RU2798786C2 - Production of human dairy oligosaccharides in microbial producers with artificial import/export - Google Patents

Abstract

FIELD: oligosaccharides.

SUBSTANCE: invention relates to methods for the production of oligosaccharides in genetically modified bacterial host cells, as well as genetically modified host cells used in the methods. The invention relates to the production of lacto-N-triose II by a genetically modified host microorganism cell. A method for producing lacto-N-triose II by a genetically modified host microorganism cell includes obtaining a genetically modified host microorganism cell that contains at least one recombinantβ -1,3-N-acetylglucosaminyltransferase, increased expression or activity of at least one sugar export protein capable of exporting lacto-N-triose II. The said cell is cultured and lacto-N-triose II is obtained from the medium. At the same time, at least one sugar exporting protein is selected from the group consisting of YjhB from Escherichia coli, ProP from Mannheimia succiniciproducens, SetA from Cedecea neteri, and functional fragments thereof. Also, a genetically modified host microorganism cell for the production of lacto-N-triose II is proposed.

EFFECT: improved export and production of an increased amount of lacto-N-triose II.

12 cl, 9 dwg, 3 tbl, 8 ex

Description

This application is separated from the application No. 2018109987 for the grant of a patent of the Russian Federation for an invention, filed on September 12, 2016, with a claim for priority by the filing date of application No. 15184968.4, filed on September 12, 2015.

PRIOR ART

Human milk is considered the best nutrition for infant development. It consists of fats, proteins, vitamins, minerals, trace elements and a complex mixture of carbohydrates, which includes lactose and approximately 150 oligosaccharides of various structures (human milk oligosaccharides, HMO).

Attempts to produce HMOs chemically or biotechnologically have attracted worldwide attention mainly due to their beneficial effects on the development of the gastrointestinal flora of infants, thus justifying their use as nutritional supplements. In addition to these prebiotic properties, HMOs have many other positive effects, which expands the scope of their application.

However, carrying out deep scientific research requires obtaining individual pure compounds, which is difficult to achieve. This is especially true for complex free neutral and acidic oligosaccharides, for which there are still no competitive large-scale production processes (for example, for lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4) Gluc), lacto-N-neotetraoses (Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Gluc), lacto-N-fucopentaose I (Fuc(α1-2) Gal(β1-3)GlcNAc (β1-3)Gal(β1-4)Gluc) lacto-N-neofucopentaose I (Fuc(α1-2) Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Gluc) (lacto-N -sialylpentaose a (LST-a; Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Gluc)). the methods are rather inefficient for the production of these molecules on a multi-ton scale.

For simpler HMOs, such as 2'-fucosyllactose, 3-fucosyllactose, or 3'-sialyllactose, several enzymatic approaches have already been developed, mainly using metabolically engineered strains of Escherichia coli.

However, on a large scale, they can only be obtained by increasing the export of oligosaccharides from the bacterial cell, thus (i) increasing productivity and (ii) ensuring the release of the target oligosaccharide from the culture medium. The need to resolve the export problem increases as the size of the sugar produced increases. In addition, when producing more complex oligosaccharides using existing enzymatic processes, there is a problem of undesirable export of oligosaccharide precursors from the producer cell, resulting in undesirable mixing of the product and oligosaccharide precursors in the fermentation medium. Although many transport proteins are known to transport mono- or disaccharides across the membrane, the transport of larger oligosaccharides (eg, trisaccharides and larger oligosaccharides) remains unexplored.

For example, the genome of E. coli, a model organism often used for fermentation, encodes over 500 different transport proteins (Busch and Saier, Crit Rev Biochem Mol Biol. 2002; 37(5): 287-337). The classification of these membrane transport proteins is quite diverse, and their subgroups may differ in the mechanism of transport, protein structure, or evolutionary origin.

According to classical concepts, active carriers transfer the substrate with energy expenditure against its concentration or electrochemical gradient, while the movement and direction of substrate movement through the channels mainly occur along the gradient. Depending on the energy source used for translocation, pumps can be classified into primary and secondary active transporters using metabolic energy in the form of ATP or electrochemical potential, respectively (Davidson and Maloney, Trends Microbiol. 2007 Oct;15(10): 448-55 ; Forrest et al, Biochim Biophys Acta 2011 Feb;1807(2): 167-88). Although several membrane proteins involved in energy production, carbohydrate import, and protein and antibacterial efflux are well understood, a deep understanding of the process mechanisms or information on natural or possible substrates is currently available for only a small subset of the annotated bacterial vectors.

E. coli LacY lactose permease is perhaps the most well-described solute transporter (Guan and Kaback, Annu Rev Biophys Biomol Struct. 2006; 35: 67-91) and is a member of a large and highly diverse superfamily of major facilitators (MFS). superfamily), belonging to the class of secondary active carriers that transport sugars, drugs, hydrophobic molecules, peptides, organic ions, etc. according to the principle of uniport, symport or antiport (Saier et al., J Mol Microbiol Biotechnol. 1999 Nov; 1(2): 257-79). With a few exceptions, a common structural feature of MFS transporters is two six-helical subdomains spanning the cytoplasmic membrane. The existence of functionally homologous amino acid positions at related H ⁺ -coupled MFS symporters suggests the existence of a transfer mechanism similar to lactose permease (Madej and Kaback, Proc Natl Acad Sci US A. 2013 Dec 10;110(50): E4831-8).

Over the decades, a vast amount of information could have been accumulated on the import of carbohydrates into bacteria. However, there is very limited information regarding the export of carbohydrates, especially molecules that are not surface bound. This is not surprising, since sugars are actually a convenient source of carbon and energy, so once in the cell, they should not be released into the competitive environment.

However, the natural functioning of sugar exporters may be involved in the reduction of osmotic stress or stress resulting from intracellular accumulation of phosphorylated carbohydrates, which may indicate the adaptability of the substrate spectrum. Curiously, the export of various galactosides, such as IPTG (isopropyl-β-D-1-thiogalactopyranoside), TMG (thiomethylgalactoside) and lactose, has been shown for members of the so-called sugar efflux transporter family (SET, from the English sugar efflux transporter), belonging to the group of MFS vectors (Liu et al., J Biol Chem. 1999 Aug 13; 274(33): 22977-84; Liu et al., Mol Microbiol. 1999 Mar; 31(6): 1845-51).

E. coli transport protein SetA transfers human milk oligosaccharide 3-fucosyllactose, resulting in improved production of said compound during fermentation by a recombinant E. coli strain overexpressing setA (see our international patent application WO 2010/142305). ). Similarly, expression of the efflux sugar transporter Yersinia has been shown to allow export of the human milk oligosaccharide 2'-fucosyllactose by the modified E. coli producer strain.

In addition, from a mechanistic and energetic point of view, only ion gradient driven transport systems have the potential to transport solutes across the membrane in both directions. This is, for example, true of the aforementioned LacY, the galactoside/H ⁺ symporter, which is part of the bacterial lac operon that allows E. coli to metabolize lactose. This permease preferentially imports lactose into the cell, but it is also capable of transporting its substrate in the opposite direction.

In addition to the superfamily of major mediators, representing the largest group of vectors, bacteria have another mechanism for solute excretion—often grouped into classes of multidrug-resistance efflux pumps. As in the case of MFS, the activities of the small multidrug resistance superfamily (SMR), the multidrug and toxic compound extrusion superfamily (MATE) and the superfamily resistance-nodulation-cell division superfamily (RND) depends on the electrochemical gradient. The fifth class is a superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) carriers, which uses ATP energy as an energy source to move molecules out of the cell. As with MFS, members of SMR, MATE, RND, and ABC carry structurally dissimilar molecules. In addition, most of their substrates identified so far are not natural, and therefore, their preferences are difficult to predict.

Although processes for the chemical synthesis of human milk oligosaccharides are known in the art, they are very costly and do not provide satisfactory quantities. On the other hand, a disadvantage of fermentation processes using genetically modified microorganisms is still that the export of larger oligosaccharides (tetra-, penta, hexasaccharides) is a major obstacle to establishing a cost-effective production process. Thus, there continues to be a need for improved processes for the production of human oligosaccharides on a large scale.

SUMMARY OF THE INVENTION

The invention provides methods and host microbial cells for this and other purposes as described in the appended claims.

The methods and host cells of the invention produce the target oligosaccharide, preferably an oligosaccharide that is not produced by the unmodified host cell, and preferably an oligosaccharide related to human milk oligosaccharides that can be obtained from the environment in large quantities. Thus, the oligosaccharide, so to speak, is obtained from the environment in a free form; it is not associated with a surface protein or a membrane protein or other protein on the surface of the host cell.

According to the invention, a method for producing a target oligosaccharide by a genetically modified host microorganism cell includes the steps in which: a) a genetically modified host microorganism cell is obtained that contains at least one recombinant glycosyltransferase, and in which the expression or activity of at least one endogenous protein exporting protein is modified such that the expression or activity of the sugar exporting protein is either (i) increased, or (ii) decreased or inactivated compared to the non-genetically modified host cell, such that (i) export of the oligosaccharide precursor to the environment either reduced or absent, or (ii) the transport of the target oligosaccharide is increased, respectively, compared to the non-genetically modified host cell, b) the host cell is cultured in the medium under conditions that ensure the production of the target oligosaccharide, due to which the target oligosaccharide is transported into the medium. The method may further include step c) wherein the target oligosaccharide is obtained from the medium.

Preferably in the method of the invention, the target oligosaccharide is a human milk oligosaccharide containing lacto-N-triose II (LNT-II; GlcNAc(β1-3)Gal(β1-4)Gluc) as the main trisaccharide. Thus, an oligosaccharide having a "basic trisaccharide" contains a specific trisaccharide representing the reducing end of the target oligosaccharide and, where appropriate, contains additional saccharide moieties, with the specific trisaccharide being the predominant moiety.

Accordingly, in an embodiment of the method and host cell of the invention, the target oligosaccharide is selected from the group consisting of: lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N -fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexaose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, disialyllacto -N-neotetraoses.

To overcome the above disadvantages associated with limited export of oligosaccharides, to solve the problem, a method according to the invention is also proposed, where the host cell contains: at least one homologous or heterologous nucleic acid sequence encoding a protein that ensures the export of the target oligosaccharide into the culture medium, where the specified the host cell has been modified such that the homologous or heterologous nucleic acid sequence is overexpressed or under the control of a promoter that overexpresses the nucleic acid sequence; and/or has a deletion, disruption, reduction in expression, or inactivation of at least one endogenous nucleic acid sequence encoding an export protein that exports the target oligosaccharide precursors from the host cell; and/or at least one homologous or heterologous nucleic acid sequence encoding a protein mediating import of the target oligosaccharide precursor into said host cell, wherein the nucleic acid sequence is preferably overexpressed and where the precursor is preferably larger than the disaccharide.

Genetically modified host microorganism cells having these characteristics are cultured in the presence of glucose, sucrose, glycerol, or a combination thereof, these substrates serve as carbon and energy sources, as well as in the presence of lactose or oligosaccharides larger than disaccharides, for example, LNT-II.

In a preferred embodiment of said method and host cell, said target oligosaccharide export protein belongs to the class of secondary active transporters, and more preferably exports an oligosaccharide containing at least three moieties.

According to preferred embodiments, in addition to the genes responsible for intracellular oligosaccharide biosynthesis, a suitable exporter is expressed to export the target oligosaccharides.

According to one aspect of the method and host cell of the invention, at least one nucleic acid sequence encoding a target oligosaccharide export protein is an endogenous or recombinant nucleic acid.

According to a preferred embodiment of the method and the host cell of the invention, the nucleic acid sequence encoding the target oligosaccharide export protein is from a bacterium, archaebacterium, plant, yeast or animal; preferably, at least one nucleic acid sequence encoding a target oligosaccharide export protein is a gene selected from the group consisting of yebQ and yjhB from Escherichia coli, proP from Mannheimia succiniciproducens, and setA from Cedecea neteri, or functional fragments thereof.

Preferably, the oligosaccharide exporter is a protein selected from at least one of the following: SetA, SetB, SetC, YdeA, Cmr, YnfM, MdtD, YfcJ, YhhS, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr and YdeA from E. coli, or ProP from Mannheimia succiniciproducens and SetA from Cedecea neteri, or variants or homologues thereof.

In another preferred embodiment, the recombinant glycosyltransferase is selected from at least one of the following: galactosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase, and fucosyltransferase, and is preferably selected from at least one of the following: β-1,3-N-acetylglucosaminyltransferase, β-1 ,3-galactosyltransferases, β-1,4-galactosyltransferases, β-1,6-galactosyltransferases, α-2,3-sialyltransferases, α-2,6-sialyltransferases, α-1,2-fucosyltransferases or α-1,3 -fucosyltransferase.

A preferred embodiment of the method and host cell of the invention relates to a host cell or provision thereof, wherein the host cell contains as glycosyltransferases (i) β-1,3-N-acetylglucosaminyltransferase and (ii) β-1,3-galactosyltransferase or β -1,4-galactosyltransferase. In this regard, it is preferable that said β-1,3-N-acetylglucosaminyltransferase has an activity that allows N-acetylglucosamine to be ligated with lactose to form lacto-N-triose II, and that said β-1,3-galactosyltransferase or said β-1 ,4-galactosyltransferase, respectively, had an activity that provides galactosylation of lacto-N-triose II with the formation of lacto-N-tetraose or lacto-N-neotetraose, respectively. The developed system can be easily adapted for even more complex oligosaccharides due to the expression of additional glycosyltransferases.

Due to the microorganism cell and the method of the invention, it is possible to obtain a target oligosaccharide in large amounts by fermentation, in particular an oligosaccharide containing LNT-II as a main structure, and to separate it from the culture liquid.

In a preferred embodiment, said β-1,3-N-acetylglucosaminyltransferase is of the lgtA class from Neisseria meningitides or PmnagT from Pasteurella multocida, or variants thereof.

Preferably, the glycosyltransferase is selected from galactosyltransferase, sialyltransferase, N-acetylglucosaminyltransferase and fucosyltransferase.

In yet another preferred embodiment, the lacto-N-tetraose-forming β-1,3-galactosyltransferase is WbdO or a functional variant thereof. According to an aspect of the invention, β-1,3-galactosyltransferase is a β-1,3-galactosyltransferase derived from Salmonella enterica (wbdO, accession number AY730594) and is preferably encoded by a gene selected from the group consisting of wbgO from Escherichia coli O55:H7 or furA from Lutiella nitroferrum or their functional fragments.

The invention also relates to a genetically modified microorganism host cell, preferably a bacterial host cell, as described above, in which an endogenous β-galactosidase gene has been inactivated or deleted and a functional lactose permease gene is present.

Accordingly, in a preferred embodiment of the method and host cell of the invention, there is provided a genetically modified host cell which, where applicable, has inactivated or deleted an endogenous β-galactosidase gene and a glucosamine-6-phosphate deaminase gene, and wherein said genetically modified cell the host contains a nucleic acid sequence encoding a functional lactose permease protein, preferably LacY.

In a preferred embodiment, the genetically modified host cell has an increased ability to produce UDP-N-acetylglucosamine and UDP-galactose, GDP-fucose, or CMP-N-acetylneuraminic acid compared to the non-genetically modified host cell.

In an improved embodiment of the method and host cell of the invention, said increased ability to produce UDP-N-acetylglucosamine and UDP-galactose comprises overexpression of one or more genes encoding proteins having the following activity: L-glutamine: D-fructose-6-phosphate -aminotransferase, N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase, glucose-1-phosphate uridylyltransferase.

Synthesis of, for example, LNT requires UDP-galactose and UDP-N-acetylglucosamine. UDP-galactose can be produced by supplying galactose to a BT-producing bacterial host cell in a fermentation medium. Then galactose enters the cell, is phosphorylated to galactose-1-phosphate and then converted to UDP-galactose. The genes encoding this enzymatic activity are well known in the literature (Grossiord et al., J. Bacteriol 2003 185(3) 870-878). The cell can also obtain UDP-galactose from its own metabolism, and metabolism can be improved by additional genetic modification, such as overexpression of UDP-galactose-4'-epimerase or UDP-galactose-4'-epimerase in combination with glucose-1-phosphate uridinyltransferase. . UDP-N-acetylglucosamine can also be produced from the intrinsic metabolism of UDP-N-acetylglucosamine in the bacterial host cell. It is possible to improve the availability of UDP-N-acetylglucosamine for the synthesis of oligosaccharides containing N-acetylglucosamine by inactivating the catabolism of N-acetylglucosamine in the producer cell.

According to one aspect of the invention, the genetically modified host cell is cultured in the presence of glucose, sucrose, glycerol, or a combination thereof, but not with or in the presence of N-acetylglucosamine or galactose, or a combination thereof.

In a preferred embodiment of the method and host cell of the invention, the target oligosaccharide is lacto-N-triose II, which is formed from a simple carbon source by complete fermentation in the host cell by the expressed heterologous glycosyltransferases β-1,4-galactosyltransferase and β- 1,3-N-acetylglucosaminyltransferase.

The present invention, as mentioned above, also relates to a genetically modified host cell for the production of the target oligosaccharide, which contains as the main trisaccharide lacto-N-triose II (LNT-II; GlcNAc(β1-3)Gal(β1-4)Gluc ), where the host cell contains at least one recombinant glycosyltransferase, which is preferably selected from galactosyltransferase, sialyltransferase and N-acetylglucosaminyltransferase, and has a modified expression or activity of at least one endogenous sugar transport protein, such that the endogenous sugar transport protein, functionally inactivated in terms of export of the precursor of the target oligosaccharide.

A preferred embodiment relates to a host cell as described above, comprising as glycosyltransferases (i) an expressed heterologous β-1,3-N-acetylglucosaminyltransferase and (ii) an expressed heterologous β-1,3-galactosyltransferase or an expressed heterologous β-1,4-galactosyltransferase where the host cell preferably additionally contains at least one homologous or heterologous nucleic acid sequence encoding a protein that ensures the export of the oligosaccharide into the culture medium in which the host cell is cultivated, where the specified protein that ensures the export of the target oligosaccharide belongs to the class of secondary active carriers wherein said host cell has been modified such that the homologous or heterologous nucleic acid sequence is overexpressed or under the control of a promoter that overexpresses the nucleic acid sequence. In a preferred embodiment of the host cell, said at least one nucleic acid sequence encoding the target oligosaccharide export protein is an endogenous or recombinant nucleic acid sequence.

As already mentioned with respect to the method of the invention, it is also preferred for the host cell that said nucleic acid sequence encoding the target oligosaccharide export protein is from a bacterium, archaebacterium, plant, yeast or animal.

According to another aspect of the invention, the host cell described above further has: deletion, disruption, reduction in expression, or inactivation of at least one endogenous nucleic acid sequence encoding an export protein that exports the target oligosaccharide precursors from the host cell; and/or at least one homologous or heterologous nucleic acid sequence encoding a protein allowing import of the target oligosaccharide precursor into said host cell, where preferably the nucleic acid sequence is overexpressed and where preferably the precursor is larger than the disaccharide.

When overexpressing at least one homologous or heterologous nucleic acid sequence encoding a protein that ensures the import of the precursor of the target oligosaccharide into the specified host cell, it is possible to supply the culture medium with precursors of the target oligosaccharide that will be imported into the host cell, for example, as LNT-II .

According to one aspect of the invention, in a host cell, said at least one nucleic acid sequence encoding a target oligosaccharide export protein is a gene selected from the group consisting of yebQ and yjhB from Escherichia coli, proP from Mannheimia succiniciproducens, and setA from Cedecea neteri or their functional fragments.

According to another preferred embodiment, the target oligosaccharide is lacto-N-triose II and the protein that exports the oligosaccharide to the culture medium in which the host cell is cultured is YjhB from Escherichia coli, ProP from Mannheimia succiniciproducens and SetA from Cedecea neteri, or their functional fragments.

According to a preferred embodiment, the host microorganism of the invention is further modified not to express proteins that export precursors of the target oligosaccharide.

In a preferred embodiment of the host cell, the target oligosaccharide is lacto-N-tetraose, the precursor is lacto-N-triose II, and the host cell has a deletion, disruption, or inactivation of at least one nucleic acid sequence encoding an exporter protein capable of exporting lacto-N-triose II from the host cell.

In this regard, it is preferable if the lacto-N-tetraose export protein is selected from YebQ from Escherichia coli BL21(DE3), SpoVB from Bacillus amyloliquefaciens, YabM from Erwinia pyrilfolia, Bcr from E. coli MG1655, YdeA from E. coli MG1655, ProP2 from Haemophilus parainfluenzae, SetA from Pectobacterium carotovorum, FucP from E. coli MG1655, MdeA from Staphylococcus aureus Bmb9393, ImrA from Lactococcus lactis, SetA from Pseudomonas sp. MT-1 and SetA from Beauveria bassiana D1-5.

