GAATAGTCTT ATAAATAATT CAAACGGGAT GTTTTTATCT


 


 241
GCGTTACATT AATTTTTCGC AATAGTTAAT TATTCCGTTA ATTATGGTAA TGATGAGGCA


 


 301
CAAAGAGAAA ACCCTGCCAT TTTCCCCTAC TTTCAATCCT GTGATAGGAT GTCACTGATG


 


 361
ATGTTAATCA CACTGACCTT ACAGAATGGG CCTTATGAAC GCATTTGATT CGCAAACCGA


 


 421
AGATTCTTCA CCTGCAATTG GTCGCAACTT GCGTAGCCGC CCGCTGGCGC GTAAAAAACT


 


 481
CTCCGAAATG GTGGAAGAAG AGCTGGAACA GATGATCCGC CGTCGTGAAT TTGGCGAAGG


 


 541
TGAACAATTA CCGTCTGAAC GCGAACTGAT GGCGTTCTTT AACGTCGGGC GTCCTTCGGT


 


 601
GCGTGAAGCG CTGGCAGCGT TAAAACGCAA AGGTCTGGTG CAAATAAACA ACGGCGAACG


 


 661
CGCTCGCGTC TCGCGTCCTT CTGCGGACAC TATCATCGGT GAGCTTTCCG GCATGGCGAA


 


 721
AGATTTCCTT TCTCATCCCG GTGGGATTGC CCATTTCGAA CAATTACGTC TGTTCTTTGA


 


 781
ATCCAGTCTG GTGCGCTATG CGGCTGAACA TGCCACCGAT GAGCAAATCG ATTTGCTGGC


 


 841
AAAAGCACTG GAAATCAACA GTCAGTCGCT GGATAACAAC GCGGCATTCA TTCGTTCAGA


 


 901
CGTTGATTTC CACCGCGTGC TGGCGGAGAT CCCCGGTAAC CCAATCTTCA TGGCGATCCA


 


 961
CGTTGCCCTG CTCGACTGGC TTATTGCCGC ACGCCCAACG GTTACCGATC AGGCACTGCA


 


1021
CGAACATAAC AACGTTAGTT ATCAACAGCA TATTGCGATC GTTGATGCGA TCCGCCGTCA


 


1081
TGATCCTGAC GAAGCCGATC GTGCGTTGCA ATCGCATCTC AACAGCGTCT CTGCTACCTG


 


1141
GCACGCTTTC GGTCAGACCA CCAACAAAAA GAAATAATGC CACTTTAGTG AAGCAGATCG


 


1201
CATTATAAGC TTTCTGTATG GGGTGTTGCT TAATTGATCT GGTATAACAG GTATAAAGGT


 


1261
ATATCGTTTA TCAGACAAGC ATCACTTCAG AGGTATTTAT GGCAACGAAT TTACGTGGCG


 


1321
TAATGGCTGC ACTCCTGACT CCTTTTGACC AACAACAAGC ACTGGATAAA GCGAGTCTGC


 


1381
GTCGCCTGGT TCAGTTCAAT ATTCAGCAGG GCATCGACGG TTTATACGTG GGTGGTTCGA


 


1441
CCGGCGAGGC CTTTGTACAA AGCCTTTCCG AGCGTGAACA GGTACTGGAA ATCGTCGCCG


 


1501
AAGAGGCGAA AGGTAAGATT AAACTCATCG CCCACGTCGG TTGCGTCAGC ACCGCCGAAA


 


1561
GCCAACAACT TGCGGCATCG GCTAAACGTT ATGGCTTCGA TGCCGTCTCC GCCGTCACGC


 


1621
CGTTCTACTA TCCTTTCAGC TTTGAAGAAC ACTGCGATCA CTATCGGGCA ATTATTGATT


 


1681
CGGCGGATGG TTTGCCGATG GTGGTGTACA ACATTCCAGC CCTGAGTGGG GTAAAACTGA


 