Preferably, the oligosaccharide exporter is a protein selected from at least one of the following: SetA, SetB, SetC, YdeA, Cmr, YnfM, MdtD, YfcJ, YhhS, EmrD, YdhC, YbdA, YdeE, MhpT, YebQ, YjhB, Bcr and YdeA from E. coli or ProP from Mannheimia succiniciproducens and SetA from Cedecea neteri or variants or homologues thereof.

As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded macromolecule composed of deoxyribonucleotides or ribonucleotides and encompasses known natural or artificial nucleotide analogs that hybridize to a desired nucleic acid and encode a polypeptide.

The term "recombinant" or "genetically modified" as used herein in relation to a host microorganism cell indicates that the host microorganism cell replicates a heterologous nucleic acid or expresses a peptide or protein encoded by a heterologous nucleic acid (i.e., a "foreign" sequence). for the specified cell). Recombinant cells may contain genes that are not found in native (non-recombinant) forms of the cell. Recombinant cells may also contain genes that occur in the native form of the cell, where the genes are modified and brought back into the cell by artificial means. The term also encompasses cells that contain the endogenous nucleic acid of the cell that has been modified without removing the nucleic acid from the cell; said modifications include those obtained by gene substitution, site-specific mutation, and the like. Accordingly, a "recombinant polypeptide" is a polypeptide produced by a recombinant cell. A "heterologous sequence" or "heterologous nucleic acid" as used herein is derived from a source foreign to a particular host cell (e.g. from a different species) or modified from its original form if originating from the same source. Thus, a heterologous nucleic acid operably linked to a promoter originates from a source different from that from which the promoter originates, or is modified from its original form if it originates from the same source. The heterologous sequence can be stably inserted, for example, by transfection, transformation, conjugation or transduction into the genome of a host microorganism cell, which thus represents the genetically modified host cell. The methods used will depend on the host cell into which the sequence is inserted. Various techniques are known to those skilled in the art and are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, ^2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

Accordingly, "host microorganism cell" as used herein means a microbial, preferably bacterial, cell that has been transformed or transfected or is capable of being transformed or transfected with an exogenous polynucleotide sequence.

Thus, the nucleic acid sequences of the invention may, for example, be incorporated into a vector for stably transforming/transfecting or otherwise introducing into a cell of a host microorganism.

As used herein, the term "operably linked" means a functional relationship between a nucleic acid expression control sequence (such as a promoter, a signal sequence, or a set of transcription factor binding sites) and a second nucleic acid sequence, where expression of the control sequence affects transcription and/or translation of the nucleic acid. acid corresponding to the second sequence. Accordingly, the term "promoter" refers to DNA sequences that are usually located before the gene in the DNA polymer and represent the site that provides the initiation of transcription in mRNA. "Regulatory" DNA sequences are also usually located in the "-" (i.e., located in front of) the gene in a given DNA polymer, bind proteins that determine the frequency of transcription initiation. Collectively referred to as a "promoter/regulator" or "control" DNA sequence, these sequences, located upstream of a selected gene (or set of genes) in a functional DNA polymer, collectively determine whether transcription (and hence expression) of the gene will occur. DNA sequences that are usually located after a gene in a DNA polymer and provide a signal for terminating transcription in mRNA are referred to as transcription "terminator" sequences.

A wide variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, but are not limited to, chromosomal vectors, episomal vectors, and virus-based vectors, such as vectors based on bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosome elements, viruses, and vectors based on combinations thereof, such as based on plasmids and bacteriophage genetic elements such as cosmids and phagemids. Expression system constructs may contain control regions that regulate as well as trigger expression. In general, any system or vector suitable for maintaining, reproducing or expressing polynucleotides and synthesizing a polypeptide in a host cell can be used for expression. The appropriate DNA sequence can be inserted into the expression system by any of the many known and routine methods, such as those described by Sambrook et al., supra.

There are many patents and publications in the art relating to "recombinant DNA" technology for isolating, synthesizing, purifying and amplifying genetic material for use in the transformation of selected host organisms. Thus, it is common knowledge to transform a host organism with a "hybrid" viral or circular plasmid DNA that includes selected exogenous (ie, foreign or "heterologous") DNA sequences. Procedures known in the art primarily involve the creation of a transforming vector by enzymatic cleavage of circular viral or plasmid DNA to form linear strands of DNA. Selected foreign DNA strands, which typically include sequences encoding the desired protein product, are prepared in linear form using the same/similar enzymes. Linear viral or plasmid DNA is incubated with foreign DNA in the presence of ligating enzymes capable of carrying out the repair process, and "hybrid" vectors are formed that include the selected exogenous DNA segment "spliced" into viral or circular plasmid DNA.

In this description, the term "cultivation" means the cultivation of a bacterial cell in a medium under conditions that provide and are suitable for the production of the target(s) oligosaccharide(s). Upon reading this specification and based on the knowledge known in the art, one skilled in the art will have at his disposal a pair of suitable bacterial host cells, as well as their culture media and conditions.

As used herein, the term "isolation" or "obtaining" means isolating, collecting, purifying, collecting, or otherwise separating an oligosaccharide produced by a host cell of the invention from a host cell culture.

The "host microorganism" cell of the invention, as well as the common meaning of the term, means any microorganism, including bacteria, yeasts and archaebacteria, which is usually suitable for cultivation in large quantities, and which can be genetically modified according to the invention to produce the target oligosaccharide. Preferred microorganisms are bacteria such as Escherichia coli, Corynebacterium glutamicum and yeast Saccharomyces sp., which have the advantage that these microorganisms can be easily and inexpensively cultivated in the laboratory, and bacteria and yeast have been extensively studied for many years.

In the technical field and in the description of this invention, the term "glycosyl transferase activity" or "glycosyl transferase" refers to and encompasses enzymes that are responsible for the biosynthesis of disaccharides, oligosaccharides and polysaccharides and catalyze the transfer of monosaccharide groups from an activated nucleotide monosaccharide/sugar ("glycosyl donor") to glycosyl acceptor molecule.

In the technical field and in the description of this invention, the terms "exporter" or "export protein" or "protein that ensures the export of the target oligosaccharide" are used interchangeably and refer to one or more polypeptides that, alone or as part of a complex of several proteins, transport the oligosaccharide from the intracellular environment of the bacterial cell into the periplasm of said cell or into the culture supernatant, thereby allowing passage of the oligosaccharide through the cell membrane and/or cell wall of said cell.

Within the scope of this invention, these terms also cover polymorphic variants, alleles, mutants and cross-species homologs of nucleic acids/polynucleotides and polypeptides having an amino acid sequence that is more than 60%, 65%, 70%, 75%, 80%, 85 identical. %, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, preferably over a length of at least about 25, 50, 100, 200, 500 or 1000 or more amino acids, with a polypeptide encoded by a wild-type gene, or with a wild-type protein showing glycosyl transferase activity or exporting an oligosaccharide.

"Option(s)" in this description is a polynucleotide or polypeptide that differs from the reference polynucleotide or polypeptide, respectively, but retains significant properties. An exemplary polynucleotide variant differs in nucleic acid sequence from another reference polynucleotide. Changes in the nucleic acid sequence of a variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide substitutions can result in substitutions, insertions, amino acid deletions, fusions, and truncations of the polypeptide encoded by the reference sequence, as will be discussed below. An exemplary polypeptide variant differs in amino acid sequence from another reference polypeptide. Typically, differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and identical in many areas. The amino acid sequence of a variant polypeptide and a reference polypeptide may differ by one or more substitutions, insertions, deletions, in any combination thereof. The substituted or inserted amino acid residue may or may not be the residue encoded by the genetic code. The polynucleotide or polypeptide variant may be a naturally occurring variant, such as an allelic variant, or it may be a variant not known to occur naturally. Variants of polynucleotides and polypeptides that are not naturally occurring can be obtained by mutagenesis, direct synthesis, or other recombinant methods known to those skilled in the art.

Accordingly, a "functional fragment" of any of the genes/proteins described herein refers to sequence variants of the genes/proteins that still retain the same or somewhat less activity of the gene or protein from which the corresponding fragment is derived.

In this regard, the term "nucleic acid sequence encoding" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, and usually represents a gene that encodes a particular polypeptide or protein.

In this context, the term "polypeptide(s)" refers to any peptide or protein containing two or more amino acids connected to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers, and longer chains, commonly referred to as "proteins". The polypeptides may contain amino acids that are not among the 20 amino acids encoded by the genes. "Polypeptide(s)" includes those modified naturally, such as by processing and other post-translational modifications, as well as by chemical modification. It should be appreciated that the same type of modification of the same or varying degrees of severity may occur at multiple sites on a given polypeptide without significantly altering the activity of the polypeptide. Also, a given polypeptide may contain several types of modifications. Modifications can occur anywhere on the polypeptide, including the polypeptide backbone, amino acid side chains, and amino or carboxy termini.

Further, the expression "predecessor" encompasses compounds involved in biosynthetic processes that produce the oligosaccharide of the invention, or that are produced and naturally present in the host cell.

"Precursor larger than disaccharide" as used herein means a carbohydrate moiety that contains more than two monosaccharide residues.

The term "target oligosaccharide" refers to a carbohydrate polymer consisting of at least three groups, thus encompassing trisaccharides, tetrasaccharides, pentasaccharides, etc., preferably an oligosaccharide selected from at least one of the following: lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexaose I, lacto -N-difucosylhexaose II, lacto-N-sialylpentaose LSTa, LSTb, LSTc, disialyllacto-N-tetraose, disialyllacto-N-neotetraose.

Currently, in accordance with conventional understanding in the field of technology, the expression "homologous" refers to a nucleic acid sequence that encodes a specific product or products and is derived from the same biological species in which the specified nucleic acid is inserted. Accordingly, the term "heterologous" refers to a nucleic acid sequence that encodes a specific product or products and is derived from species other than those into which said nucleic acid is inserted.

The term "endogenous" in this document and in the technical field means that the nucleic acid encoding the enzyme of interest originates from a bacterial host cell, and has not been integrated into said host cell, while "recombinant" means that the nucleic acid has been inserted into said host cell and is not derived from said host cell.

The expression "over-expression" or "over-expressed" or "under the control of a promoter sequence allowing over-expression of said nucleic acid sequence" is currently understood to mean the expression of a gene in excess of normal, i. in an increased amount, which leads to an increase in the amount of protein encoded by the nucleic acid sequence.

In some embodiments, the nucleic acid sequence is under the control of an inducible promoter, which is a promoter that directs gene expression, where the level of expression changes under the influence of environmental or developmental factors, such as temperature, pH, anaerobic or aerobic conditions, lighting. , transcription factors and chemicals. Such promoters are referred to herein as "inducible" promoters, which control the timing of expression of the proteins used in this invention. Inducible promoters for E. coli and other microbial host cells are well known to those skilled in the art.

The following advantages will be apparent from the description and accompanying drawings.

It is self-evident that the characteristics mentioned above, as well as the characteristics explained below, can be used not only in the respective combinations given, but also in other combinations or by themselves, while remaining within the scope of the present invention.

Examples and drawings are provided for the purpose of explaining the essence of the invention in more detail.

On FIG. 1 is a schematic representation of the production of lacto-N-triose II or lacto-N-tetraose in a media cultured host cell;

On FIG. Figure 2 shows the results of thin-layer chromatography (TLC) analysis of culture extracts of E. coli BL21(DE3) strains producing lacto-N-triose II (LNT II) overexpressing the β-1,3-N-acetylglucosaminyltransferase PmnagT(13, 14) gene. ;

On FIG. Figure 3 shows the results of TLC analysis of culture extracts of E. coli BL21(DE3) strains producing lacto-N-tetraose (LNT), overexpressing BfgalT2 (1), PmgalT7 (3), MsgalT8 (6), gatD (7), lex1 genes. (9), lgtB (11) or lsgD (13), encoding β-1,4-galactosyltransferase;

On FIG. Figure 4 shows the results of TLC analysis of culture extracts of E. coli BL21(DE3) strains producing lacto-N-tetraose (LNT), overexpressing the KdgalT10 (1), cpsI14J (7), cpsIaJ (8, 9), HpgalT (12) genes. encoding β-1,4-galactosyltransferase;

On FIG. 5 shows the results of TLC analysis of culture extracts of lacto-N-tetraose-producing E. coli BL21(DE3) strains overexpressing the waaX gene (5) encoding β-1,4-galactosyltransferase;

On FIG. 6 shows the results of TLC analysis of culture extracts of lacto-N-tetraose-producing E. coli strains BL21(DE3) overexpressing the wbdO or furA genes encoding β-1,3-galactosyltransferase;

On FIG. 7 shows the results of a high performance liquid chromatography (HPLC) analysis of a culture supernatant of lacto-N-tetraose producing strain E. coli BL21(DE3). (A) Supernatant of E. coli BL21(DE3) 1353 and 1431 cultured in the presence of glucose and lactose after 24 hours of incubation. (B) Supernatant of E. coli BL21(DE3) 1353 and 1431 cultured in the presence of glucose and lactose after 48 hours of incubation.

On FIG. 8 is a graph showing the relative concentration of lacto-N-tetraose in the supernatant of E. coli strains BL21 (DE3) overexpressing efflux sugar transporters compared to the control strain 1353; And

On FIG. 9 is a graph showing the concentration of lacto-N-triose II in the supernatant of E. coli strains BL21 (DE3) overexpressing the TP11 (2), YjhB (3), or TP70 (4) efflux sugar transporters.

DESCRIPTION OF EXAMPLES OF CARRYING OUT THE INVENTION

On FIG. 1 shows a schematic representation of an exemplary host cell 10 of the invention importing lactose and synthesizing lacto-N-triose II (LNT II) and lacto-N-tetraose (LNT). Lactose is imported into the cell from the medium in which the host cell is cultured using transporter 1. The enzyme N-acetylglucosaminyltransferase NacGlcT ligates N-acetylglucosamine with the acceptor substrate lactose to form LNT-II. LNT-II is exported from the cell via exporter protein 20. Since LNT-II is the precursor of LNT or LNnT, the LNT-II exporter is the exporter protein of the precursors of said oligosaccharides. As shown in FIG. 1, the cell contains a protein having β-1,3-galactosyltransferase activity, which provides galactosylation of LNT-II for intracellular formation of LNT; the cell may also and/or alternatively have a β-1,4-galactosyltransferase activity allowing galactosylation of LNT-II to form lacto-N-neotetraose LNnt intracellularly. The LNT, or in some cases LNnt, is then exported by the oligosaccharide exporter from the cell to the culture medium in which the cell is cultured.

Exporters are membrane-bound and may be overexpressed, LNT-II exporter overexpression increases LNT-II export and decreases LNT export, while deletion or other inactivation of the LNT-II exporter protein improves LNT export. . Preferably, the LNT-II exporter is an endogenous export protein, while the LNT export protein is preferably a heterologous export protein.

Example 1

Creation of an E. coli strain producing lacto-N-triose II

Escherichia coli BL21(DE3) was used to construct a lacto-N-triose II (LNT-2) producing strain. Metabolic engineering included mutagenesis and deletions of certain genes, respectively, and integration into the genome of heterologous genes. The lacZ and araA genes were inactivated by mutagenesis using non-complementary oligonucleotides as described by Ellis et al., “High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides”, Proc. Natl. Acad. sci. USA 98: 6742-6746 (2001).

Deletions in the genome were carried out according to the method proposed by Datsenko and Warner (Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000)). In the genome of E. coli strain BL21 (DE3), the genes encoding N-acetylglucosamine-6-phosphate deacetylase (nagA) and glucosamine-6-phosphate deaminase (nagB) were deleted to prevent intracellular degradation of N-acetylglucosamine. The wzxC-wcaJ genes were also deleted. WcaJ encodes a UDP-glucose:undecaprenyl phosphate glucose-1-phosphate transferase that catalyzes the first step in the synthesis of colanoic acid (Stevenson et al., J. Bacteriol. 1996, 178: 4885-4893). In addition, the fucI and fucK genes encoding L-fucose isomerase and L-fuculose kinase, respectively, were removed.

Integration into the genome of heterologous genes was carried out by transposition. The transposase EZ-Tn5™ (Epicentre, USA) was used for integration of linear DNA fragments or the hyperactive C9 mutant of transposase mariner Himar1 (Lampe et al., Proc. Natl. Acad. Sci. 1999, USA 96: 11428-11433) was used for transposition. . To generate EZ-Tn5 transposons, the gene of interest, together with an antibiotic resistance marker flanked by FRT sites, was amplified using primers 1119 and 1120 (all primers used are listed in Table 3 below); The resulting PCR product had 19-bp Mosaic End recognition sites for EZ-Tn5 transposase at both ends. For integration using the Himar1 transposase, the expressed constructs (operons) of interest were similarly cloned together with an antibiotic resistance marker flanked by FRT sites into the pEcomar vector. The pEcomar vector encodes a hyperactive mariner Himar1 transposase C9 mutant under the control of the arabinose-inducible P _araB promoter. The expressed fragment <P _tet -lacY-FRT-aadA-FRT> (SeqID1) was integrated using EZ-Tn5 transposase. After successful integration of the lactose importer gene LacY from E. coli K12 TG1 (accession number ABN72583), the resistance gene was removed from streptomycin-resistant clones using FLP recombinase encoded by plasmid pCP20 (Datsenko and Warner, Proc. Natl. Acad. Sci. 2000, USA 97: 6640-6645). The N-acetylglucosaminyltransferase lgtA gene from Neisseria meningitidis MC58 (accession number NP_274923) was codon-optimized for expression in E. coli and artificially synthesized by gene synthesis. Along with the galT gene encoding galactose-1-phosphate uridyltransferase from E. coli K-12 substrain MG1655 (accession number NP_415279), similarly obtained by gene synthesis, lgtA was inserted via transposition (SeqID2) using the pEcomar-lgtA-galT plasmid. To enhance the de novo synthesis of UDP-N-acetylglucosamine, genes encoding L-glutamine: D-fructose-6-phosphate aminotransferase (glmS), phosphoglucosamine mutase from E. coli K-12 substrain MG1655 (glmM) and N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase (glmU) from E. coli K-12 substrain MG1655 (accession numbers NP_418185, NP_417643, NP_418186, respectively) was codon optimized and obtained by gene synthesis. The glmUM operon was cloned under the control of the constitutive tetracycline P _tet promoter, and glmS was cloned under the control of the constitutive P _T5 promoter. A transposon comprising the <P _tet -glmUM-P _T5 -glmS-FRT-dhfr-FRT> (SeqID3) cassette flanked by inverted terminal repeats specifically recognized by the mariner-like element by Himar1 transposase was inserted from pEcomar-glmUM-glmS to obtain strain producing lacto-N-triose II. In addition, the expressed fragment <P _tet -lacY(6HIS)-FRT-aadA-FRT> (SeqID4) was integrated using EZ-Tn5 transposase.

The gal-operon (galETKM) from E. coli K12 TG1 (SeqID6) was amplified using primers 605 and 606 and inserted into the galM ybhJ locus of E. coli strain BL21 (DE3) by homologous recombination using the helper plasmid pKD46 encoding red recombinase ( Datsenko and Warner, Proc Natl Acad Sci 2000, USA 97: 6640-6645). Sequences of heterologous genes and gene clusters are given in Appendix 1.

Example 2

Cultivation of E. coli BL21 (DE3) 707 batchwise screening for various β-1,3-N-acetylglucosaminyltransferases

The β-1,3-N-acetylglucosaminyltransferase PmnagT gene from Pasteurella multocida subsp. multocida strain HN06 (accession number PMCN06_0022) was codon optimized and artificially synthesized at GenScript (Piscataway, USA). Gene cloning was performed by sequence and ligation independent cloning into the pET-DUET plasmid (Merck KGaA, Darmstadt, Germany). All primers used for cloning are listed in Table 3 below.

E. coli strain BL21(DE3) 707 (Table 2 below) carrying the pET-PmnagT plasmid encoding β-1,3-N-acetylglucosaminyltransferase was cultured at 30°C in a medium with inorganic salts (Samain et al., J. Biotech 1999, 72: 33-47) supplemented with 2% (w/v) glucose and 100 μg/ml ampicillin. When the cultures reached an optical density of 0.1 at 660 nm, gene expression was induced by adding 0.3 mM IPTG. After 4 hours of incubation, 1.5 mM lactose was added. After an additional incubation for 24 hours at 30° C., cells were harvested in shake flasks. For the detection of LNT-2 used thin layer chromatography. To do this, the cells were mechanically destroyed in a certain volume using glass beads. Then, for TLC, the samples were applied to TLC Silica Gel 60 F ₂₅₄ silica gel plates (Merck KGaA, Darmstadt, Germany). The mobile phase consisted of acetone:butanol:acetic acid:water (35:35:7:23).

The results of TLC analysis are shown in FIG. 2. The formation of a compound migrating at the same rate as the standard LNT-II trisaccharide was observed when the PmnagT gene was overexpressed. Strain 724 producing LNT-II was used as a control (19). Standards for lactose (1) and LNT-II (2) are given. The formation of the LNT-II product in the samples is marked with asterisks.

Example 3

Creation of a strain of E. coli producing lacto-N-triose II, overexpressing a homologous efflux sugar transporter

The export of oligosaccharides produced in E. coli has been proven to be the limiting factor in the fermentation process. However, trisaccharides such as 2'-fucosyllactose and LNT-2 pass to some extent into the culture supernatant, thus probably encoding a functioning sugar efflux transporter. To improve lacto-N-triose II (LNT-II; GluNAc(β1-3)Gal(β1-4)Glc) efflux, E. coli strain BL21(DE3) 1326 (Table 2 below) was used to screen a library of efflux sugar transporters ( SET). The candidate SET proteins from E. coli were amplified from E. coli BL21 (DE3) genomic DNA and integrated into the pINT vector by sequence independent cloning and ligation. When using, for example, the yjhB gene, primers 2567, 2568, 2526 and 2443 were used to obtain plasmid pINT-yjhB. The primer sequences used for cloning are shown in Table 3 below.

E. coli strain BL21(DE3) 1326 carrying plasmids encoding 20 different E. coli transporters was cultured at 30° C. in inorganic salt medium (Samain et al., J. Biotech. 1999, 72: 33-47) with addition of 2% (w/v) glucose, 100 μg/ml ampicillin and 40 μg/ml zeocin. When the cultures reached an optical density of 0.1 at 660 nm, gene expression was induced by adding 200 ng/ml of anhydrotetracycline. After 4 hours of incubation, 2.5 mM lactose was added. After additional incubation for 24 hours and 48 hours at 30°C in shake flasks, the concentration of LNT-II in the supernatant was determined.

Mass analysis was performed by detecting characteristic fragment (fragment) ions using a triple quadrupole mass spectrometric detection system for liquid chromatography. Precursor ions were sampled and analyzed in quadrupole 1, fragmentation was performed in a collision cell using nitrogen as the gas for collision-induced dissociation, fragment (fragmentation) ions were selected in quadrupole 3.

Lacto-N-tetraose (LNT (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc)), LNT-II and maltotriose (internal standard for quantitation) were analyzed in electrospray positive ionization mode. LNT forms an ion with m/z 708.3 M+H ⁺ ], LNT-II forms an ion with m/z 546.1 [M+H ⁺ ], and maltotriose forms an ion with m/z 522.0 [M+NH ₄ ⁺ ]. The formation of adducts of this carbohydrate [m/z 504.0] with the ammonium ion (NH4 ⁺ ) occurs, which leads to a mass shift of +18. Thus, the maltose precursor ion m/z 522.0 was isolated. Further fragmentation of the precursor ion occurred in the collision cell with the formation of characteristic fragment ions with m/z 487.1, m/z 325.0, and m/z 163.2. The molecular ion LNT (m/z 708.3) formed fragments with m/z 546.3, m/z 528.3, m/z 366.2, and m/z 204.0. LNT-II (m/z 546.1) formed fragments with m/z 204.2, m/z 186.0, 138.0 and m/z 126.0 (see method description).

Chromatographic separation of LNT and LNT-II was carried out on a Luna NH ₂ HPLC column (Phenomenex, Aschaffenburg, Germany). This was necessary due to partial fragmentation of the LNT during ionization, whereby the signals due to LNT-II affected the results of the quantification of individual carbohydrates.

An increased amount of LNT-2 in the culture supernatant was observed only for the strain expressing the yjhB gene (see Table 1 below).

Table 1. Calculated concentrations of LNT-II in the culture supernatant of E. coli strain BL21 (DE3) overexpressing yjhB and the reference strain Sample Calc. conc. after 24 h incubation [µM] Calc. conc. after 48 h incubation [µM] Analyte retention time 1326 751 1265 0.616 1326 pint-yjhB 413 1975 0.609

Example 4

Batch cultivation of E. coli BL21 (DE3) 724, screening for various β-1,4-galactosyltransferases

Genes of β-1,4-galactosyltransferases lex1 from Aggregatibacter aphrophilus NJ8700 (accession number YP_003008647), PmgalT7 from Pasteurella multocida subsp. multocida strain HN06 (accession number PMCN06_0021), MsgalT8 from Myxococcus stipitatus DSM14675 (accession number MYSTI_04346), KdgalT10 from Kingella denitrificans ATCC 33394 (accession number HMPREF9098_2407), gatD from Pasteurella multocida M1404 (accession number GQ444331), BfgalT2 from Bacterioidis fragilis NCTC9343 (accession number accession BF9343_0585), lsgD from Haemophilus influenza (accession number AAA24981) and HpgalT from Helicobacter pylori (accession number AB035971) were codon optimized and artificially synthesized by GenScript Cooperation (Piscataway, USA). Gene cloning was performed by sequence and ligation independent cloning (Li and Elledge, Nat Methods. 2007 Mar; 4(3): 251-6.). Thus, the pINT plasmid carrying the malE gene under the control of an anhydrotetracycline-activated promoter was used, which made it possible to carry out the N-terminal fusion of the β-1,4-galactosyltransferase and malE genes. The waaX gene from Pectobacterium atrosepticum JG10-08 (accession number ECA0154) encoding β-1,4-galactosyltransferase was cloned into the pACYC-Duet plasmid (Merck KGaA, Darmstadt, Germany) alone. All primers used for cloning are listed in Table 3 below.

E. coli strain BL21(DE3) 724 (Table 2 below) carrying the plasmid pCDF-galE and the plasmid encoding the fusion gene of malE and β-1,4-galactosyltransferase was cultured at 30° C. in a medium with inorganic salts (Samain et al ., J. Biotech. 1999, 72: 33-47) supplemented with 2% (w/v) glucose, 100 μg/ml ampicillin and 40 μg/ml zeocin. When the cultures reached an optical density of 0.1 at 660 nm, expression of the galE gene and β-1,4-galactosyltransferase was induced by the addition of 0.3 mm IPTG and 200 ng/ml anhydrotetracycline. The E. coli strain BL21(DE3) 534 (Table 2 below) carrying the plasmids pET-lgtA, pCOLA-glmUM-glmS, pCDF-galT-galE, and pACYC-waaX was cultured at 30°C in a medium with inorganic salts supplemented with 2 % (w/v) glucose, 100 μg/ml ampicillin, 34 μg/ml chloramphenicol, 50 μg/ml streptomycin and 30 μg/ml kanamycin. When the cultures reached an optical density of 0.1 at 660 nm, gene expression was induced by adding 0.3 mM IPTG. 4 hours after the induction of gene expression, 2 mM lactose was added. After an additional incubation for 48 hours at 30° C. in shake flasks, the cells were harvested and mechanically disrupted. Lacto-N-neotetraose (LNnT (Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc)) was determined by thin layer chromatography. To do this, the cells were mechanically destroyed in a certain volume using glass beads. Then, for TLC, the samples were applied to TLC Silica Gel 60 F ₂₅₄ silica gel plates (Merck KGaA, Darmstadt, Germany). The mobile phase consisted of acetone:butanol:acetic acid:water (35:35:7:23).

The results of the TLC analysis are shown in FIG. 3-5. On FIG. Figure 3 shows the results of TLC analysis of culture extracts of E. coli BL21(DE3) strains producing lacto-N-tetraose (LNT), overexpressing BfgalT2 (1), PmgalT7 (3), MsgalT8 (6), gatD (7), lex1 genes. (9), lgtB (11) or lsgD (13), encoding β-1,4-galactosyltransferase. Standards for lactose (15), LNT-II (16) and LNnT (17) are shown. The formation of the LNnT product in the samples is marked with asterisks.

On FIG. Figure 4 shows the results of TLC analysis of culture extracts of E. coli BL21(DE3) strains producing lacto-N-tetraose (LNT), overexpressing the KdgalT10 (1), cpsI14J (7), cpsIaJ (8, 9), HpgalT (12) genes. encoding β-1,4-galactosyltransferase. Standards for lactose (3), LNT-II (4, 16) and LNnT (5, 17) are shown. The formation of the LNnT product in the samples is marked with asterisks.

On FIG. 5 shows the results of TLC analysis of culture extracts of lacto-N-tetraose-producing E. coli BL21(DE3) strains overexpressing the waaX (5) gene encoding β-1,4-galactosyltransferase. The standards for lactose (1) LNT-II and LNnT (2) are shown. The formation of the LNnT product in the samples is also marked with asterisks.

The formation of a compound migrating at the same rate as the standard tetrasaccharide LNnT was observed upon overexpression of the following genes: lex1, PmgalT7, MsgalT8, BfgalT2, gatD, lsgD, KdgalT10, HpgalT, waaX.

β-1,4-galactosyltransferases cpsIaJ and cpsI14J, which are known in the literature to produce LNnT (Watanabe et al., J Biochem. 2002 Feb;131(2): 183-91; Kolkman et al., J Bacteriol. 1996 Jul ;178(13): 3736-41) were also included in the activity screen and served as a positive control. Using the expression system described herein, LNnT formation was observed for CpsIaJ and CpsI14J (FIG. 3). Only 11 out of 30 tested genes provided LNnT production from LNT-II and UDP-galactose.

Example 5

Cultivation of E. coli BL21 (DE3) 534 in batch mode, screening for various β-1,3-galactosyltransferases

Using E. coli K12 DH5α genomic DNA as a template, galE was amplified using primers 1163 and 1162. The PCR product was purified, restricted with restriction endonucleases NdeI and XhoI, and ligated into the second multiple cloning site of the pCDFDuet vector (Merck KGaA, Darmstadt, Germany), which was cut with the same enzymes. GalE was expressed under the control of the IPTG-inducible T7 promoter. The E. coli K12 galT gene was amplified from genomic DNA and inserted into the plasmid pCDF-galE by sequence independent cloning and ligation using primers 991-994 to obtain plasmid pCDF-galT-galE.

Using the codon-optimized lgtA gene as a template, amplification was carried out using primers 688 and 689. PCR product was purified, restriction endonucleases NdeI and AatII were carried out, and ligated into the multiple cloning site of the pETDuet vector (Merck KGaA, Darmstadt, Germany), which was cut with those the same enzymes, to obtain the plasmid pET-lgtA.

Cloning of the codon-optimized glmUM gene construct was performed by sequence and ligation independent cloning into the pCOLA-Duet plasmid (Merck KGaA, Darmstadt, Germany) using primers 848-851. The codon-optimized form of glmS was amplified using primers 852 and 853. The PCR product was purified, restricted with restriction endonucleases NdeI and AatII, and ligated into the second multiple cloning site of the pCOLA-glmUM vector, which was cut with the same enzymes, to obtain plasmid pCOLA-glmUM-glmS .

Genes of β-1,3-galactosyltransferases wbdO from Salmonella enterica subsp. salamae serovar Greenside (accession number AY730594) and furA from Lutiella nitroferrum 2002 (FuraDRAFT_0419) were also codon-optimized and artificially synthesized at GenScript Cooperation (Piscataway, USA). Gene cloning was performed by sequence and ligation independent cloning into the pACYC-Duet plasmid (Merck KGaA, Darmstadt, Germany). All primers used for cloning are listed in Table 3 below.

E. coli strain BL21(DE3) 534 carrying plasmids pET-lgtA, pCOLA-glmUM-glmS, pCDF-galT-galE and encoding β-1,3-galactosyltransferase plasmid pACYC-furA or pACYC-wbdO was cultured at 30°C in inorganic salt medium (Samain et al., J. Biotech. 1999, 72: 33-47) supplemented with 2% (w/v) glucose, 100 μg/ml ampicillin, 34 μg/ml chloramphenicol, 50 μg /ml streptomycin and 30 µg/ml kanamycin. When the cultures reached an optical density of 0.1 at 660 nm, gene expression was induced by adding 0.3 mM IPTG. After 4 hours of incubation, 2 mM lactose was added. After an additional incubation for 48 hours at 30° C., the cells were harvested in shake flasks. LNT was determined by thin layer chromatography. To do this, the cells were mechanically destroyed in a certain volume using glass beads. Then, for TLC, the samples were applied to TLC Silica Gel 60 F ₂₅₄ silica gel plates (Merck KGaA, Darmstadt, Germany). The mobile phase consisted of acetone:butanol:acetic acid:water (35:35:7:23).

On FIG. 6 shows the results of TLC analyzes of culture extracts of lacto-N-tetraose producing E. coli strains BL21(DE3) overexpressing the wbdO or furA genes encoding β-1,3-galactosyltransferase. The formation of the LNT product in the samples is labeled. Among the 12 candidate β-1,3-galactosyltransferases tested, the formation of a compound migrating at the same rate as the LNT tetrasaccharide standard was observed only when the wbdO and furA genes were overexpressed.

Example 6

Creation of an improved E. coli strain producing lacto-N-tetraose

Escherichia coli BL21 ₍ DE3 ₎ 724 strain was used to construct a strain producing lacto-N-tetraose (LNT). -FRT> (SeqID5) flanked by inverted terminal repeats specifically recognized by the mariner-like element Himar1 transposase, which was inserted from pEcomar-wbdO-galE. The resulting 1353 strain was subjected to further metabolic engineering to increase the intracellular pool of LNT-II, resulting in increased LNT production. Thus, the yjhB major mediator superfamily carrier (accession number YP_003001824) was deleted in the genome of the E. coli strain to obtain strain 1431 (Table 2 below).

E. coli BL21(DE3) strains 1353 and 1431 were cultured batchwise for 48 h at 30°C in inorganic salt medium (Samain et al., J. Biotech. 1999, 72: 33-47) containing 2% ( wt./about.) glucose as a source of carbon and energy. When the cultures reached an optical density of 0.5 at 660 nm, 2.5 mM lactose was added. The presence of LNT-II and LNT in the culture supernatant was determined by high performance liquid chromatography (HPLC).

HPLC analysis was performed using a refractive index detector (RID-10A) (Shimadzu, Duisburg, Germany) and a ReproSil Carbohydrate, 5 µm (250 mm × 4.6 mm) column (Dr. Maisch GmbH, Germany) connected to an HPLC system ( Shimadzu, Duisburg, Germany). Elution was carried out in isocratic mode at 35°C using as eluent a mixture of acetonitrile : H ₂ O (68/32 (v/v)) at a flow rate of 1.4 ml/min, 40 μl of sample was applied to the column. Samples were filtered (pore size 0.22 μm) and clarified by solid phase extraction on an ion exchange matrix (Strata ABW, Phenomenex, Aschaffenburg, Germany).

On FIG. 7 shows the results of HPLC analysis of the culture supernatant of E. coli strain BL21(DE3) producing lacto-N-tetraose. (A) Supernatant of E. coli strains BL21(DE3) 1353 (black graph) and 1431 (pink graph) cultured in the presence of glucose and lactose after 24 hours of incubation. (B) Supernatant of E. coli strains BL21(DE3) 1353 (blue graph) and 1431 (brown graph) cultured in the presence of glucose and lactose after 48 h of incubation. As shown by HPLC analysis, the deletion of yjhB in the strain producing LNT increased the accumulation of LNT in the culture supernatant.

Example 7

Creation of a lacto-N-tetraose-producing E. coli strain that overexpresses the efflux sugar transporter

Since the export of lacto-N-tetraose to the medium of the producing strains is only moderate, a library of efflux sugar transporters was screened. According to Example 3, candidate SET proteins were either amplified from E. coli genomic DNA or codon optimized and artificially synthesized by GenScript Cooperation (Piscataway, USA). After amplification, the genes were inserted into the pINT vector by sequence independent cloning and ligation. E. coli gene cloning primers were designed as described in Example 3. Synthesized genes were synthesized with standard nucleotide overhangs and similarly inserted into an expression vector using primers 2527, 2444, 2526, and 2443. The sequences of the primers used for cloning are given in Table 3 below.

E. coli strain BL21(DE3) 1353 (Table 2 below) carrying plasmids encoding 66 different transporters was cultured at 30° C. in inorganic salt medium (Samain et al., J. Biotech. 1999, 72: 33-47 ) supplemented with 3% (w/v) glucose, 5 g/l NH ₄ Cl ₂ , 100 μg/ml ampicillin and 15 μg/ml kanamycin. Pre-culture was carried out in 96-well plates containing a total volume of 200 μl. After 24 hours of incubation at 30° C. with continuous shaking, 50 μl from each well was transferred to 96-well deep well plates containing 400 μl of inorganic salt medium supplemented with 200 ng/ml anhydrotetracycline and 10 mM lactose. After an incubation period of 24 h to 48 h, the LNT concentrations in the supernatant were determined by LC-MS. Mass analysis was performed as described in Example 3.

On FIG. 8 shows the relative concentration of lacto-N-tetraose in the supernatant of E. coli strains BL21 (DE3) overexpressing efflux sugar transporters compared to the control strain 1353. The LNT titer of strain 1353 was taken as 100%. As shown in FIG. 8, overexpression of 11 of 66 genes resulted in a doubling of LNT production. Among them, the protein encoded by the E. coli genome BL21 (DE3) was shown to enhance LNT export (TP37, yebQ, accession number NC_012971). YebQ is a putative MFS transporter possibly mediating multiple drug efflux, which may be the transporter protein responsible for the major LNT efflux observed during 1353 fermentation.

In addition, exporters encoded by spoVB genes from Bacillus amyloliquefaciens (TP1, accession number AFJ60154), yabM from Erwinia pyrilfoliae (TP2, accession number CAY73138), bcr from E. coli MG1655 (TP18, accession number AAC75243), ydeA from E. coli MG1655 (TP20, accession number AAC74601), proP2 from Haemophilus parainfluenzae (TP54, accession number EGC72107), setA from Pectobacterium carotovorum (TP55, accession number ZP_03829909), fucP from E. coli MG1655 (TP59, accession number AIZ90162), mdeA from Staphylococcus aureus Bmb9393 (TP61, accession number SABB_01261), lmrA from Lactococcus lactis (TP62, accession number L116532), setA from Pseudomonas sp. MT-1 (TP72, accession number BAP78849) and setA from Beauveria bassiana D1-5 (TP73, accession number KGQ13398) increased LNT production when overexpressed in E. coli 1353 production strain.

Example 8

Creation of a lacto-N-triose II E. coli strain by overexpressing heterologous efflux sugar transporters

Curiously, the LNT exporter screen described in Example 6 revealed two proteins, TP11 from Mannheimia succiniciproducens MBEL55E (proP, accession number AAU37785) and TP70 from Cedecea neteri M006 (setA, accession number WP_039290253), whose overexpression resulted in a significant increase in LNT production. -II and, accordingly, a decrease in the production of LNT (results not shown). This observation was confirmed in the experiment described in Example 3. Overexpression of the efflux sugar transporter YjhB served as a positive control. Overexpression of TP11 as well as TP70 resulted in an approximately 4-fold increase in LNT-II production, which was even somewhat superior to that of YjhB: in FIG. 9 is a graph showing the concentrations of lacto-N-triose II in the supernatant of E. coli BL21 (DE3) strains overexpressing the TP11 (2), YjhB (3), or TP70 (4) efflux sugar transporters. Strain 1326 carrying an empty control plasmid served as control (1). In summary, 3 sugar efflux transporters that export LNT-II have been identified, the overexpression of which may be effective in engineering a strain that produces LNT-II.