1741
CCCTGGATCA GATCAACACA CTTGTTACAT TGCCTGGCGT AGGTGCGCTG AAACAGACCT


 


1801
CTGGCGATCT CTATCAGATG GAGCAGATCC GTCGTGAACA TCCTGATCTT GTGCTCTATA


 


1861
ACGGTTACGA CGAAATCTTC GCCTCTGGTC TGCTGGCGGG CGCTGATGGT GGTATCGGCA


 


1921
GTACCTACAA CATCATGGGC TGGCGCTATC AGGGGATCGT TAAGGCGCTG AAAGAAGGCG


 


1981
ATATCCAGAC CGCGCAGAAA CTGCAAACTG AATGCAATAA AGTCATTGAT TTACTGATCA


 


2041
AAACGGGCGT ATTCCGCGGC CTGAAAACTG TCCTCCATTA TATGGATGTC GTTTCTGTGC


 


2101
CGCTGTGCCG CAAACCGTTT GGACCGGTAG ATGAAAAATA TCTGCCAGAA CTGAAGGCGC


 


2161
TGGCCCAGCA GTTGATGCAA GAGCGCGGGT GAGTTGTTTC CCCTCGCTCG CCCCTACCGG


 


2221
GTGAGGGGAA ATAAACGCAT CTGTACCCTA CAATTTTCAT ACCAAAGCGT GTGGGCATCG


 


2281
CCCACCGCGG GAGACTCACA ATGAGTACTA CAACCCAGAA TATCCCGTGG TATCGCCATC


 


2341
TCAACCGTGC ACAATGGCGC GCATTTTCCG CTGCCTGGTT GGGATATCTG CTTGACGGTT


 


2401
TTGATTTCGT TTTAATCGCC CTGGTACTCA CCGAAGTACA AGGTGAATTC GGGCTGACGA


 


2461
CGGTGCAGGC GGCAAGTCTG ATCTCTGCAG CCTTTATCTC TCGCTGGTTC GGCGGCCTGA


 


2521
TGCTCGGCGC TATGGGTGAC CGCTACGGGC GTCGTCTGGC AATGGTCACC AGCATCGTTC


 


2581
TCTTCTCGGC CGGGACGCTG GCCTGCGGCT TTGCGCCAGG CTACATCACC ATGTTTATCG


 


2641
CTCGTCTGGT CATCGGCATG GGGATGGCGG GTGAATACGG TTCCAGCGCC ACCTATGTCA


 


2701
TTGAAAGCTG GCCAAAACAT CTGCGTAACA AAGCCAGTGG TTTTTTGATT TCAGGCTTCT


 


2761
CTGTGGGGGC CGTCGTTGCC GCTCAGGTCT ATAGCCTGGT GGTTCCGGTC TGGGGCTGGC


 


2821
GTGCGCTGTT CTTTATCGGC ATTTTGCCAA TCATCTTTGC TCTCTGGCTG CGTAAAAACA


 


2881
TCCCGGAAGC GGAAGACTGG AAAGAGAAAC ACGCAGGTAA AGCACCAGTA CGCACAATGG


 


2941
TGGATATTCT CTACCGTGGT GAACATCGCA TTGCCAATAT CGTAATGACA CTGGCGGCGG


 


3001
CTACTGCGCT GTGGTTCTGC TTCGCCGGTA ACCTGCAAAA TGCCGCGATC GTCGCTGTTC


 


3061
TTGGGCTGTT ATGCGCCGCA ATCTTTATCA GCTTTATGGT GCAGAGTGCA GGCAAACGCT


 


3121
GGCCAACGGG CGTAATGCTG ATGGTGGTCG TGTTGTTTGC TTTCCTCTAC TCATGGCCGA


 


3181
TTCAGGCGCT GCTGCCAACG TATCTGAAAA CCGATCTGGC TTATAACCCG CATACTGTAG


 


3241
CCAATGTGCT GTTCTTTAGT GGCTTTGGCG CGGCGGTGGG ATGCTGCGTA GGTGGCTTCC


 


3301
TCGGTGACTG GCTGGGAACC CGCAAAGCGT ACGTTTGTAG CCTGCTGGCC TCGCAGCTGC


 


3361
TGATTATTCC GGTATTTGCG ATTGGCGGCG CAAACGTCTG GGTGCTCGGT CTGTTACTGT


 


3421
TCTTCCAGCA AATGCTTGGA CAAGGGATCG CCGGGATCTT ACCAAAACTG ATTGGCGGTT


 


3481
ATTTCGATAC CGACCAGCGT GCAGCGGGCC TGGGCTTTAC CTACAACGTT GGCGCATTGG


 


3541
GCGGTGCACT GGCCCCAATC ATCGGCGCGT TGATCGCTCA ACGTCTGGAT CTGGGTACTG


 


3601
CGCTGGCATC GCTCTCGTTC AGTCTGACGT TCGTGGTGAT CCTGCTGATT GGGCTGGATA


 


3661
TGCCTTCTCG CGTTCAGCGT TGGTTGCGCC CGGAAGCGTT GCGTACTCAT GACGCTATCG


 


3721
ACGGTAAACC ATTCAGCGGT GCCGTGCCGT TTGGCAGCGC CAAAAACGAT TTAGTCAAAA


 


3781
CCAAAAGTTA ATCCTGTTGC CCGGTCTATG TACCGGGCCT TTCGCTAAGG GAAGATGTAT


 


3841
GTCGTTACTT GCACAACTGG ATCAAAAAAT CGCTGCTAAC GGTGGCCTGA TTGTCTCCTG


 


3901
CCAGCCGGTT CCGGACAGCC CGCTCGATAA ACCCGAAATC GTCGCCGCCA TGGCATTAGC


 


3961
GGCAGAACAG GCGGGCGCGG TTGCCATTCG CATTGAAGGT GTGGCAAATC TGCAAGCCAC


 


4021
GCGTGCGGTG GTGAGCGTGC CGATTATTGG AATTGTGAAA CGCGATCTGG AGGATTCTCC


 


4081
GGTACGCATC ACGGCCTATA TTGAAGATGT TGATGCGCTG GCGCAGGCGG GCGCGGACAT


 


4141
TATCGCCATT GACGGCACCG ACCGCCCGCG TCCGGTGCCT GTTGAAACGC TGCTGGCACG


 


4201
TATTCACCAT CACGGTTTAC TGGCGATGAC CGACTGCTCA ACGCCGGAAG ACGGCCTGGC


 


4261
ATGCCAAAAG CTGGGAGCCG AAATTATTGG CACTACGCTT TCTGGCTATA CCACGCCTGA


 


4321
AACGCCAGAA GAGCCGGATC TGGCGCTGGT GAAAACGTTG AGCGACGCCG GATGTCGGGT


 


4381
GATTGCCGAA GGGCGTTACA ACACGCCTGC TCAGGCGGCG GATGCGATGC GCCACGGCGC


 


4441
GTGGGCGGTG ACGGTCGGTT CTGCAATCAC GCGTCTTGAG CACATTTGTC AGTGGTACAA


 


4501
CACAGCGATG AAAAAGGCGG TGCTATGACC ACACTGGCGA TTGATATCGG CGGTACTAAA


 


4561
CTTGCCGCCG CGCTGATTGG CGCTGACGGG CAGATCCGCG ATCGTCGTGA ACTTCCTACG


 


4621
CCAGCCAGCC AGACACCAGA AGCCTTGCGT GATGCCTTAT CCGCATTAGT CTCTCCGTTG


 


4681
CAAGCTCATG CGCAGCGGGT TGCCATCGCT TCGACCGGGA TAATCCGTGA CGGCAGCTTG


 


4741
CTGGCGCTTA ATCCGCATAA TCTTGGTGGA TTGCTACACT TTCCGTTAGT CAAAACGCTG


 