Table 2. Strains and plasmids Strain Genotype Link E. coli BL21(DE3) F- ompT hsdSB(rB-, mB-) gal dcm (DE3) Merck KGaA, Darmstadt, Germany E. coli BL21(DE3) 534 E. coli BL21(DE3) ΔlacZ Δara ΔwcaJ ΔfucIK ΔnagAB carrying genome-integrated: galETKM, lacy This study E. coli BL21(DE3) 724 E. coli BL21(DE3) ΔlacZ Δara ΔwcaJ ΔfucIK ΔnagAB carrying genome-integrated: galETKM, lacY, lgtA-galT-kanR, glmUM-glmS-dhfr This study E. coli BL21(DE3) 1326 E. coli BL21(DE3) ΔlacZ Δara ΔwcaJ ΔfucIK ΔnagAB carrying genome-integrated: galETKM, lacY, lgtA-galT-kanR, glmUM-glmS-dhfr, lacy(6HIS)-aadA This study E. coli BL21(DE3) 707 E. coli BL21(DE3) ΔlacZ Δara ΔwcaJ ΔfucIK ΔnagAB carrying genome-integrated: galETKM, lacY, glmUM-glmS-dhfr This study E. coli BL21(DE3) 1353 E. coli BL21(DE3) ΔlacZ Δara ΔwcaJ ΔfucIK ΔnagAB carrying genome-integrated: galETKM, lacY, lgtA-galT-kanR, glmUM-glmS-dhfr, wbdO-galE-cat This study E. coli BL21(DE3) 1431 E. coli BL21(DE3) ΔlacZ Δara ΔwcaJ ΔfucIK ΔnagAB carrying genome-integrated: galETKM, lacY, lgtA-galT-kanR, glmUM-glmS-dhfr, wbdO-galE-cat, ΔyjhB-aacC1 This study pCDF-galE galE from E. coli K12 embedded in pCDFDuet vector EP 14 162 869.3 pET-lgtA (SeqID7) lgtA from Neisseria meningitidis embedded in pETDuet vector This study pCDF-galT-galE (SeqID8) galT and galE from Escherichia coli K12 embedded in pCDFDuet vector This study pCOLA-glmUM-glmS (SeqID9) glmU, glmM and glmS from Escherichia coli K12 embedded in pCOLADuet vector This study pINT-malE-lex1 Merged gene from malE with lex-1 from Aggregatibacter aphrophilus NJ8700 inserted into pINT vector EP 14 162 869.3 pINT-malE-PmgalT7 (SeqID10) The fusion gene from PmgalT7 from Pasteurella multocida subsp. multocida str. HN06 embedded in vector pINT This study pINT-malE-MsgalT8 (SeqID11) Fusion gene from MsgalT8 from Myxococcus stipitatus DSM14675 inserted into pINT vector This study pINT-malE-KdgalT10 (SeqID12) Fusion gene from KdgalT10 from Kingella denitrificans ATCC 33394 inserted into pINT vector This study pINT-malE-gatD (SeqID13) Fusion gene from gatD from Pasteurella multocida M1404 inserted into pINT vector This study pINT-malE-BFgalT2 (SeqID14) Fusion gene from BfgalT2 from Bacterioidis fragilis NCTC9343 inserted into pINT vector This study pINT-malE-lsgD (SeqID15) Merged gene from lsgD from Haemophilus influenza embedded in pINT vector This study pINT-malE-HPgalT (SeqID16) A fusion gene from HpgalT from Helicobacter pylori inserted into a pINT vector This study pACYC-waaX (SeqID17) waaX from Pectobacterium atrosepticum JG10-08 embedded in pACYCDuet vector This study pACYC-wbdO (SeqID18) wbdO from Salmonella enterica subsp. salamae serovar Greenside embedded in vector pACYCDuet This study pACYC-furA (SeqID19) furA from Lutiella nitroferrum 2002 embedded in pACYCDuet vector This study pET-PmnagT (SeqID20) PmnagT from Pasteurella multocida subsp. multocida str. HN06 embedded in pETDuet vector This study pINT-yjhB (SeqID21) yjhB from E. coli BL21 DE3 embedded in pINT vector This study pINT-yebQ (SeqID22) yebQ from E. coli BL21 DE3 embedded in pINT vector This study pINT-proP (SeqID23) proP from Mannheimia succiniciproducens MBEL55E embedded in pINT vector This study pINT-Cn-setA (SeqID24) setA from Cedecea neteri M006 embedded in vector pINT This study pINT-spoVB (SeqID25) spoVB from Bacillus amyloliquefaciens embedded in pINT vector This study pINT-yabM (SeqID26) yabM from Erwinia pyrifoliae embedded in pINT vector This study pINT-ydeA (SeqID27) ydeA from E. coli MG1655 embedded in pINT vector This study pINT-proP2 (SeqID28) proP2 from Haemophilus parainfluenzae embedded in pINT vector This study pINT-Pc-setA (SeqID29) setA of Pectobacterium carotovorum embedded in pINT vector This study pINT-fucP (SeqID30) fucP from Escherichia coli BL21 (DE3) embedded in pINT vector This study pINT-mdeA (SeqID31) mdeA from Staphylococcus aureus Bmb9393 embedded in pINT vector This study pINT-lmrA (SeqID32) lmrA from Lactococcus lactis embedded in pINT vector This study pINT-Ps-setA (SeqID33) setA from Pseudomonas sp. MT-1 embedded in vector pINT This study pINT-Bb-setA (SeqID34) setA from Beauveria bassiana D1-5 embedded in vector pINT This study

Table 3. Oligonucleotides used for PCR primer Sequence 5'-3' 605 KI gal fwd TTACTCAGCAATAAACTGATATTCCGTCAGGCTGG (SeqID35) 606 KI gal rev TTGTAATTCTCGCGCTCTTCACATCAGACTTTCCATATAGAGCGTAATTTCCGTTAACGTCGGTAGTGCTGACCTTGCCGGAGG (SeqID36) 1119 ME for CTGTCTCTTATCACATCTCCTGAAATGGCCAGATGTAATTCCTAATTTTTGTTG (SeqID37) 1120ME rev CTGTCTCTTATCACATCTCACATTACATCTGAGCGATTGTTAGG (SeqID38) 1163 galE_NdeI-for GATCACATATGAGAGTTCTGGTTACCGGTG (SeqID39) 1164 galE_XhoI-rev GATCACTCGAGTCATTAATCGGGATATCCCTGTGGATGGC (SeqID40) 5176 lex1 pINT-f GTCGATGAAGCCCTGAAAGACCGCGCAGACTATGCACTTCATTGAAAACAAAAACTTCGTC (SeqID41) 5177 lex1 pint-r GATGGCCTTTTTGCGTGTCGACGCGGCCGCCTAGATAAACAGGATGATATTTTTGCCTTG (SeqID42) 5178 pINT lex1-f CAAGGCAAAAATATCATCCTGTTTATCTAGGCGGCCGCGTCGACACGCAAAAAGGCCATC (SeqID43) 5179 pINT lex1-r GACGAAGTTTTTGTTTTCAATGAAGTGCATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID44) 5192 waaX pint for GTCGATGAAGCCCTGAAAGAACGCGCAGACTATGATTGATAACCTGATTAGCGTACCCCG (SeqID45) 5193 waaX pint rev ATGGCCTTTTTGCGTGTCGACGCGGCCGCTTAATTCGAGCGGGTAAAGATCTTCATCAGG (SeqID46) 5194 pint waaX for CTGATGAAGATCTTTACCCGCTCGAATTAAGCGGCCGCGTCGACACGCAAAAAGGCCATC (SeqID47) 5195 pint waaX rev CGGGGTACGCTTAATCAGGTTATCAATCATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID48) 5164 PmgalT7 pINT for GTCGATGAAGCCCTGAAAGACGCGCAGACTATGAGCGGTGAACACTATGTCATTAGCCTG (SeqID49) 5165 PmgalT7 pINT rev GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCATTTAAATTCGATGATCATCTTGTCGTT (SeqID50) 5166 pINT PmgalT7 for AACGACAAGATGATCATCGAATTTAAATGAGCGGCCGCGTCGACACGCAAAAAGGCCATC (SeqID51) 5167 pINT PmgalT7 rev CAGGCTAATGACATAGTGTTCACCGCTCATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID52) 5168 MsgalT8 pINT for GTCGATGAAGCCCTGAAAGACGCGCAGACTATGGATGAAATCAAACTGTCGGTGGTTATG (SeqID53) 5169 MsgalT8 pINT rev GATGGCCTTTTTGCGTGTCGACGCGGCCGCTTGGCGACGCCAATCGAACGCAACGCG (SeqID54) 5170 pINT MsgalT8 for CGCGTTGCGTTCGATTGGCGTCGCCAATGAGCGGCCGCGTCGACACGCAAAAAAGGCCATC (SeqID55) 5171 pINT MsgalT8 rev CATAACCACCGACAGTTTGATTTCATCCATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID56) 5561 KdgalT10 pINT for GTCGATGAAGCCCTGAAAGACGCGCAGACTATGGAAAACTATGTCGTCTCTATCCGCACC (SeqID57) 5562 KdgalT10 pINT-rev GATGGCCTTTTTGCGTGTCGACGCGGCCGCTTTGAACGGAACAATCTTTTTGTCATC (SeqID58) 5563 pINT-KdgalT10 for GATGACAAAAAGATTGTTCCGTTCAAATGAGCGGCCGCGTCGACACGCAAAAAGGCCATC (SeqID59) 5564 pINT-KdgalT10 rev GGTGCGGATAGAGACGACATAGTTTTCCATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID60) 5172 gatD pINT for GTCGATGAAGCCCTGAAAGACGCGCAGACTATGTCCTCAGCTTTCCATTACGTCATTAGC (SeqID61) 5173 gatD pINT rev GATGGCCTTTTTGCGTGTCGACGCGGCCGCTTCAAATTCGATAATCATGGTGATTTTT (SeqID62) 5174 pINT gatD for AAAATCACCATGATTATTCGAATTTGAATGAGCGGCCGCGTCGACACGCAAAAAGGCCATC (SeqID63) 5175 pINT gatD rev GCTAATGACGTAATGGAAAGCTGAGGACATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID64) 5160 BfglaT2 pINT for GTCGATGAAGCCCTGAAAGACCGCGCAGACTATGAACGTGAATAAGCCGACCACCGAAAAG (SeqID65) 5161 BfgalT2 pINT rev GATGGCCTTTTTGCGTGTCGACGCGGCCGCTCAGTATTTCTTCAATTTTGTCCAGTTGATA (SeqID66) 5162 pINT BfgalT2 for TATCAACTGGACAAAATTGAAGAATACTGAGCGGCCGCGTCGACACGCAAAAAGGCCATC (SeqID67) 5163 pINT BfgalT2 rev CTTTTCGGTGGTCGGCTTATTCACGTTCATAGTCTGCGCGTCTTTCAGGGCTTCATCGAC (SeqID68) 5746 GTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACT (SeqID69) 5747 GCGGCCGCGTCGACACGCAAAAAAGGCCATCCATCCGTCAGGATGGCCTTCTGCTTAATTT (SeqID70) 5748 AAATTAAGCAGAAGGCCATCCTGACGGATGGATGGCCTTTTTGCGTGTCGACGCGGCCGC (SeqID71) 5749 AGTCTGCGCGTCTTTCAGGGCTTCATCGACAGTCTGACGACCGCTGGCGGCGTTGATCAC (SeqID72) 1886 SLIC wbdO pACYC for GTTTAACTTTAATAAGGAGATATACCATGCTGACGGAAGTGCGCCCGGTCTCTACGACGAAACCGC (SeqID73) 1887 SLIC wbdO pACYC rev CGACCTGCAGGCGCCGAGCTCGAATTCATTTGATGTATTTGCAATAGAACACAGAAAAGACCGT (SeqID74) 1888 SLIC pACYC wbdo rev GTGTTCTATTGCAAATACATCAAATGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGG (SeqID75) 1889 SLIC pACYC WbdO For GAGACCGGGCGCACTTCCGTCAGCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGG (SeqID76) 1890 SLIC pACYC furA rev GTATGGTGACCCTGTGGCGCAAATGAGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCT (SeqID77) 1891 SLIC pACYC furA for GCGCTGCCCTGTTTGATTTTATCCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCT(SeqID78) 1892 SLIC furA pACYC rev CCTGCAGGCGCGCCGAGCTCGAATTCTCATTTGCGCCACAGGGTCACCATACGTGCCGGCAGG (SeqID79) 1893 SLIC furA pACYC for GTTTAACTTTAATAAGGAGATATACCATGGGATAAAATCAAACAGGGCAGCGCCTCTCTGGTTGTCG (SeqID80) 3055 SLIC PmnagT pET rev CAGACTCGAGGGTACCGACGTCCTAATAAGTAGATGAATATTTATCAGGACGAAGAT (SeqID81) 3056 SLIC pET PmnagT for AACTAAAGGTTTATTTTCCATATGTATATCTCCTTCTTATACTTAACTAATATAC (SeqID82) 3057 SLIC pET PmnagT rev TAAATATTCATCTACTTATTAGGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCG (SeqID83) 3058 SLIC PmnagT pET for GTATAAGAAGGAGATATACATATGGAAAATAAACCTTTAGTTTCAGTTTTGATTTGTGC (SeqID84) 2567_SLIC_yjhB-for TAACTTTAAGAAGGAGATATACAAGAGCTCGAGTCGAAGGAGATAGAACCATGGCAACAGCATGGTATAAACAAG (SeqID85) 2568_SLIC_yjhB-rev GCGTGTCGACGCGTTTAGAGGCCCCAAGGGGTTATGCTAGTATCGATTTATCATTTAGCCACGGATAGTTTATAAATTTTAC (SeqID86) 2526_SLIC_pINT_TP-rev GGTTCTATCTCCTTCGACTCGAGCTCTTGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGATTTTTGTCGAAC (SeqID87) 2443_SLIC_pINT_TP-forw TAAATCGATACTAGCATAACCCCTTGGGGCCTCTAAACGCGTCGACACGCAAAAAAGGCCATCC (SeqID88) 2527_SLIC_TP_pINT-forw GTTCGACAAAAAATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAAGAGCTCGAGTCGAAGGAGATAGAACC (SeqID89) 2444_SLIC_TP_pINT-rev GGATGGCCTTTTTGCGTGTCGACGCGTTTAGAGGCCCCAAGGGGTTATGCTAGTATCGATTTA (SeqID90) 688 lgtA AatII rev ATATGACGTCTCATTAGCGGTTTTTCAGGAGACG (SeqID91) 689 lgtA NdeI for ATATCATATGCCGTCCGAAGCATTCCGTCGTCACC (SeqID92) 991 galT-pCDF for TAACTTTAATAAGGAGATATACCATGACGCAATTTAATCCCGTTGATCATCCACATCGCCGC (SeqID93) 992 pCDF-galT for ATTTTCGCGAATCCGGAGTGTAAAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCG (SeqID94) 993 galT-pCDF rev AAGCATTATGCGGCCGCAAGCTTTTACACTCCGGATTCGCGAAAATGGATATCGCTGACTGCGCGCAAACGC (SeqID95) 994 pCDF-galT rev TCAACGGGATTAAATTGCGTCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGGGG (SeqID96) 848 glmM pCOLA SLIC rev ATGGTGATGGCTGCTGCCCATTTAAACCGCTTTGACTGCGTCGGCAATACGGTGCGC (SeqID97) 849 glmU pCOLA SLIC for GTTTAACTTTAATAAGGAGATATACCATGCTGAACAACGCGATGTCTGTTGTTATCCTGG (SeqID98) 850 pCOLA glmM SLIC rev CGCAGTCAAAGCGGTTTAAATGGGCAGCAGCCATCACCATCATCACCACAGCC (SeqID99) 851 pCOLA glmU SLIC for TCGCGTTGTTCAGCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGG (SeqID100) 852 glmSco pCOLA for NdeI ATATCATATGTGCGGTATCGTTGGTGCTATTCGC (SeqID101) 853 glmSco pCOLA rev Aat II ATATATGACGTCTTATTCCACGGTCACGGATTTCGC (SeqID102)

--->

Sequence listing

<110> JENNEWEIN BIOTECHNOLOGY GMBH

<120> PRODUCTION OF HUMAN MILK OLIGOSACCHARIDES

IN HOST MICROORGANISMS WITH MODIFIED IMPORT/EXPORT

<130> 2827P111EP

<160> 102

<170>PatentIn version 3.5

<210> 1

<211> 2851

<212> DNA

<213> Artificial sequence

<220>

<223> construct Ptet-lacY-FRT-add1-FRT

<400> 1

tggccagatg attaattcct aatttttgtt gacactctat cattgataga gttatttttac 60

cactccctat cagtgataga gaaaagtgaa atgaatagtt cgacaaaaat ctagaaataa 120

ttttgtttaa ctttaagaag gagatataca aatgtactat ttaaaaaaca caaacttttg 180

240

cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta ttatttttgc 300

cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt ctgacaaact 360

cgggctgcgc aaatacctgc tgtggattat taccggcatg ttagtgatgt ttgcgccgtt 420

ctttattttt atcttcgggc cactgttaca atacaacatt ttagtaggat cgattgttgg 480

tggtatttat ctaggctttt gttttaacgc cggtgcgcca gcagtagagg catttattga 540

gaaagtcagc cgtcgcagta atttcgaatt tggtcgcgcg cggatgtttg gctgtgttgg 600

ctgggcgctg tgtgcctcga ttgtcggcat catgttcacc atcaataatc agtttgtttt 660

ctggctgggc tctggctgtg cactcatcct cgccgtttta ctctttttcg ccaaaacgga 720

tgcgccctct tctgccacgg ttgccaatgc ggtaggtgcc aaccattcgg catttagcct 780

taagctggca ctggaactgt tcagacagcc aaaactgtgg tttttgtcac tgtatgttat 840

tggcgtttcc tgcacctacg atgtttttga ccaacagttt gctaatttct ttacttcgtt 900

ctttgctacc ggtgaacagg gtacgcgggt atttggctac gtaacgacaa tgggcgaatt 960

acttaacgcc tcgattatgt tctttgcgcc actgatcatt aatcgcatcg gtgggaaaaa 1020

cgccctgctg ctggctggca ctattatgtc tgtacgtatt attggctcat cgttcgccac 1080

ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg tttgaagtac cgttcctgct 1140

ggtggggctgc tttaaatata ttaccagcca gtttgaagtg cgtttttcag cgacgattta 1200

1260

1320

gggcttcacc ttaatttccg tgttcacgct tagcggcccc ggcccgcttt ccctgctgcg 1380

tcgtcaggtg aatgaagtcg ctgggagcta agcggccgcg tcgacacgca aaaaggccat 1440

ccgtcaggat ggccttctgc ttaatttgat gcctggcagt ttatggcggg cgtcctgccc 1500

1560

tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag 1620

cctttcgttt tatttgatgc ctggcagttc cctactctcg catggggaga ccccacacta 1680

ccatcatgta tgaatatcct ccttagttcc tattccgaag ttcctattct ctagaaagta 1740

taggaacttc ggcgcgtcct acctgtgaca cgcgtgccgc agtctcacgc ccggagcgta 1800

1860

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgaggga 1920

agcggtgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca 1980

tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa 2040

gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg 2100

gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct 2160

ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc 2220

taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga 2280

gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt 2340

tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt 2400

tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga 2460

gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc 2520

gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt 2580

catacttgaa gctagacagg cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc 2640

agatcagttg gaagaatttg tccactacgt gaaaggcgag atcaccaagg tagtcggcaa 2700

ataatgtcta acaattcgtt caagccgagg ggccgcaaga tccggccacg atgacccggt 2760

cgtcgggtac cggcagggcg gggcgtaagg cgcgccattt aaatgaagtt cctattccga 2820

agttcctatt ctctagaaag tataggaact t 2851

<210> 2

<211> 4568

<212> DNA

<213> Artificial sequence

<220>

<223> construction Ptet-lgtA-PT5-galT-FRT-kanR-FRT

<400> 2

acaggttggc tgataagtcc ccggtctagc ttgcatgcag attgcagcat tacacgtctt 60

gagcgattgt gtaggctgga gctgcttcga agttcctata ctttctagag aataggaact 120

tcggaatagg aacttcattt aaatggcgcg ccttacgccc cgccctgccg gtaccgagag 180

cgcttttgaa gctggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca 240

tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg 300

aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg aaccccagag 360

tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc 420

ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat 480

atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc 540

gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg 600

ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc 660

tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat 720

ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg 780

atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc 840

aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc 900

cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga 960

tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa 1020

aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt 1080

ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg 1140

caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcttga 1200

tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcaggggctt 1260

cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg tccataaaac 1320

cgccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc tctttgcgct 1380

tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt 1440

ctgcggactg gctttctacg tgttccgctt cctttagcag cccttgcgcc ctgagtgctt 1500

1560 gcggcagcgt gaggggatct tgacgcgtgt cacaggtagg

tttctagaga ataggaactt cggaatagga actaaggagg atattcatac atgatggtag 1620

tgttcgaaat taatacgact cactataggg gaattgattc tggtaccaaa tgagtcgacc 1680

ggccagatga ttaattccta atttttgttg acactctatc attgatagag ttattttacc 1740

actccctatc agtgatagag aaaagtgaaa tgaaatagttc gacaaaaatc tagaaataat 1800

tttgtttaac tttaagaagg agatatacaa atgccgtccg aagcattccg tcgtcaccgt 1860

gcttatcgcg aaaacaaact gcagccactg gtctctgtcc tgatctgcgc atacaacgtt 1920

gagaaatact tcgcacagtc tctggcagct gtagttaacc agacctggcg taacctggat 1980

atcctgatcg tagatgacgg ctctacggat ggtacgctgg cgatcgcaca gcgtttccag 2040

gaacaggacg gtcgtatccg cattctcgct cagccgcgta actctggtct gatcccgtct 2100

ctgaacatcg gtctggacga actggccaaa tctggtggtg gtggcgaata catcgcccgt 2160

actgacgccg acgacattgc ggccccggat tggatcgaaa aaatcgtagg tgaaatggag 2220

aaagaccgct ctatcatcgc gatgggtgct tggctggaag ttctgtccga agagaaagac 2280

ggtaaccgtc tggcccgtca ccatgaacac ggcaaaatct ggaaaaaacc gacccgtcac 2340

gaagatatcg cggacttctt cccgttcggt aacccgatcc ataacaacac catgatcatg 2400

taatcgacgg tggtctgcgt tacaacaccg aacgtgattg ggcagaagac 2460

taccagtttt ggtatgacgt gtctaaactg ggtcgtctgg cttactaccc agaagcgctg 2520

gttaaatacc gtctgcacgc caaccaggtt agctccaaat actccatccg tcagcacgaa 2580

atcgcacagg gtatccagaa aacggctcgt aacgacttcc tgcagtccat gggtttcaaa 2640

acccgtttcg actctctgga gtaccgtcag atcaaagcgg ttgcgtatga gctgctggag 2700

aaacacctgc cggaagagga ctttgaacgt gcgcgtcgtt tcctgtacca gtgcttcaaa 2760

cgtaccgaca ctctgccggc gggtgcatgg ctcgactttg cagcggatgg tcgtatgcgt 2820

cgtctgttta ccctgcgtca gtacttcggt atcctgcatc gtctcctgaa aaaccgctaa 2880

tgatttcgtc gacacacagg aaacatatta aaaattaaaa cctgcaggag tttaaacgcg 2940

gccgcgatat cgttgtaaaa cgacggccag tgcaagaatc ataaaaaatt tatttgcttt 3000

caggaaaatt tttctgtata atagattcat aaatttgaga

taacaattcc ccatcttagt atattagtta agtataaata cacaaggaga tataccatga 3120

cgcaatttaa tcccgttgat catccacatc gccgctacaa cccgctcacc gggcaatgga 3180

ttctggtttc accgcaccgc gctaagcgcc cctggcaggg ggcgcaggaa acgccagcca 3240

aacaggtgtt acctgcgcac gatccagatt gcttcctctg cgcaggtaat gtgcgggtga 3300

3360

tgatgtctga cacgccagat gcgccagaaa gtcacgatcc gctgatgcgt tgccagagcg 3420

3480

tcagcgttgc agcattgacg gaaatcgtca aaacctggca ggagcaaacc gcagaactgg 3540

3600

acccgcatcc gcacggtcag atttgggcaa atagcttcct gcctaacgaa gctgagcgcg 3660

aagaccgcct gcaaaaagaa tattttgccg aacagaaatc accaatgctg gtggattatg 3720

ttcagcgcga gctggcagac ggtagccgta ccgttgtcga aaccgaacac tggttagccg 3780

3840

tacggatcac cgatttgacc gacgcccagc gcagcgatct ggcgctggcg ttgaaaaagc 3900

tgaccagtcg ttatgacaac ctcttccagt gctccttccc ctactctatg ggctggcacg 3960

gcgcgccatt taatggcgaa gagaatcaac actggcagct gcacgcgcac ttttatccgc 4020

ctctgctgcg ctccgccacc gtacgtaaat ttatggttgg ttatgaaatg ctggcagaga 4080

cccagcgaga cctgaccgca gaacaggcag cagagcgttt gcgcgcagtc agcgatatcc 4140

attttcgcga atccggagtg taacgcggag gcgcgccatt taaatcaacc tcagcggtca 4200

tagctgtttc ctgtgactga gcaataacta gcataacccc ttggggcctc taaacgggtc 4260

ttgaggggtt ttttgctgaa accaatttgc ctggcggcag tagcgcggtg gtcccacctg 4320

accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc 4380

atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg 4440

gcctttcggg atccaggccg gcctgttaac gaattaatct tccgcggcaa caaaaattag 4500

gaattaatca tctggccaat ttcaggtggc acttttcgggg cagaccgggg acttatcagc 4560

caacctgt 4568

<210> 3

<211> 6521

<212> DNA

<213> Artificial sequence

<220>

<223> construction Ptet-glmUM-PT5-glmS-FRT-dhfr-FRT

<400> 3

acaggttggc tgataagtcc ccggtctagc ttgcatgcag attgcagcat tacacgtctt 60

gagcgattgt gtaggctgga gctgcttcga aattaatacg actcactata ggggaattga 120

ttctggtacc aaatgagtcg accggccaga tgattaattc ctaatttttg ttgacactct 180

atcattgata gagttatttt accactccct atcagtgata gagaaaagtg aaatgaatag 240

ttcgacaaaa atctagaaat aattttgttt aactttaaga aggagatata caaatgctga 300

acaacgcgat gtctgttgtt atcctggcgg cgggtaaagg tacccgtatg tactctgacc 360

tgccgaaagt tctgcacacc ctggcgggta aagcgatggt tcagcacgtt atcgacgcgg 420

cgaacgaact gggtgcggcg cacgttcacc tggtttacgg tcacggtggt gacctgctga 480

aacaggcgct gaaagacgac aacctgaact gggttctgca ggcggaacag ctgggtaccg 540

gtcacgcgat gcagcaggcg gcgccgttct tcgcggacga cgaagacatc ctgatgctgt 600

acggtgacgt tccgctgatc tctgttgaaa ccctgcagcg tctgcgtgac gcgaaaccgc 660

agggtggtat cggtctgctg accgttaaac tggacgaccc gaccggttac ggtcgtatca 720

cccgtgaaaa cggtaaagta accggtatcg ttgaacacaa agacgcgacc gacgaacagc 780

gtcagatcca ggagatcaac accggtatcc tgatcgcgaa cggtgcagac atgaaacgtt 840

ggctggcgaa actgaccaac aacaacgcgc agggtgaata ctacatcacc gacatcatcg 900

cgctggcgta cgggaaggt cgtgaaatcg ttgcggttca cccgcagcgt ctgtctgaag 960

ttgaaggtgt taacaaccgt ctgcagctgt ctcgtctgga acgtgtttac cagtctgaac 1020

aggcggaaaa actgctgctg gcgggtgtta tgctgcgtga cccggcgcgt ttcgacctgc 1080

gtggtaccct gacccacggt cgtgacgttg aaatcgacac caacgttatc atcgaaggta 1140

acgttaccct gggtcaccgt gtaaaaatcg gcaccggttg cgttatcaaa aactctgtta 1200

tcggtgacga ctgcgaaatc tctccgtaca ccgttgttga agacgcgaac ctggcggcgg 1260

cgtgcaccat cggtccgttc gcgcgtctgc gtccgggtgc ggaactgctg gaaggtgcgc 1320

acgttggtaa cttcgttgaa atgaaaaaag cgcgtctggg taaaggttct aaagcgggtc 1380

acctgaccta cctgggtgac gcggaaatcg gtgacaacgt taacatcggt gcgggtacca 1440

tcacctgcaa ctacgacggt gcgaacaaat tcaaaaccat catcggtgac gacgttttcg 1500

ttggttctga cacccagctg gttgcgccgg ttaccgttgg taaaggtgcg accatcgcgg 1560

cgggtaccac cgttacccgt aacgttggtg aaaacgcgct ggcgatctct cgtgttccgc 1620

agaccagaa agaaggttgg cgtcgtccgg ttaaaaaaaa ataacgaagg agatagaacc 1680

atgtccaacc gtaaatactt cggtacggac ggtatccgtg gtcgtgtagg tgatgctccg 1740

attacgccgg atttcgtcct gaaactcggt tgggcagcgg gtaaagttct cgcacgtcac 1800

ggctctcgta aaatcatcat cggtaaagac acccgtatct ctggttacat gctcgaatct 1860

gcactggaag cgggtctggc tgcagctggt ctgtctgcac tgttcacgggg tccgatgcca 1920

accccagctg tagcgtacct gactcgcact ttccgtgcag aagcaggtat cgtgatctct 1980

gcctctcaca acccgttcta cgacaacggt atcaaattct tcagcatcga tggtaccaaa 2040

ctcccagacg cggttgaaga ggctatcgaa gcggaaatgg agaaagaaat ctcttgtgta 2100

gactctgccg aactcggtaa agcgtctcgt atcgttgatg cagcgggtcg ttacatcgag 2160

ttctgcaaag ccaccttttcc gaacgaactg agcctgtctg agctgaaaat cgtcgtagac 2220

tgtgccaacg gtgcgactta ccacattgcc ccaaacgtac tgcgtgagct gggtgctaac 2280

gtcatcgcga tcggttgtga accgaacggt gtcaacatca acgcggaagt aggtgcgacc 2340

gatgttcgtg cactgcaggc tcgtgtactc gcggagaaag cggatctcgg tatcgccttt 2400

gacggtgatg gtgaccgtgt tatcatggtt gaccacgaag gtaacaaagt ggatggtgac 2460

cagatcatgt acatcattgc ccgtgaaggt ctgcgtcagg gtcagctgcg tggtggtgca 2520

gtaggtaccc tcatgagcaa catgggtctg gaactggccc tgaaacagct gggtatccca 2580

ttcgctcgtg ctaaagtagg cgaccgttac gttctggaga aaatgcagga gaaaggttgg 2640

cgtatcggtg ccgaaaactc tggtcacgtc atcctgctgg acaaaaccac taccggtgac 2700

ggtatcgtag caggtctgca ggtactcgcc gctatggccc gtaaccacat gtccctccat 2760

gacctctgct ctggtatgaa aatgttcccg cagatcctgg ttaacgttcg ttacaccgca 2820

ggttctggtg atccgctgga acacgagtct gtgaaagccg ttaccgcaga agtggaagcg 2880

gccctgggta accgtggtcg tgtactgctg cgtaaatccg gtactgagcc actgatccgt 2940

gttatggttg agggcgaaga tgaagcccag gtcaccgaat ttgcgcaccg tattgccgac 3000

3060

aggagtttaa acgcggccgc gatatcgttg taaaacgacg gccagtgcaa gaatcataaa 3120

3180

gtttttgtga gcggataaca attccccatc ttagtatatt agttaagtat aaatacacaa 3240

ggagatatac atatgtgcgg tatcgttggt gctatcgcac agcgtgatgt agcggagatc 3300

ctcctggaag gtctgcgtcg tctcgaatac cgtggttacg actctgccgg tctggcagta 3360

gtggatgcag aaggtcacat gactcgtctg cgtcgtctgg gtaaagtgca gatgctcgcg 3420

caggcggcgg aagaacaccc actccacggt ggtacggggta tcgcacacac tcgttgggca 3480

acccacggtg aaccgtctga ggtcaacgca cacccgcatg ttagcgagca catcgtagtc 3540

3600

tacaccttcg taagcgaaac cgacacggaa gttatcgccc acctcgttaa ctgggaactc 3660

aaacagggtg gtactctgcg tgaagcagtt ctgcgtgcca ttccacagct gcgtggtgca 3720

tacggtaccg tgatcatgga ctctcgtcat ccggataccc tgctcgccgc acgttctggt 3780

tctccactcg ttatcggtct gggtatggggt gagaacttca tcgcctctga tcagctggcc 3840

ctgctcccag ttacccgtcg cttcatcttc ctggaagagg gtgacatcgc cgaaatcacc 3900

3960

tctaacctgc agtatgacgc tggtgacaaa ggcatctacc gtcactacat gcagaaagag 4020

atctacgaac agccgaacgc gatcaaaaac accctgaccg gtcgtatctc tcacggtcag 4080

gttgacctgt ctgagctggg tccaaacgcg gacgaactcc tgtccaaagt cgagcacatc 4140

cagatcctgg cttgtggtac ctcttacaac tccggtatgg tttctcgtta ctggttcgaa 4200

tctctggcag gtatcccatg cgacgttgaa atcgcctccg aattccgtta tcgtaaatct 4260

gcggtacgtc gtaactccct catgatcacc ctgtctcagt ctggtgaaac cgctgatact 4320

ctggcaggtc tgcgtctcag caaagaactg ggttacctgg gttctctggc catctgcaac 4380

gttccgggtt ctagcctggt tcgtgagtct gacctggctc tgatgaccaa cgcgggtacg 4440

gagatcggtg ttgcctctac caaagcgttc actacccagc tcactgtcct gctgatgctg 4500

gttgccaaac tgtctcgtct caaaggcctc gacgctagca tcgaacacga catcgtacac 4560

ggtctgcagg ccctcccatc tcgtatcgag cagatgctgt ctcaggacaa acgtatcgaa 4620

gcactggcag aagacttcag cgacaaacac cacgcgctgt ttctgggtcg tggtgaccag 4680

tacccaattg cgctggaagg tgccctgaaa ctgaaagaga tcagctacat ccatgcagag 4740

gcatacgcag cgggtgagct gaaacatggt ccactggccc tgatcgacgc agatatgccg 4800

gttattgtgg ttgctccgaa caacgaactg ctggagaaac tgaaatccaa catcgaggaa 4860

gtacgtgcgc gtggtggtca gctgtacgtg tttgctgacc aggacgcggg tttcgtttcc 4920

agcgacaaca tgcacatcat cgaaatgccg catgttgaag aggtaatcgc gccaatcttc 4980

tacaccgtac cgctgcagct gctggcgtac catgtagccc tgatcaaagg tacggacgtt 5040

gaccagccgc gtaacctggc gaaatccgtg accgtggaat aacgcgggagg cgcgccattt 5100

aaatcaacct cagcggtcat agctgtttcc tgtgactgag caataactag cataacccct 5160

tggggcctct aaacgggtct tgaggggttt tttgctgaaa ccaatttgcc tggcggcagt 5220

agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat 5280

ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa 5340

ggctcagtcg aaagactgggg cctttcggga tccaggccgg cctgttaacg aattaatctt 5400

ccgcggcggt atcgataagc ttgatatcga attccgaagt tcctattctc tagaaagtat 5460

aggaacttca ggtctgaaga ggagtttacg tccagccaag ctagcttggc tgcaggtcgt 5520

cgaaattcta cggggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag 5580

cagccccgct gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat 5640

ccaccggtag gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc 5700

ctcccctagt caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa 5760

tggaagtagc acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc 5820

gggtaggcct ttggggcagc ggccaaatagc agctttgctc cttcgctttc tgggctcaga 5880

ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg 5940

cccgaaggtc ctccgggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc 6000

tgttctcctc ttcctcatct ccgggccttt cgacctgcag cctgttgaca attaatcatc 6060

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tgggtcaaag 6120

tagcgatgaa gccaacgctc ccgttgcagg gcagtttgcg cttcccctga gtgccacctt 6180

tggcttaggg gatcgcgtac gcaagaaatc tggtgccgct tggcagggtc aagtcgtcgg 6240

ttggtattgc acaaaactca ctcctgaagg ctatgcggtc gagtccgaat cccacccagg 6300

ctcagtgcaa atttatcctg tggctgcact tgaacgtgtg gcctaatgag gggatcaatt 6360

ctctagagct cgctgatcag aagttcctat tctctagaaa gtataggaac ttcgatggcg 6420

cctcatccct gaagccaata caacaaaaat taggaattaa tcatctggcc aatttcaggt 6480

ggcacttttc gggcagaccg gggacttatc agccaacctg t 6521

<210> 4

<211> 2937

<212> DNA

<213> Artificial sequence

<220>

<223> construct <Ptet-lacY(6HIS)-FRT-aadA-FRT>

<400> 4

ggccagatga ttaattccta atttttgttg acactctatc attgatagag ttattttacc 60

actccctatc agtgatagag aaaagtgaaa tgaaatagttc gacaaaaatc tagaaataat 120

tttgtttaac tttaagaagg agatatacaa atgggctact atttaaaaaa cacaaacttt 180

240

ttcccgattt ggctacatga catcaaccat atcagcaaaa gtgatacgggg tattattttt 300

gccgctattt ctctgttctc gctattattc caaccgctgt ttggtctgct ttctgacaaa 360

ctcgggctgc gcaaatacct gctgtggatt attaccggca tgttagtgat gtttgcgccg 420

ttctttattt ttatcttcgg gccactgtta caatacaaca ttttagtagg atcgattgtt 480

ggtggtattt atctaggctt ttgttttaac gccggtgcgc cagcagtaga ggcattttatt 540

gagaaagtca gccgtcgcag taatttcgaa tttggtcgcg cgcggatgtt tggctgtgtt 600

ggctgggcgc tgtgtgcctc gattgtcggc atcatgttca ccatcaataa tcagtttgtt 660

ttctggctgg gctctggctg tgcactcatc ctcgccgttt tactcttttt cgccaaaacg 720

gatgcgccct cttctcatca ccatcaccat cacgccacgg ttgccaatgc ggtaggtgcc 780

aaccattcgg catttagcct taagctggca ctggaactgt tcagacagcc aaaactgtgg 840

tttttgtcac tgtatgttat tggcgtttcc tgcacctacg atgtttttga ccaacagttt 900

gctaatttct ttacttcgtt ctttgctacc ggtgaacagg gtacgcgggt atttggctac 960

gtaacgacaa tgggcgaatt acttaacgcc tcgattatgt tctttgcgcc actgatcatt 1020

aatcgcatcg gtgggaaaaa cgccctgctg ctggctggca ctattatgtc tgtacgtatt 1080

attggctcat cgttcgccac ctcagcgctg gaagtggtta ttctgaaaac gctgcatatg 1140

tttgaagtac cgttcctgct ggtgggctgc tttaaatata ttaccagcca gtttgaagtg 1200

cgtttttcag cgacgattta tctggtctgt ttctgcttct ttaagcaact ggcgatgatt 1260

tttatgtctg tactggcggg caatatgtat gaaagcatcg gtttccaggg cgcttatctg 1320

gtgctgggtc tggtggcgct gggcttcacc ttaatttccg tgttcacgct tagcggcccc 1380

ggcccgcttt ccctgctgcg tcgtcaggtg aatgaagtcg cttaagcggc cgcgtcgaca 1440

cgcaaaaagg ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg 1500

1560

cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca 1620

gtctttcgac tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg 1680

gagaccccac actaccatca tgtatgaata tcctccttag ttcctattcc gaagttccta 1740

ttctctagaa agtataggaa cttcggcgcg tcctacctgt gacacgcgtg ccgcagtctc 1800

acgcccggag cgtagcgacc gagtgagcta gctatttgtt tatttttcta aatacattca 1860

aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 1920

aagagtatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc 1980

gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg 2040

gatggcggcc tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt 2100

gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga 2160

gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg 2220

tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt 2280

gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca 2340

agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct 2400

gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac 2460

tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta 2520

accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc 2580

2640