4801
GAACAACTTA CCAATTTGCC GACCATTGCC ATTAACGACG CGCAGGCCGC AGCATGGGCG


 


4861
GAGTTTCAGG CGCTGGATGG CGATATAACC GATATGGTCT TTATCACCGT TTCCACCGGC


 


4921
GTTGGCGGCG GTGTAGTGAG CGGCTGCAAA CTGCTTACCG GCCCTGGCGG TCTGGCGGGG


 


4981
CATATCGGGC ATACGCTTGC CGATCCACAC GGCCCAGTCT GCGGCTGTGG ACGCACAGGT


 


5041
TGCGTGGAAG CGATTGCTTC TGGTCGCGGC ATTGCAGCGG CAGCGCAGGG GGAGTTGGCT


 


5101
GGCGCGGATG CGAAAACTAT TTTCACGCGC GCCGGGCAGG GTGACGAGCA GGCGCAGCAG


 


5161
CTGATTCACC GCTCCGCACG TACGCTTGCA AGGCTGATCG CTGATATTAA AGCCACAACT


 


5221
GATTGCCAGT GCGTGGTGGT CGGTGGCAGC GTTGGTCTGG CAGAAGGGTA TCTGGCGCTG


 


5281
GTGGAAACGT ATCTGGCGCA GGAGCCAGCG GCATTTCATG TTGATTTACT GGCGGCGCAT


 


5341
TACCGCCATG ATGCAGGTTT ACTTGGGGCT GCGCTGTTGG CCCAGGGAGA AAAATTATGA


 


5401
TGATGGGTGA AGTACAGTCA TTACCGTCTG CTGGGTTACA TCCTGCGTTA CAGGACGCGT


 


5461
TAACGCTGGC ATTAGCTGCC AGACCGCAAG AAAAAGCGCC GGGTCGTTAC GAATTACAGG


 


5521
GCGACAATAT CTTTATGAAT GTCATGACGT TTAACACTCA ATCGCCCGTC GAGAAAAAAG


 


5581
CGGAATTGCA CGAGCAATAC ATTGATATCC AGCTGTTATT AAACGGTGAG GAACGGATTC


 


5641
TGTTTGGCAT GGCAGGCACT GCGCGTCAGT GTGAAGAGTT CCACCATGAG GATGATTATC


 


5701
AGCTTTGCAG CACCATTGAT AACGAGCAAG CCATCATCTT AAAACCGGGA ATGTTCGCCG


 


5761
TGTTTATGCC AGGTGAACCG CATAAACCAG GATGCGTTGT CGGCGAGCCT GGAGAGATTA


 


5821
AAAAGGTTGT GGTGAAGGTT AAGGCTGATT TAATGGCTTA A 


 


//







Other Embodiments
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method for producing a sialylated oligosaccharide in an Escherichia coli (E. coli) bacterium, wherein said sialylated oligosaccharide comprises 3′-sialyllactose (3′-SL) or 6′-sialyllactose (6′-SL), said method comprising:
(i) providing an E. coli bacterium, said bacterium comprising
an exogenous sialyl-transferase comprising an α(2,3) sialyl-transferase, an α(2,6) sialyl-transferase, or an α(2,8) sialyltransferase,
a mutation in an endogenous N-acetylneuraminate lyase gene (nanA), wherein said bacterium comprises an endogenous N-acetylmannosamine kinase gene (nanK) that is not mutated,
an increased UDP-GlcNAc production capability comprising overexpression of nagC, such that the bacterium produces at least 10% more UDP-GlcNAc than a native E. coli bacterium,
a sialic acid synthetic capability, and
a functional lactose permease gene,

and
(ii) culturing said bacterium in the presence of lactose.

2. The method of claim 1, wherein said bacterium comprises a null mutation in any one of the genes selected from endogenous N-acetylneuraminate lyase gene (nanA), endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE), and endogenous N-acetylneuraminic acid transporter gene (nanT), or any combination thereof.