ttggcctcgc gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc 2700

aaggtagtcg gcaaataatg tctaacaatt cgttcaagcc gaggggccgc aagatccggc 2760

cacgatgacc cggtcgtcgg gtaccggcag ggcggggcgt aaggcgcgcc atttaaatga 2820

agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcgaag cagctccagc 2880

ctacacaatc gctcaagacg tgtaatgctg caatctgcat gcaagcttgg cactggc 2937

<210> 5

<211> 3856

<212> DNA

<213> Artificial sequence

<220>

<223> construct <Ptet-wbdO-PT5-galE-FRT-cat-FRT>

<400> 5

acaggttggc tgataagtcc ccggtctgcc cgaaaagtgc cacctgaaat tggccagatg 60

attaattcct aatttttgtt gattctggta ccaaatgagt cgaccggcca gatgattaat 120

tcctaatttt tgttgacact ctatcattga tagagttatt ttaccactcc ctatcagtga 180

240

gaaggagata tacaaatgct gacggaagtg cgcccggtct ctacgacgaa accgctggtg 300

tctgtgattc tgccggtgaa caaattcaac ccgtatctgg atcgtgcaat tcattcaatc 360

ctgagtcagt cctatccgtc gattgaactg attatcattg caaacaattg caccaatgac 420

tttttcgatg ctctgaaaaa acgtgaatgt gaaaccatta aagtgctgcg cacgaacatc 480

gcgtatctgc cgtactgcct gaataaaggc ctggatctgt gtaacggtga ctttgttgcc 540

cgcatggatt cagatgacat ttcgcacccg gaacgtatcg atcgccaggt cgacttcctg 600

attaacaatc cggacatcga tgtggttggc accaatgcag tctatattga tgaagatgac 660

atcgaactgg aaaaaagcaa cctgccggtg aacaataacg ctattcgtaa aatgctgccg 720

tataaatgct gtctggtgca tccgtctgtt atgtttcgca aaaatgtcgt gatcaccagc 780

ggcggttaca tgttcgcgaa ttattctgaa gattacgaac tgtggaaccg tctggccgtt 840

gaaggccgca atttttataa cctgagcgaa tacctgctgt attaccgtct gcacaataac 900

caatcaacgt cgaaaaataa cctgtttatg gtgatggcga acgatgtcgc cattaaagtg 960

aaatatttcc tgctgaccaa gaaaattagc tacctgctgg gtatcattcg cacggtcttt 1020

1080

attaaaacct gcaggagttt aaacgcggcc gcgatatcgt tgtaaaacga cggccagtgc 1140

aagaatcata aaaaatttat ttgctttcag gaaaattttt ctgtataata gattcataaa 1200

tttgagagag gagtttttgt gagcggataa caattcccca tcttagtata ttagttaagt 1260

ataaatacac cgcgggaggcg tcgaaggaga tacaaccatg agagttctgg ttaccggtgg 1320

tagcggttac attggaagtc atacctgtgt gcaattactg caaaacggtc atgatgtcat 1380

cattcttgat aacctctgta acagtaagcg cagcgtactg cctgttatcg agcgtttagg 1440

1500

cctgcacgat cacgctatcg acaccgtgat ccacttcgcc gggctgaaag ccgtgggcga 1560

atcggtacaa aaaccgctgg aatattacga caacaatgtc aacggcactc tgcgcctgat 1620

tagcgccatg cgcgccgcta acgtcaaaaa ctttattttt agctcctccg ccaccgttta 1680

tggcgatcag cccaaaattc catacgttga aagcttcccg accggcacac cgcaaagccc 1740

ttacggcaaa agcaagctga tggtggaaca gatcctcacc gatctgcaaa aagcccagcc 1800

ggactggagc attgccctgc tgcgctactt caacccggtt ggcgcgcatc cgtcggggcga 1860

tatgggcgaa gatccgcaag gcattccgaa taacctgatg ccatacatcg cccaggttgc 1920

tgtaggccgt cgcgactcgc tggcgatttt tggtaacgat tatccgaccg aagatggtac 1980

tggcgtacgc gattacatcc acgtaatgga tctggcggac ggtcacgtcg tggcgatgga 2040

aaaactggcg aacaagccag gcgtacacat ctacaacctc ggcgctggcg taggcaacag 2100

cgtgctggac gtggttaatg ccttcagcaa agcctgcggc aaaccggtta attatcattt 2160

tgcaccgcgt cgcgagggcg accttccggc ctactgggcg gacgccagca aagccgaccg 2220

tgaactgaac tggcgcgtaa cgcgcacact cgatgaaatg gcgcaggaca cctggcactg 2280

gcagtcacgc catccacagg gatatcccga ttaacgccat ttaaatcaac ctcagcggtc 2340

atagctgttt cctgtgactg agcaataact agcataaccc cttggggcct ctaaacgggt 2400

cttgaggggt tttttgctga aaccaatttg cctggcggca gtagcgcggt ggtcccacct 2460

gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc 2520

catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg 2580

ggcctttcgg gatccaggcc ggcctgttaa cgaattaatc ttccgcggcg gtatcgataa 2640

gcttgatatc gaggctgaca tgggaattag ccatggtcca tatgaatatc ctccttagtt 2700

2760

ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg ggaagccctg 2820

ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca actttcacca 2880

taatgaaata agatcactac cgggcgtatt ttttgagttg tcgagatttt caggagctaa 2940

ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca 3000

tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt 3060

tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc 3120

ggcctttatt cacattcttg ccgcctgat gaatgctcat ccggaattac gtatggcaat 3180

gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga 3240

gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct 3300

3360

gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga 3420

tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta 3480

3540

tggcttccat gtcggcagat gcttaatgaa tacaacagta ctgcgatgag tggcagggcg 3600

3660

tataggaact tcgaagcagc tccagcctac acaatcgctc aagacgtgta atgctgcaat 3720

ctgcatgcaa gcttggcact ggcgatggcg cctcatccct gaagccaata agcagctcca 3780

gcctacacaa tcgctcaaga cgtgtaatgc tgcaatctgc atgcaagcta gaccggggac 3840

ttatcagcca acctgt 3856

<210> 6

<211> 4259

<212> DNA

<213> Artificial sequence

<220>

<223> construction galMKTE

<400> 6

ttactcagca ataaactgat attccgtcag gctggaatac tcttcgccag gacgcaggaa 60

gcagtccggt tgcggccatt cagggtggtt cgggctgtcc ggtagaaact cgctttccag 120

agccagccct tgccagtcgg cgtaaggttc ggttccccgc gacggtgtgc cgccgaggaa 180

gttgccggag tagaattgca gagccggagc ggtggtgtag accttcagct gcaatttttc 240

atctgctgac cagacatgcg ccgccacttt cttgccatcg cctttggcct gtaacaagaa 300

tgcgtgatcg taacctttca ctttgcgctg atcgtcgtcg gcaagaaact cactggcgat 360

gattttggcg ctgcggaaat caaaagacgt tccggcgaca gatttcaggc cgtcgtgcgg 420

aatgccgcct tcatcaaccg gcagatattc gtccgccaga atctgcaact tgtgattgcg 480

cacgtcagac tgctcgccgt caagattgaa atagacgtga ttagtcatat tcaccgggca 540

600

ttgcaccgtc gcgccgagat tacccgggaa gccctgatca ccatcatctg aactcagggc 660

aaacagcacc tgacgatcgt tctggttcac aatctgccag cgacgtttgt cgaacccttc 720

cggcccgccg tgcagctggt taacgccctg acttggcgaa agcgtcacgg tttcaccgtc 780

aaaggtataa cggctattgg cgatacggtt ggcataacga ccaatagagg cccccagaaa 840

cgcggcctga tcctgatagc attccgggct ggcacagccg agcagcgcct cgcggacgct 900

gccatcggaa agcggaatac gggcggaaag taaagtcgca ccccagtcca tcagcgtgac 960

taccatccct gcgttgttac gcaaagttaa cagtcggtac ggctgaccat cgggtgccag 1020

tgcgggagtt tcgttcagca ctgtcctgct ccttgtgatg gtttacaaac gtaaaaagtc 1080

tctttaatac ctgtttttgc ttcatattgt tcagcgacag cttgctgtac ggcaggcacc 1140

agctcttccg ggatcagcgc gacgatacag ccgccaaatc cgccgccggt catgcgtacg 1200

1260

gtgatttcga aatcatcgcg catagaggca tgagactccg ccatcaactc gcccatacgt 1320

ttcaggtcgc cttgctccag cgcgctggca gcttcaacgg tgcgggcgtt ttcagtcagt 1380

atatgacgca cgcgttttgc cacgatcggg tccagttcat gcgcaacagc gttgaactct 1440

tcaatggtga catcacgcag ggctggctgc tggaagaaac gcgcaccggt ttcgcactgt 1500

tcacgacggg tgttgtattc gctgccaacc agggtacgtt tgaagttact gttgatgatg 1560

acgacagcca cacctttgggg catggaaact gctttggtcc ccagtgagcg gcaatcgatc 1620

agcaaggcat gatctttctt gccgagcgcg gaaattagct gatccatgat cccgcagtta 1680

cagcctacaa actggttttc tgcttcctga ccgttaagcg cgatttgtgc gccgtccagc 1740

ggcagatgat aaagctgctg caatacggtt ccgaccgcga cttccagtga agcggaagaa 1800

cttaacccgg caccctgcgg cacattgccg ctgatcacca tgtccacgcc gccgaagctg 1860

ttgttacgca gttgcagatg tttcaccacg ccacgaacgt agttagccca ttgatagttt 1920

tcatgtgcga caatgggcgc atcgagggaa aactcgtcga gctgattttc ataatcggct 1980

gccatcacgc gaactttacg gtcatcgcgt ggtgcacaac tgatcacggt ttgataatca 2040

atcgcgcagg gcagaacgaa accgtcgttg tagtcggtgt gttcaccaat caaattcacg 2100

cggccaggcg cctgaatggt gtgagtggca gggtagccaa atgcgttggc aaacagagat 2160

tgtgtttttt ctttcagact catttcttac actccggatt cgcgaaaatg gatatcgctg 2220

actgcgcgca aacgctctgc tgcctgttct gcggtcaggt ctcgctgggt ctctgccagc 2280

atttcataac caaccataaa tttacgtacg gtggcggagc gcagcagagg cggataaaag 2340

tgcgcgtgca gctgccagtg ttgattctct tcgccattaa atggcgcgcc gtgccagccc 2400

atagagtagg ggaaggagca ctggaagagg ttgtcataac gactggtcag ctttttcaac 2460

gccagcgcca gatcgctgcg ctgggcgtcg gtcaaatcgg tgatccgtaa aacgtgggct 2520

ttgggcagca gtagcgtttc gaacggccag gcagcccagt aaggcacgac ggctaaccag 2580

tgttcggttt cgacaacggt acggctaccg tctgccagct cgcgctgaac ataatccacc 2640

agcattggtg atttctgttc ggcaaaatat tctttttgca ggcggtcttc gcgctcagct 2700

2760

atcgccgcgc ctttgttttc aaaaacctgc acccatgggt acgttttccc cagttctgcg 2820

gtttgctcct gccaggtttt gacgatttcc gtcaatgctg caacgctgag ctctggcagc 2880

gttttactgt gatccggtga aaagcagatc acccggctgg tgccgcgcgc gctctggcaa 2940

cgcatcagcg gatcgtgact ttctggcgca tctggcgtgt cagacatcaa agccgcaaag 3000

tcattagtga aaacgtaagt cccggtgtaa tcggggtttt tatcgcctgt cacccgcaca 3060

ttacctgcgc agaggaagca atctggatcg tgcgcaggta acacctgttt ggctggcgtt 3120

3180

gtgagcgggt tgtagcggcg atgtggatga tcaacgggat taaattgcgt catggtcgtt 3240

ccttaatcgg gatatccctg tggatggcgt gactgccagt gccaggtgtc ctgcgccatt 3300

tcatcgagtg tgcgcgttac gcgccagttc agttcacggt cggctttgct ggcgtccgcc 3360

3420

caggctttgc tgaaggcatt aaccacgtcc agcacgctgt tgcctacgcc agcgccgagg 3480

ttgtagatgt gtacgcctgg cttgttcgcc agtttttcca tcgccacgac gtgaccgtcc 3540

gccagatcca ttacgtggat gtaatcgcgt acgccagtac catcttcggt cggataatcg 3600

ttaccaaaaa tcgccagcga gtcgcgacgg cctacagcaa cctgggcgat gtatggcatc 3660

aggttattcg gaatgccttg cggatcttcg cccatatcgc ccgacggatg cgcgccaacc 3720

gggttgaagt agcgcagcag ggcaatgctc cagtccggct gggctttttg cagatcggtg 3780

3840

3900

ataaagtttt tgacgttagc ggcgcgcatg gcgctaatca ggcgcagagt gccgttgaca 3960

ttgttgtcgt aatattccag cggtttttgt accgattcgc ccacggcttt cagcccggcg 4020

aagtggatca cggtgtcgat agcgtgatcg tgcaggatct cggtcatcaa cgcttcgtta 4080

4140

acgctgcgct tactgttaca gaggttatca agaatgatga catcatgacc gttttgcagt 4200

aattgcacac aggtatgact tccaatgtaa ccgctaccac cggtaaccag aactctcat 4259

<210> 7

<211> 6431

<212> DNA

<213> Artificial sequence

<220>

<223> construct pET-lgtA

<400> 7

ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag 60

gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag 120

ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag 180

taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 240

gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 300

tgccgtccga agcattccgt cgtcaccgtg cttatcgcga aaacaaactg cagccactgg 360

tctctgtcct gatctgcgca tacaacgttg agaaatactt cgcacagtct ctggcagctg 420

tagttaacca gacctggcgt aacctggata tcctgatcgt agatgacggc tctacggatg 480

gtacgctggc gatcgcacag cgtttccagg aacaggacgg tcgtatccgc attctcgctc 540

agccgcgtaa ctctggtctg atcccgtctc tgaacatcgg tctggacgaa ctggccaaat 600

ctggtggtgg tggcgaatac atcgcccgta ctgacgccga cgacattgcg gccccggatt 660

ggatcgaaaa aatcgtaggt gaaatggaga aagaccgctc tatcatcgcg atgggtgctt 720

ggctggaagt tctgtccgaa gagaaagacg gtaaccgtct ggcccgtcac catgaacacg 780

gcaaaatctg gaaaaaaccg acccgtcacg aagatatcgc ggacttcttc ccgttcggta 840

acccgatcca taacaacacc atgatcatgc gtcgtagcgt aatcgacggt ggtctgcgtt 900

acaacaccga acgtgattgg gcagaagact accagttttg gtatgacgtg tctaaactgg 960

gtcgtctggc ttactaccca gaagcgctgg ttaaataccg tctgcacgcc aaccaggtta 1020

gctccaaata ctccatccgt cagcacgaaa tcgcacaggg tatccagaaa acggctcgta 1080

acgacttcct gcagtccatg ggtttcaaaa cccgtttcga ctctctggag taccgtcaga 1140

tcaaagcggt tgcgtatgag ctgctggaga aacacctgcc ggaagaggac tttgaacgtg 1200

cgcgtcgttt cctgtaccag tgcttcaaac gtaccgacac tctgccggcg ggtgcatggc 1260

tcgactttgc agcggatggt cgtatgcgtc gtctgtttac cctgcgtcag tacttcggta 1320

tcctgcatcg tctcctgaaa aaccgctaat gagacgtcgg taccctcgag tctggtaaag 1380

aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat 1440

taacctagggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac 1500

gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc 1560

gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 1620

1680

cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 1740

tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 1800

gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 1860

cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 1920

gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 1980

gaattttaac aaaatattaa cgtttacaat ttctggcggc acgatggcat gagattatca 2040

2100

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 2160

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 2220

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 2280

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 2340

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 2400

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 2460

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 2520

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 2580

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 2640

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 2700

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 2760

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 2820

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 2880

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 2940

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 3000

3060 caatcatgat tgaagcattt atcagggtta ttgtctcatg

tatttagaaa aataaacaaa taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc 3120

actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 3180

3240

3300

atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 3360

ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 3420

gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 3480

cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 3540

tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 3600

3660

ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 3720

gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggcctttt ttacggttcc 3780

tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 3840

ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 3900

3960 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat

atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc 4020

cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc 4080

4140

tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 4200

ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag 4260

atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg 4320

cttctgataa agcggggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc 4380

gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc 4440

acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa 4500

ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc 4560

gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg 4620

aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg 4680

aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt 4740

cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg 4800

gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg 4860

actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa 4920

cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 4980

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt 5040

ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg 5100

agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat 5160

ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga 5220

gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat 5280

ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt 5340

ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg 5400

attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa 5460

tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag 5520

tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc 5580

aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc 5640

cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc 5700

tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg 5760

atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga 5820

ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg 5880

aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg 5940

gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac 6000

atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt cggggcgcta 6060

tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc 6120

6180

ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc 6240

ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt 6300

ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc 6360

cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac 6420

gactcactat a 6431

<210> 8

<211> 5739

<212> DNA

<213> Artificial sequence

<220>

<223> construct pCDF-galT-galE

<400> 8

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atgacgcaat ttaatcccgt tgatcatcca catcgccgct acaacccgct 120

caccgggcaa tggattctgg tttcaccgca ccgcgctaag cgcccctggc agggggcgca 180

ggaaacgcca gccaaacagg tgttacctgc gcacgatcca gattgcttcc tctgcgcagg 240

taatgtgcgg gtgacaggcg ataaaaaccc cgattacacc gggacttacg ttttcactaa 300

tgactttgcg gctttgatgt ctgacacgcc agatgcgcca gaaagtcacg atccgctgat 360

gcgttgccag agcgcgcgcg gcaccagccg ggtgatctgc ttttcaccgg atcacagtaa 420

aacgctgcca gagctcagcg ttgcagcatt gacggaaatc gtcaaaacct ggcaggagca 480

aaccgcagaa ctggggaaaa cgtacccatg ggtgcaggtt tttgaaaaca aaggcgcggc 540

gatgggctgc tctaacccgc atccgcacgg tcagatttgg gcaaaatagct tcctgcctaa 600

cgaagctgag cgcgaagacc gcctgcaaaa agaatatttt gccgaacaga aatcaccaat 660

gctggtggat tatgttcagc gcgagctggc agacggtagc cgtaccgttg tcgaaaccga 720

acactggtta gccgtcgtgc cttactggggc tgcctggccg ttcgaaacgc tactgctgcc 780

caaagcccac gttttacgga tcaccgattt gaccgacgcc cagcgcagcg atctggcgct 840

ggcgttgaaa aagctgacca gtcgttatga caacctcttc cagtgctcct tcccctactc 900

tatgggctgg cacggcgcgc catttaatgg cgaagagaat caacactggc agctgcacgc 960

1020

aatgctggca gagacccagc gagacctgac cgcagaacag gcagcagagc gtttgcgcgc 1080

agtcagcgat atccattttc gcgaatccgg agtgtaaaag cttgcggccg cataatgctt 1140

aagtcgaaca gaaagtaatc gtattgtaca cggccgcata atcgaaatta atacgactca 1200

ctatagggga attgtgagcg gataacaatt ccccatctta gtatattagt taagtataag 1260

1320

agtcatacct gtgtgcaatt actgcaaaac ggtcatgatg tcatcattct tgataacctc 1380

tgtaacagta agcgcagcgt actgcctgtt atcgagcgtt taggcggcaa acatccaacg 1440

tttgttgaag gcgatattcg taacgaagcg ttgatgaccg agatcctgca cgatcacgct 1500

atcgacaccg tgatccactt cgccgggctg aaagccgtgg gcgaatcggt acaaaaaccg 1560

ctggaatatt acgacaacaa tgtcaacggc actctgcgcc tgattagcgc catgcgcgcc 1620

1680

attccatacg ttgaaagctt cccgaccggc acaccgcaaa gcccttacgg caaaagcaag 1740

ctgatggtgg aacagatcct caccgatctg caaaaagccc agccggactg gagcattgcc 1800

ctgctgcgct acttcaaccc ggttggcgcg catccgtcgg gcgatatggg cgaagatccg 1860

caaggcattc cgaataacct gatgccatac atcgcccagg ttgctgtagg ccgtcgcgac 1920

tcgctggcga tttttggtaa cgattatccg accgaagatg gtactggcgt acgcgattac 1980

atccacgtaa tggatctggc ggacggtcac gtcgtggcga tggaaaaact ggcgaacaag 2040

ccaggcgtac acatctacaa cctcggcgct ggcgtaggca acagcgtgct ggacgtggtt 2100

aatgccttca gcaaagcctg cggcaaaccg gttaattatc attttgcacc gcgtcgcgag 2160

ggcgaccttc cggcctactg ggcggacgcc agcaaagccg accgtgaact gaactggcgc 2220

gtaacgcgca cactcgatga aatggcgcag gacacctggc actggcagtc acgccatcca 2280

cagggatatc ccgattaatg actcgagtga tctcgagtct ggtaaagaaa ccgctgctgc 2340

gaaatttgaa cgccagcaca tggactcgtc tactagcgca gcttaattaa cctaggctgc 2400

tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacgggg tcttgagggg 2460

ttttttgctg aaacctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa 2520

taaaccggta aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac 2580

gaccgggtca tcgtggccgg atcttgcggc ccctcggctt gaacgaattg ttagacatta 2640

tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc 2700

tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat 2760

gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg 2820

cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg 2880

ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc 2940

aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc 3000

aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg 3060

ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt 3120

agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg 3180

gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg 3240

ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg 3300

cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag 3360

atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc 3420

3480

gagcggatac atatttgaat gtatttagaa aaataaacaa atagctagct cactcggtcg 3540

ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt tacccacaga 3600

ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc gccggtggcg 3660

tttttccata ggctccgcc tcctgccaga gttcacataa acagacgctt ttccggtgca 3720

tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac ccgacaggac 3780

ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg ttccgaccct 3840

gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc gctttctcat 3900

agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg ggctgtaagc 3960

aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca cttgagtcca 4020

acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg gagttcgcag 4080

aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt ccggctacac 4140

tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt taagcagttc 4200

cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg tttacagggc 4260

aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctactgaac 4320

cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac 4380

gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg 4440

gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac 4500

attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 4560

ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt 4620

ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga 4680

gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg 4740

ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt 4800

ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat 4860

cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt 4920

gaaaaccggga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc 4980

5040

ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg 5100

taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa 5160

ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg 5220

gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac 5280

aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg 5340

cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg 5400

5460

aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg 5520

cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt 5580

ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg 5640

ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta 5700

tgcgactcct gcattaggaa attaatacga ctcactata 5739

<210> 9

<211> 8232

<212> DNA

<213> Artificial sequence

<220>

<223> construct pCOLA-glmUM-glmS

<400> 9

cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc 60

gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat 120

cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag 180

caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc 240

catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac 300

gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg 360

tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa 420

ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca 480

ttaggaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctgtag 540

aaataatttt gtttaacttt aataaggaga tataccatgc tgaacaacgc gatgtctgtt 600

gttatcctgg cggcgggtaa aggtacccgt atgtactctg acctgccgaa agttctgcac 660

accctggcgg gtaaagcgat ggttcagcac gttatcgacg cggcgaacga actgggtgcg 720

gcgcacgttc acctggttta cggtcacggt ggtgacctgc tgaaacaggc gctgaaagac 780

gacaacctga actgggttct gcaggcggaa cagctgggta ccggtcacgc gatgcagcag 840

gcggcgccgt tcttcgcgga cgacgaagac atcctgatgc tgtacggtga cgttccgctg 900

atctctgttg aaaccctgca gcgtctgcgt gacgcgaaac cgcagggtgg tatcggtctg 960

ctgaccgtta aactggacga cccgaccggt tacggtcgta tcacccgtga aaacggtaaa 1020

gtaaccggta tcgttgaaca caaagacgcg accgacgaac agcgtcagat ccaggagatc 1080

aacaccggta tcctgatcgc gaacggtgca gacatgaaac gttggctggc gaaactgacc 1140

aacaacaacg cgcagggtga atactacatc accgacatca tcgcgctggc gtaccaggaa 1200

ggtcgtgaaa tcgttgcggt tcacccgcag cgtctgtctg aagttgaagg tgttaacaac 1260

1320

1380

ggtcgtgacg ttgaaatcga caccaacgtt atcatcgaag gtaacgttac cctgggtcac 1440

cgtgtaaaaa tcggcaccgg ttgcgttatc aaaaactctg ttatcggtga cgactgcgaa 1500

atctctccgt acaccgttgt tgaagacgcg aacctggcgg cggcgtgcac catcggtccg 1560

ttcgcgcgtc tgcgtccggg tgcggaactg ctggaaggtg cgcacgttgg taacttcgtt 1620

gaaatgaaaa aagcgcgtct gggtaaaggt tctaaagcgg gtcacctgac ctacctgggt 1680

gacgcggaaa tcggtgacaa cgttaacatc ggtgcgggta ccatcacctg caactacgac 1740

ggtgcgaaca aattcaaaac catcatcggt gacgacgttt tcgttggttc tgacacccag 1800

ctggttgcgc cggttaccgt tggtaaaggt gcgaccatcg cggcgggtac caccgttacc 1860

cgtaacgttg gtgaaaacgc gctggcgatc tctcgtgttc cgcagaccca gaaagaaggt 1920

tggcgtcgtc cggttaaaaa aaaataacga aggagataga accatgtcca accgtaaata 1980

cttcggtacg gacggtatcc gtggtcgtgt aggtgatgct ccgattacgc cggatttcgt 2040

cctgaaactc ggttgggcag cgggtaaagt tctcgcacgt cacggctctc gtaaaatcat 2100

catcggtaaa gacacccgta tctctggtta catgctcgaa tctgcactgg aagcgggtct 2160

ggctgcagct ggtctgtctg cactgttcac gggtccgatg ccaaccccag ctgtagcgta 2220

cctgactcgc actttccgtg cagaagcagg tatcgtgatc tctgcctctc acaacccgtt 2280

ctacgacaac ggtatcaaat tcttcagcat cgatggtacc aaactcccag acgcggttga 2340

agaggctatc gaagcggaaa tggagaaaga aatctcttgt gtagactctg ccgaactcgg 2400

taaagcgtct cgtatcgttg atgcagcggg tcgttacatc gagttctgca aagccacctt 2460

tccgaacgaa ctgagcctgt ctgagctgaa aatcgtcgta gactgtgcca acggtgcgac 2520

ttaccacatt gccccaaacg tactgcgtga gctgggtgct aacgtcatcg cgatcggttg 2580

tgaaccgaac ggtgtcaaca tcaacgcgga agtaggtgcg accgatgttc gtgcactgca 2640

ggctcgtgta ctcgcggaga aagcggatct cggtatcgcc tttgacggtg atggtgaccg 2700

tgttatcatg gttgaccacg aaggtaacaa agtggatggt gaccagatca tgtacatcat 2760

tgcccgtgaa ggtctgcgtc agggtcagct gcgtggtggt gcagtaggta ccctcatgag 2820

2880

aggcgaccgt tacgttctgg agaaaatgca ggagaaaggt tggcgtatcg gtgccgaaaa 2940

ctctggtcac gtcatcctgc tggacaaaac cactaccggt gacggtatcg tagcaggtct 3000

gcaggtactc gccgctatgg ccgtaacca catgtccctc catgacctct gctctggtat 3060

gaaaatgttc ccgcagatcc tggttaacgt tcgttacacc gcaggttctg gtgatccgct 3120

3180

tcgtgtactg ctgcgtaaat ccggtactga gccactgatc cgtgttatgg ttgagggcga 3240

agatgaagcc caggtcaccg aatttgcgca ccgtattgcc gacgcagtca aagcggttta 3300

aatgggcagc agccatcacc atcatcacca cagccaggat ccgaattcga gctcggcgcg 3360

cctgcaggtc gacaagcttg cggccgcata atgcttaagt cgaacagaaa gtaatcgtat 3420

tgtacacggc cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata 3480

acaattcccc atcttagtat attagttaag tataagaagg agatatacat atgtgcggta 3540

tcgttggtgc tatcgcacag cgtgatgtag cggagatcct cctggaaggt ctgcgtcgtc 3600

3660

ctcgtctgcg tcgtctgggt aaagtgcaga tgctcgcgca ggcggcggaa gaacacccac 3720

tccacggtgg tacgggtatc gcacacactc gttgggcaac ccacggtgaa ccgtctgagg 3780

tcaacgcaca cccgcatgtt agcgagcaca tcgtagtcgt tcacaacggt atcatcgaga 3840

accacgaacc actccgtgag gaactcaaag cccgtggtta caccttcgta agcgaaaccg 3900

acacggaagt tatcgcccac ctcgttaact gggaactcaa acagggtggt actctgcgtg 3960

aagcagttct gcgtgccatt ccacagctgc gtggtgcata cggtaccgtg atcatggact 4020

ctcgtcatcc ggataccctg ctcgccgcac gttctggttc tccactcgtt atcggtctgg 4080

gtatgggtga gaacttcatc gcctctgatc agctggccct gctcccagtt acccgtcgct 4140

tcatcttcct ggaagagggt gacatcgccg aaatcacccg tcgttccgtt aacatcttcg 4200

acaaaacggg tgcggaagtt aaacgtcagg acatcgagtc taacctgcag tatgacgctg 4260

gtgacaaagg catctaccgt cactacatgc agaaagagat ctacgaacag ccgaacgcga 4320

tcaaaaacac cctgaccggt cgtatctctc acggtcaggt tgacctgtct gagctgggtc 4380

caaacgcgga cgaactcctg tccaaagtcg agcacatcca gatcctggct tgtggtacct 4440

cttacaactc cggtatggtt tctcgttact ggttcgaatc tctggcaggt atcccatgcg 4500

acgttgaaat cgcctccgaa ttccgttatc gtaaatctgc ggtacgtcgt aactccctca 4560

tgatcaccct gtctcagtct ggtgaaaccg ctgatactct ggcaggtctg cgtctcagca 4620

aagaactgggg ttacctggggt tctctggcca tctgcaacgt tccggggttct agcctggttc 4680

gtgagtctga cctggctctg atgaccaacg cgggtacgga gatcggtgtt gcctctacca 4740

aagcgttcac tacccagctc actgtcctgc tgatgctggt tgccaaactg tctcgtctca 4800

aaggcctcga cgctagcatc gaacacgaca tcgtacacgg tctgcaggcc ctcccatctc 4860

gtatcgagca gatgctgtct caggacaaac gtatcgaagc actggcagaa gacttcagcg 4920

acaaacacca cgcgctgttt ctgggtcgtg gtgaccagta cccaattgcg ctggaaggtg 4980

ccctgaaact gaaagagatc agctacatcc atgcagaggc atacgcagcg ggtgagctga 5040

aacatggtcc actggccctg atcgacgcag atatgccggt tattgtggtt gctccgaaca 5100

acgaactgct ggagaaactg aaatccaaca tcgaggaagt acgtgcgcgt ggtggtcagc 5160

tgtacgtgtt tgctgaccag gacgcgggtt tcgtttccag cgacaacatg cacatcatcg 5220

aaatgccgca tgttgaagag gtaatcgcgc caatcttcta caccgtaccg ctgcagctgc 5280

tggcgtacca tgtagccctg atcaaaggta cggacgttga ccagccgcgt aacctggcga 5340

aatccgtgac cgtggaataa gacgtcggta ccctcgagtc tggtaaagaa accgctgctg 5400

cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg 5460

ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 5520

gttttttgct gaaacctcag gcatttgaga agcacacggt cacactgctt ccggtagtca 5580

ataaaccggt aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga 5640

cgacaagctg acgaccgggt ctccgcaagt ggcacttttc ggggaaatgt gcgcggaacc 5700

cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta 5760

gaaaaactca tcgagcatca aatgaaactg caattttattc atatcaggat tatcaatacc 5820

atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag 5880

gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat 5940

taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga 6000

atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc 6060

attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc 6120

ctgagcgaga cgaaatacgc ggtcgctgtt aaaaggacaa ttacaaacag gaatcgaatg 6180

6240

ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc 6300

aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag 6360

tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa 6420

ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt 6480

atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct 6540

agagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat attattgaag 6600

6660

acaaataggc atgctagcgc agaaacgtcc tagaagatgc caggaggata cttagcagag 6720

agacaataag gccggagcga agccgttttt ccataggctc cgcccccctg acgaacatca 6780

cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc 6840

gtttccccct gatggctccc tcttgcgctc tcctgttccc gtcctgcggc gtccgtgttg 6900

tggtggaggc tttacccaaa tcaccacgtc ccgttccgtg tagacagttc gctccaagct 6960

gggctgtgtg caagaacccc ccgttcagcc cgactgctgc gccttatccg gtaactatca 7020

tcttgagtcc aacccggaaa gacacgacaa aacgccactg gcagcagcca ttggtaactg 7080

agaattagtg gatttagata tcgagagtct tgaagtggtg gcctaacaga ggctacactg 7140

aaaggacagt atttggtc tgcgctccac taaagccagt taccaggtta agcagttccc 7200

caactgactt aaccttcgat caaaccgcct ccccaggcgg ttttttcgtt tacagagcag 7260

gagattacga cgatcgtaaa aggatctcaa gaagatcctt tacggattcc cgacaccatc 7320

actctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca gccccatacg 7380

atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga gctgactggg 7440

ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca 7500

ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 7560

taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt 7620

tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag 7680

ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt 7740

taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatgtc 7800

cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc 7860

gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg 7920

aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg 7980

agtgagatat ttatgccagc cagccagacg cagacgcgcc

8100

accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa 8160

taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg 8220

atagttaatg at 8232

<210> 10

<211> 6792

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-PmgalT7

<400> 10

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatgagc ggtgaacact 2580

atgtcattag cctgtcgtcg gcagttgaac gtcgccagca cattcgtaac cagttttcgc 2640

agaagaacat cccgtttcag tttttcgatg caatttcacc gtcgccgctg ctggaccagc 2700

tggtgctgca atttttcccg cgtctggcgg atagctctct gaccggcggt gaaaaagcct 2760

gctttatgag ccatctgtct ctgtggcaca aatgtgtgga agaaaacctg ccgtatattg 2820

tggttttga agatgacatc gttctgggca aagatgcgga caagttcctg attggtgatg 2880

aatggctgtt ttctcgtttc gacccggaag aaatctttat tatccgcctg gaaaccttcc 2940

tgcagaaagt cgtgtgcgaa agcaccata ttgccccgta tacgcaccgc gattttctga 3000

gtctgaaatc cgcacatttc ggcacggctg gttacgtcat cagtcagggc gcggccaaat 3060

ttctgctgga tattttcaag aacatctcca atgaacacat tgcgccgatc gacgaactga 3120

tttttaacca gttcctggtt aagaactcat tcaacgtcta ccaactgtcg ccggcaatct 3180

gtgttcagga actgcaactg aacaatgaaa gttccgctct gcagagccaa ctggaactgg 3240

aacgtaacaa attccgcaat aaaaagtctg aagaactgaa gcgtaaccgc aagaacttca 3300

tcgaaaagtt catctacatc ctgaaaaagc cgaagcgtat gctggataac aataagcgta 3360

agcgcgaaga gagtaagatc gaaaacgaca agatgatcat cgaatttaaa tgagcggccg 3420

cgtcgacacg caaaaaggcc atccgtcagg atggccttct gcttaattat ctagatgcct 3480

ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttcg caacgttcaa 3540

atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa 3600

acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg cagttcccta 3660

ctctcgcatg gggagacccc acactaccat catgtatgaa tatcctcctt agttcctatt 3720

ccgaagggta atggcatcag ggaatggcga acgcgctccc cacactacca tcatgtatga 3780

atatcctcct tagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcggt 3840

3900

atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact aaaccatggc 3960

caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt 4020

ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg ccggtgtggt 4080

ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc cggacaacac 4140

cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt cggaggtcgt 4200

gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg agcagccgtg 4260

ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg tggccgagga 4320

gcaggactga gtggcagggc ggggcgtaag gcgcgccatt taaatgaagt tcctattccg 4380

aagttcctat tctctagaaa gtataggaac ttcgaagcag ctccagccta cacaatcgct 4440

caagacggaa cccgcgcttg gcaggaaagt aatagggata gcagctccag cctacacaat 4500

cgctcaagac gtgtaatgct gcacaataac cctgctgcag aggcctgcat gcaagcttgg 4560

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 4620

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 4680

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 4740

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 4800

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 4860

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 4920

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 4980

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 5040

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 5100

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 5160

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 5220

gctgtgtgca cgaaccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 5280

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 5340

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 5400

gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 5460

5520

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 5580

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 5640

tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 5700

aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 5760

tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 5820

ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 5880

gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 5940

gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 6000

taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 6060

tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 6120

ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 6180

tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 6240

ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 6300

6360

ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 6420

aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 6480

actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 6540

aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 6600

6660

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 6720

ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 6780

ggccctttcg tc 6792

<210> 11

<211> 6702

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-MsgalT8

<400> 11

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatggat gaaatcaaac 2580

2640

atgcccgctt ctttagccgt catgaatatg aagtggttct gagtctggac gaaccgtccg 2700

aagaagccga tctgctgcgt gtcctgcgcg acttcccgtc tattcgttgg cgcgttctgg 2760

tcaatgacct ggatcacccg tggcgtccgc cgtgccgtgc actgaacgtt ggcatccgta 2820

atgctctggg tgaaaacgtc ctggtcgtga gccgggaatc tgcgtttgtg accgatgttc 2880

cggcacgcgc tctggatcat attgcagcaa acccgggtac cgcagctctg ggtcacgttt 2940

gttttgcaac gttcgatgcg ctggaagccc gtcaggggcag cctggaaaaa acgtgcgctc 3000

3060

gctacgacga aagcttcgat cgctggggcg gtgatgacga taacctgcgt attcgcctga 3120

tgcagaccga aacgtatctg catccgctgg acgatatgcg catcctgcac ctgagttttg 3180

aagcccgtaa agtgcgtcaa gcagcagaac cgccgtcccc ggaatacgca gaacgtattt 3240

tccagccggt gtcaccgcaa gcaaatccgg gcggttgggg tgaatcgttt cagcgcgttg 3300

cgttcgattg gcgtcgccaa tgagcggccg cgtcgacacg caaaaaggcc atccgtcagg 3360

atggccttct gcttaattat ctagatgcct ggcagtttat ggcgggcgtc ctgcccgcca 3420

ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg tcctactcag 3480

gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 3540

tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc acactaccat 3600

catgtatgaa tatcctcctt agttcctatt ccgaagggta atggcatcag ggaatggcga 3660

acgcgctccc cacactacca tcatgtatga atatcctcct tagttcctat tccgaagttc 3720

ctattctcta gaaagtatag gaacttcggt ggaacgacgc gtaactcacg ttaagggatt 3780

ttggtcatga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata 3840

atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac 3900

cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga 3960

cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc 4020

ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtggggtgc gcggcctgga 4080

cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc 4140

ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc 4200

cggcaactgc gtgcacttcg tggccgagga gcaggactga gtggcaggggc ggggcgtaag 4260

gcgcgccatt taaatgaagt tcctattccg aagttcctat tctctagaaa gtataggaac 4320

ttcgaagcag ctccagccta cacaatcgct caagacggaa cccgcgcttg gcaggaaagt 4380

aatagggata gcagctccag cctacacaat cgctcaagac gtgtaatgct gcacaataac 4440

cctgctgcag aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt 4500

gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 4560

cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 4620

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 4680

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 4740

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 4800

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 4860

aaaaggccgc gttgctggcg ttttttccata ggctccgccc ccctgacgag catcacaaaa 4920

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4980

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5040

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 5100

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5160

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5220

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5280

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 5340

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 5400

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 5460

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 5520

actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 5580

taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 5640

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 5700

tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 5760

ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 5820

accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5880

agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 5940

acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 6000

tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 6060

cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 6120

tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 6180

ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 6240

gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 6300

tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 6360

ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 6420

gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 6480

6540

gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 6600

ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga 6660

cattaaccta taaaaatagg cgtatcacga ggccctttcg tc 6702

<210> 12

<211> 6777

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-KdgalT10

<400> 12

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatggaa aactatgtcg 2580

tctctatccg caccgcagcc caacgccgcc agcatgtcgc cgccgaattc aataagcacc 2640

aaatcgcctt tcatttcttt gatgcggtga ccccggaaac gctggcggaa agcatcgcag 2700

2760

ctcatgtctg cctgtgggca aaatgtgtgg ctgatgacct gccgtatatt ggcatctttg 2820

aagatgacgt tattttcggt cagaacagct ctcgttttct gaatgatacc aaatggctgg 2880

acgaacgttt tcagaaccaa tcattcatta tccgcatgga aacgtttctg aaggcgaacc 2940

cggttgccct gagcaaatct ggcgtccgtc cgttcaatgg tcgtaagatc ctgcgcctgc 3000

agagttttgg cttcggtacc gcggcctatc tgatttccca gcaaaccgca atcacgctgc 3060

3120

atgcagcttc agaaattccg gaaatccaga tgtaccaaat ctcgccggcc ctgtgcattc 3180

aggaactgca actgaaccgc gcagatagtt ccctgtcatc gaccctggaa gacggtcgtc 3240

tggcacgtca ccagcaactg gatggcggta aaacccagcc ggaacagacg caagaaaacc 3300

gtaacatctt cgcatgggct aagaacaaga tcgtgaagga atacaagcgc gttaaacgtc 3360

3420

aggccatccg tcaggatggc cttctgctta attatctaga tgcctggcag tttatggcgg 3480

gcgtcctgcc cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg 3540

atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc 3600

3660

accccacact accatcatgt atgaatatcc tccttagttc ctattccgaa gggtaatggc 3720

atcagggaat ggcgaacgcg ctccccacac taccatcatg tatgaatatc ctccttagtt 3780

ctctagaaag tataggaact tcggtggaac gacgcgtaac 3840

tcacgttaag ggattttggt catgatcagc acgtgttgac aattaatcat cggcatagta 3900

tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc 3960

4020 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc

cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac 4080

cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg 4140

ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg 4200

ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc 4260

cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgagtggc 4320

agggcggggc gtaaggcgcg ccatttaaat gaagttccta ttccgaagtt cctattctct 4380

agaaagtata ggaacttcga agcagctcca gcctacacaa tcgctcaaga cggaacccgc 4440

gcttggcagg aaagtaatag ggatagcagc tccagcctac acaatcgctc aagacgtgta 4500

atgctgcaca ataaccctgc tgcagaggcc tgcatgcaag cttggcgtaa tcatggtcat 4560

agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 4620

gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 4680

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4740

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4800

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4860

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4920

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4980

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 5040

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 5100

catagctcac 5160

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5220

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5280

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5340

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 5400

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5460

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5520

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5580

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 5640

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 5700

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 5760

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 5820

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 5880

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 5940

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 6000

6060

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 6120

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 6180

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 6240

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 6300

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 6360

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6420

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6480

6540

agggaataag ggcgacacgg aaatgttgaa tactcatact cttcctttttt caatattatt 6600

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 6660

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 6720

ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc 6777

<210> 13

<211> 6786

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-gatD

<400> 13

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatgtcc tcagctttcc 2580

attacgtcat tagcctggca tcggcagttg aacgccgtca gcacattagc gaacagtttt 2640

cccaatacga cattccgttt cagtttttcg atgcgatcag tccgtccccg ctgctgaacc 2700

agctggtgtc tcaatttttc ccgtccctgg ccgatagctc tctgaccgac ggcgaaaaag 2760

gttgctttat ttcacatctg tcgctgtggc acaagtgtgt tgaaaagaac ctgccgtata 2820

ttgtggtttt tgaagatgac atcctgctgg gcaagaatgc agataaattc ctgattgaag 2880

acgaatggtt tttctctcgt tttaacacga atgatgtctt catcgtgcgc ctggaaacct 2940