3. The method of claim 1, wherein said bacterium comprises a null mutation in an endogenous N-acetylneuraminate lyase gene (nanA).

4. The method of claim 1, wherein said bacterium comprises an endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE) that is not mutated, and (i) a null mutation in the endogenous N-acetylneuraminate lyase gene (nanA), (ii) a null mutation in an endogenous N-acetylneuraminic acid transporter gene (nanT), or (iii) a null mutation in the endogenous N-acetylneuraminate lyase gene (nanA) and a null mutation in the endogenous N-acetylneuraminic acid transporter gene (nanT).

5. The method of claim 1, wherein said bacterium comprises a null mutation in endogenous N-acetylneuraminate lyase gene (nanA), and a null mutation in endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE).

6. The method of claim 1, wherein said sialic acid synthetic capability comprises an exogenous CMP-Neu5Ac synthetase gene (neuA), an exogenous sialic acid synthase gene (neuB), and an exogenous UDP-GlcNac 2-epimerase (neuC).

7. The method of claim 1, wherein said α(2,3) sialyl-transferase, α(2,6) sialyl-transferase, or α(2,8) sialyltransferase, comprises a sequence of a Photobacterium sp. sialyl-transferase, Campylobacter jejuni sialyl-transferase, Neisseria meningitides sialyl-transferase, or Neisseria gonorrhoeae sialyl-transferase.

8. The method claim 1, wherein said sialylated oligosaccharide comprises 6′ sialyllactose (6′-SL).

9. The method of claim 1, wherein said bacterium comprises a deleted or inactivated endogenous β-galactosidase gene.

10. The method of claim 9, wherein said deleted or inactivated β-galactosidase gene comprises an E. coli lacZ gene.

11. The method of claim 1, wherein said bacterium comprises a recombinant β-galactosidase gene providing a level of β-galactosidase activity between 0.05 and 200 units.

12. The method of claim 1, wherein said bacterium further comprises a deleted, inactivated, or mutated lacA gene.

13. The method of claim 1, wherein said E. coli bacterium comprises an increased UDP-GlcNAc production capability, such that it produces at least 20% more UDP-GlcNAc than a native E. coli bacterium.

14. The method of claim 1, wherein said increased UDP-GlcNAc production capability further comprises overexpression of a glmS gene, a glmY gene, a glmZ gene or any combination thereof.

15. The method of claim 1, wherein said increased UDP-GlcNAc production capability comprises overexpression of nagC and glmS.

16. The method of claim 1, wherein said increased UDP-GlcNAc production capability comprises overexpression of nagC and glmY.

17. The method of claim 1, wherein said increased UDP-GlcNAc production capability comprises overexpression of nagC and glmZ.

18. A method of purifying a sialylated oligosaccharide produced by the method of claim 1, comprising binding said sialylated oligosaccharide from a bacterial cell lysate or bacterial cell culture supernatant of said bacterium to a carbon column, and eluting said sialylated oligosaccharide from said column.

19. A purified sialylated oligosaccharide produced by the method of claim 1.

20. The method of claim 1, further comprising retrieving said sialylated oligosaccharide from said bacterium or from a culture supernatant of said bacterium.

21. The method of claim 1, wherein said bacterium comprises a mutation in an endogenous N-acetylmannosamine-6-phosphate epimerase gene (nanE).

22. The method of claim 1, wherein said bacterium comprises an endogenous N-acetylneuraminic acid transporter gene (nanT) gene that is not mutated.

23. The method of claim 1, wherein said sialylated oligosaccharide comprises 3′-sialyllactose (3′-SL).

24. The method of claim 1, wherein said mutation is within the coding region of nanA.

25. The method of claim 24, wherein the mutation comprises an amino acid deletion or insertion.

26. The method of claim 25, wherein the mutation causes a loss of function of a nanA gene product or loss of production of a nanA gene product.