ttctgcagaa agtgtattgc caaccgagct acatcaagtc ttactacaac cgtgaactgc 3000

tgaccctgaa aagcacgcat ttcggcaccg caggttatat tatcagtctg ggtgcggcca 3060

agtttctgct gtccctgttc aacaaaatgc acattgaaga agttgctccg atcgatgaac 3120

tgctgtttaa taagttcctg gaacgcaaag actttacggt ctaccagttc agtccggcac 3180

tgtgcattca ggaactgcaa ctgaacaaat cagatgctgt cctgctgtcg caactggaac 3240

tggaacgtag caaatgtcgc attatgaccg aatctcgtat cggccgcgaa aagaaaaaac 3300

tgaaggataa gatcatccat gttctgacga agccgaaacg tatgctggaa aagaaacgtc 3360

agcgcaatga agacaagaaa atcaccatga ttatcgaatt tgaatgagcg gccgcgtcga 3420

3480

ttatggcggg cgtcctgcc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc 3540

3600

3660

catggggaga ccccacacta ccatcatgta tgaatatcct ccttagttcc tattccgaag 3720

ggtaatggca tcagggaatg gcgaacgcgc tccccacact accatcatgt atgaatatcc 3780

tccttagttc ctattccgaa gttcctattc tctagaaagt ataggaactt cggtggaacg 3840

acgcgtaact cacgttaagg gattttggtc atgatcagca cgtgttgaca attaatcatc 3900

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 3960

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4020

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4080

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4140

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcgggagg tcgtgtccac 4200

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 4260

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 4320

ctgagtggca gggcggggcg taaggcgcgc catttaaatg aagttcctat tccgaagttc 4380

ctattctcta gaaagtatag gaacttcgaa gcagctccag cctacacaat cgctcaagac 4440

ggaacccgcg cttggcagga aagtaatagg gatagcagct ccagcctaca caatcgctca 4500

agacgtgtaa tgctgcacaa taaccctgct gcagaggcct gcatgcaagc ttggcgtaat 4560

catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 4620

gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 4680

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 4740

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 4800

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4860

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4920

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4980

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5040

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5100

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5160

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5220

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5280

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5340

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5400

ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5460

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5520

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5580

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5640

aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5700

tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5760

cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5820

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5880

cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5940

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 6000

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6060

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6120

gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6180

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6240

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6300

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6360

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6420

caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6480

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6540

ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6600

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6660

tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 6720

tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 6780

ttcgtc 6786

<210> 14

<211> 7506

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-BFgalT2

<400> 14

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatgaac gtgaataagc 2580

cgaccaccga aaagaaactg attgacctga acaacgacat tatccataac tttgatgtga 2640

gcattgtgat gagcttctat aagcgttaca ccgaatttcg caaagtgctg ccgcataacg 2700

cgccgtatct gcagcgtaat ggcattgaag tcattatcgt gctggatgac ccggatgaaa 2760

aaagcgaact gctgatgctg ctgcaaaact atccgttcat caattggaag ctgattatca 2820

acgaacgtaa acatgcaccg cgcaaccacg cttctgttct gaatgtcggt ctgaaacatg 2880

cgaccaaaaa gtatattctg cagatcgatc cggaagttga atttctgacg gatattatct 2940

ggcaaatgcg tgacgccatt gaaaaatatc cgatgcacta catcctggcg atgatggcct 3000

atgtcccgta cgaacaggaa ctgaccgaaa acaacatcaa ggaactggat ttcatcccgt 3060

ggggcaacct gatggtggaa cgcaatcatc tgtataaact gcacggttac gatgaaacct 3120

3180

aaaagtttat cctgccggaa gccaagacca tccatcgtga aaagaactat gatccgaatg 3240

aacgttcgaa gcgcattaat aaacacagta tctccgactg gcgcaaaatg aactacccgt 3300

3360

ggcaggacaa tcaatacgcc aaagatctgt gctataccta cctgcagcaa tttattggtt 3420

tcgaaatccg tcatccggcg gcctttcgta aacgccacaa aaagattgtc ctgtgtcagg 3480

catataacga agaaaaactg atcgaaggct tcctgacgaa catggctaat tactttgatg 3540

gtattatcct gctggatgac gaaagtaccg atcgcacgtg ggacctggca atccatgata 3600

agatcatcct gaaggtgaaa aagaaacgtt ccggttttaa tgatctggaa aaccgcaata 3660

ttctgctgga cctgtcagcg ttttttccagt cggaatggtt ttgcttcatg gatatcgacg 3720

3780

tggtttcttt tcgtggcgtg tatctgtgga atgatgaaca gagctacaag ggcgacattc 3840

cgaactctaa taaaggtatc ctgaccgttt atcgtatgtt ccgcccgatt ggtcataccc 3900

acatcaacac gcataagaaa ctgcacttca ttgcgacgcc gtattttacc aacacgtggc 3960

agagtaatat cctgtttaag gattacggct ccatgaaaga aaatgaccgt attcgcaagt 4020

atgaacgcta catccaggaa gatcagcaaa aagacatgag ctctggttat gattacctgc 4080

tgaacagcga aaatctgtat caactggaca aaattgaaga atactgagcg gccgcgtcga 4140

cacgcaaaaa ggccatccgt caggatggcc ttctgcttaa ttatctagat gcctggcagt 4200

ttatggcggg cgtcctgcc gccaccctcc gggccgttgc ttcgcaacgt tcaaatccgc 4260

tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa 4320

ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg 4380

catggggaga ccccacacta ccatcatgta tgaatatcct ccttagttcc tattccgaag 4440

ggtaatggca tcagggaatg gcgaacgcgc tccccacact accatcatgt atgaatatcc 4500

tccttagttc ctattccgaa gttcctattc tctagaaagt ataggaactt cggtggaacg 4560

acgcgtaact cacgttaagg gattttggtc atgatcagca cgtgttgaca attaatcatc 4620

ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4680

gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4740

cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4800

cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4860

ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 4920

gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 4980

ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5040

ctgagtggca gggcggggcg taaggcgcgc catttaaatg aagttcctat tccgaagttc 5100

ctattctcta gaaagtatag gaacttcgaa gcagctccag cctacacaat cgctcaagac 5160

ggaacccgcg cttggcagga aagtaatagg gatagcagct ccagcctaca caatcgctca 5220

agacgtgtaa tgctgcacaa taaccctgct gcagaggcct gcatgcaagc ttggcgtaat 5280

catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 5340

gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 5400

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 5460

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 5520

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 5580

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 5640

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5700

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5760

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5820

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5880

atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5940

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 6000

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 6060

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6120

ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6180

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6240

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6300

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 6360

6420

aatgcttaat cagtgaggca cctatctcag 6480

cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 6540

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 6600

6660

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 6720

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 6780

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 6840

gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6900

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6960

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 7020

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 7080

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 7140

caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 7200

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 7260

ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 7320

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 7380

tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 7440

tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 7500

ttcgtc 7506

<210> 15

<211> 6717

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-lsgD

<400> 15

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatgctg aagaagtacc 2580

tgattagcct ggataaggac attcaacgcc gcaagctgtt tttctcgcag aagaacacgg 2640

aagattttca aattttctca gcgatcaaca ccatgcagaa agattgggac gaactggcat 2700

cgatcttcaa catcgaacaa ttcaaggctc attacttccg taacgtcacc aagggcgaaa 2760

ttggttgcac gctgagtcac ctgtccgtct atcagaaaat tgtggaagat aacgacatcg 2820

cagagattc atacgctctg gtttgtgaag atgacgccct gtttcatctg gatttccagc 2880

aaaatctgac cgcactgctg agtgaaaaac tggaagctga aattatcctg ctgggccagt 2940

ccaacattaa caattttaat gatacggacc tggaaatcaa ttacccgacc acgtttagct 3000

tcctgtgcaa aaagaccggt aacgtgaatt atgcgttccc gtataaatct tactttgccg 3060

gcacggttgg ttacctgatt aaaaagagcg cggcccgtcg cttcattcag caaatctctc 3120

agaacaaacc gttttggctg gcggatgact ttctgctgtt cgaacaaaac ttcaatatcc 3180

3240

aatcggtgcg cggcagcctg tctaacaatc tgctgaaaaa gctgatgaaa tatccgctga 3300

aaaagatttt tgcgatcaaa aagaacctgg ccaattaagc ggccgcgtcg acacgcaaaa 3360

aggccatccg tcaggatggc cttctgctta attatctaga tgcctggcag tttatggcgg 3420

gcgtcctgcc cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg 3480

3540

tttcgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcatggggag 3600

accccacact accatcatgt atgaatatcc tccttagttc ctattccgaa gggtaatggc 3660

atcagggaat ggcgaacgcg ctccccacac taccatcatg tatgaatatc ctccttagtt 3720

3780

tcacgttaag ggattttggt catgatcagc acgtgttgac aattaatcat cggcatagta 3840

tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc 3900

3960

cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac 4020

cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg 4080

ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg 4140

ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc 4200

cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgagtggc 4260

agggcggggc gtaaggcgcg ccatttaaat gaagttccta ttccgaagtt cctattctct 4320

agaaagtata ggaacttcga agcagctcca gcctacacaa tcgctcaaga cggaacccgc 4380

gcttggcagg aaagtaatag ggatagcagc tccagcctac acaatcgctc aagacgtgta 4440

atgctgcaca ataaccctgc tgcagaggcc tgcatgcaag cttggcgtaa tcatggtcat 4500

agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 4560

gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 4620

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4680

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4740

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4800

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4860

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4920

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4980

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 5040

catagctcac 5100

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5160

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5220

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5280

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 5340

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5400

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5460

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5520

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 5580

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 5640

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 5700

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 5760

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 5820

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 5880

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 5940

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 6000

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 6060

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 6120

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 6180

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 6240

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 6300

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 6360

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 6420

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 6480

agggaataag ggcgacacgg aaatgttgaa tactcatact cttcctttttt caatattatt 6540

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 6600

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 6660

ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc 6717

<210> 16

<211> 6769

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-malE-HPgalT

<400> 16

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caaatgaaaa tcgaagaagg taaactggta atctggatta 1500

acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg 1560

gaattaaagt caccgttgag catccggata aactggaaga gaaattccca caggttgcgg 1620

caactggcga tggccctgac attatcttct gggcacacga ccgctttggt ggctacgctc 1680

aatctggcct gttggctgaa atcaccccgg acaaagcgtt cgggacaag ctgtatccgt 1740

ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc gctgttgaag 1800

1860

tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc 1920

aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg 1980

aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc 2040

tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc gattactcca 2100

tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat 2160

ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg 2220

gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga 2280

acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa ggtctggaag 2340

cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg 2400

cgaaagatcc acgtattgcc gccaccatgg aaaacgccca gaaaggtgaa atcatgccga 2460

acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca 2520

gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactatgcgt gtgtttatta 2580

tttccctgaa tcaaaaagtg tgtgatacct tcggtctggt gttccgtgat acgacgaccc 2640

tgctgaacaa cattaacgcg acccatcacc aggcccaaat ttttgatgca atctactcca 2700

aaacgttcga aggcggtctg catccgctgg ttaaaaaaca tctgcacccg tactttatta 2760

cccagaacat caaagacatg ggcattacca cgaatctgat cagcgaagtc tctaaattct 2820

actacgctct gaaataccat gcgaaattca tgagcctggg cgaactgggt tgctatgcta 2880

gtcactactc cctgtgggaa aaatgcattg aactgaacga agcgatttgt atcctggaag 2940

atgacatcac gctgaaagaa gattttaaag aaggcctgga cttcctggaa aaacatattc 3000

aggaactggg ttatgtgcgt ctgatgcacc tgctgtacga tccgaatgtt aaaagcgaac 3060

cgctgaacca taaaaatcac gaaatccagg aacgcgtggg cattatcaaa gcctattctc 3120

atggcgttgg cacccaaggt tacgtcatta cgccgaaaat cgcaaaagtc ttcaaaaaac 3180

atagtcgtaa atgggtggtt ccggtggata ccattatgga cgcgacgttt atccacggtg 3240

tcaaaaatct ggtgctgcaa ccgttcgtta ttgccgatga cgaacaaatt tcaaccatcg 3300

3360

acctgaaata ctggcaattc gtctaagcgg ccgcgtcgac acgcaaaaag gccatccatc 3420

cgtcaggatg gccttctgct taattatcta gatgcctggc agtttatggc gggcgtcctg 3480

cccgccaccc tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc 3540

tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact 3600

gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg agaccccaca 3660

ctaccatcat gtatgaatat ccctccttagt tcctattccg aagggtaatg gcatcaggga 3720

atggcgaacg cgctccccac actaccatca tgtatgaata tcctccttag ttcctattcc 3780

3840 gaagttccta ttctctagaa agtataggaa cttcggtgga acgacgcgta

agggattttg gtcatgatca gcacgtgttg acaattaatc atcggcatag tatatcggca 3900

tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg 3960

tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct 4020

cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca 4080

tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg 4140

gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct 4200

ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg 4260

acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgagtg gcagggcggg 4320

gcgtaaggcg cgccatttaa atgaagttcc tattccgaag ttcctattct ctagaaagta 4380

taggaacttc gaagcagctc cagcctacac aatcgctcaa gacggaaccc gcgcttggca 4440

ggaaagtaat agggatagca gctccagcct acacaatcgc tcaagacgtg taatgctgca 4500

caataaccct gctgcagagg cctgcatgca agcttggcgt aatcatggtc atagctgttt 4560

cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4620

tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 4680

cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4740

gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4800

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4860

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4920

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4980

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 5040

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 5100

tacctgtccg ccttttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 5160

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 5220

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 5280

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5340

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 5400

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5460

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5520

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5580

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5640

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5700

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5760

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5820

tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5880

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5940

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 6000

6060

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 6120

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6180

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6240

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6300

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6360

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6420

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6480

6540

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6600

6660

ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 6720

atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 6769

<210> 17

<211> 4714

<212> DNA

<213> Artificial sequence

<220>

<223> construct pACYC-waaX

<400> 17

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atgaaagtgt ttgtggtcaa cctggataag gataaggata aaaaagaaaa 120

aatcaagaat gaatgccgca acgcagaact ggactatgaa attatctcag cagttgatgg 180

ccgtgaactg agctacaacg aactgaaatc taaggtccat ccggtgtcac tgaattatct 240

gtcgaaaggc gaaattggtt gcgtcctgtc ccaccagcgt atttacaaac gcatcctgga 300

tgacgatatt gactatgctc tgatcctgga agacgatgtg gaactgagtc aagatatcaa 360

ggtttttctg aaggaattcc tgtccgtcaa agacaagaac aaaggcgatg tgtttctgct 420

gtacccgtca ggtctgcgtt tcctgaaccg tcgcatcaac gtgtcgcatg attatttctt 480

ttatgaagcg tacaacagct cttgtgccca cggttatatt atcagcaaca aagcggccaa 540

aaagctgatt cgcatcaata ccccgattat cctggttgca gatgcttggc tgtggtttta 600

ccagatttct ctgctgaaag tgtatgttct gaacaaagaa ctggttcgtg catatgacgt 660

cgataaaagt ctgtccacca tcgaaacgga acgcagcctg ctgctggacg aaaaggaaaa 720

gcatcagatg caaatcatca aaaagcaacc gctgtactac ctgatcaagt actaccacaa 780

gtacatccgt cgcctgttca tcaataagga taaataagaa ttcgagctcg gcgcgcctgc 840

aggtcgacaa gcttgcggcc gcataatgct taagtcgaac agaaagtaat cgtattgtac 900

acggccgcat aatcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat 960

1020

tggatatcgg ccggccacgc gatcgctgac gtcggtaccc tcgagtctgg taaagaaacc 1080

gctgctgcga aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc 1140

taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacggggtc 1200

ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg 1260

gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct 1320

gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc attcatccgc ttattatcac 1380

ttattcaggc gtagcaccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc 1440

cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa 1500

gccatcacag acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg 1560

1620

taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat 1680

aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat 1740

1800

ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc 1860

tttcattgcc atacggaact ccggatgagc attcatcagg cgggcaagaa tgtgaataaa 1920

ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag 1980

ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt 2040

acgatgccat tgggatatat caacggtggt atatccagtg atttttttct ccattttagc 2100

ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc 2160

attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt 2220

ggcccagggc ttcccggtat caacagggac accaggattt attttattctg cgaagtgatc 2280

ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttactgat 2340

ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc tgtttctatc agctgtccct 2400

cctgttcagc tactgacggg gtggtgcgta acggcaaaag caccgccgga catcagcgct 2460

agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg aagtgcttca 2520

2580

tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg 2640

gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg 2700

aggggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa 2760

tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc 2820

ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 2880

gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccgggg taggcagttc 2940

gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 3000

gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 3060

ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 3120

aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 3180

3240

attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 3300

tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 3360

aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga 3420

aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa 3480

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3540

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccaggggt ggtttttctt 3600

3660

3720

ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca 3780

ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg 3840

3900

ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg 3960

agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct 4020

aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg 4080

tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac 4140

gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag 4200

ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct 4260

tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga 4320

gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg 4380

ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc 4440

agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg 4500

ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac 4560

gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata 4620

ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga 4680

ctcctgcatt aggaaattaa tacgactcac tata 4714

<210> 18

<211> 4760

<212> DNA

<213> Artificial sequence

<220>

<223> construct pACYC-wbdO

<400> 18

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atgctgacgg aagtgcgccc ggtctctacg acgaaaccgc tggtgtctgt 120

gattctgccg gtgaacaaat tcaacccgta tctggatcgt gcaattcatt caatcctgag 180

tcagtcctat ccgtcgattg aactgattat cattgcaaac aattgcacca atgacttttt 240

cgatgctctg aaaaaacgtg aatgtgaaac cattaaagtg ctgcgcacga acatcgcgta 300

360

ggattcagat gacatttcgc acccggaacg tatcgatcgc caggtcgact tcctgattaa 420

caatccggac atcgatgtgg ttggcaccaa tgcagtctat attgatgaag atgacatcga 480

actggaaaaa agcaacctgc cggtgaacaa taacgctatt cgtaaaatgc tgccgtataa 540

atgctgtctg gtgcatccgt ctgttatgtt tcgcaaaaat gtcgtgatca ccagcggcgg 600

ttacatgttc gcgaattatt ctgaagatta cgaactgtgg aaccgtctgg ccgttgaagg 660

ccgcaatttt tataacctga gcgaatacct gctgtattac cgtctgcaca ataaccaatc 720

aacgtcgaaa aataacctgt ttatggtgat ggcgaacgat gtcgccatta aagtgaaata 780

tttcctgctg accaagaaaa ttagctacct gctgggtatc attcgcacgg tcttttctgt 840

gttctattgc aaatacatca aatgaattcg agctcggcgc gcctgcaggt cgacaagctt 900

gcggccgcat aatgcttaag tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc 960

gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc catcttagta 1020

tattagttaa gtataagaag gagatataca tatggcagat ctcaattgga tatcggccgg 1080

ccacgcgatc gctgacgtcg gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt 1140

tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac 1200

cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 1260

gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc 1320

ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg 1380

gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag 1440

caccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg ccctgccact 1500

catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacagacgg 1560

catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc 1620

ccatagtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg 1680

tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga 1740

aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc 1800

1860

cggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac 1920

ggaactccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact 1980

tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt 2040

tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg 2100

atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg 2160

2220

tggaacctct tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc 2280

cggtatcaac agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta 2340

tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg 2400

tgtttttgag gtgctccagt ggcttctgtt tctatcagct gtccctcctg ttcagctact 2460

gacggggtgg tgcgtaacgg caaaagcacc gccggacatc agcgctagcg gagtgtatac 2520

tggcttacta tgttggcact gatgaggggtg tcagtgaagt gcttcatgtg gcaggagaaa 2580

aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct 2640

cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg 2700

2760

aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat 2820

cagtggtggc gaaacccgac aggactataa agataccagg cgtttcccct ggcggctccc 2880

2940

gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac 3000

tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt 3060

3120

agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg 3180

tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt 3240

cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc 3300

3360

atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc 3420

atgttagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc 3480

atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca 3540

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 3600

3660

gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac 3720

3780

3840

ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa cgcatcgc 3900

agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact 3960

ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca 4020

4080

ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa 4140

aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt 4200

gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc 4260

actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg 4320

ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc 4380

gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga 4440

ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc 4500

cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga 4560

aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac 4620

attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 4680

gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga 4740

aattaatacg actcactata 4760

<210> 19

<211> 4795

<212> DNA

<213> Artificial sequence

<220>

<223> construct pACYC-furA

<400> 19

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggataaaa tcaaacaggg cagcgcctct ctggttgtcg gtgaccagca 120

agaaaaacat ccggtggttt cagtgctgct gccggttaat cgtgtcgatc gctttttcat 180

tccggcagtt gaatcgatcc tgacccaaac gctgcaggat tttgaactga tcattatcgc 240

taatggctgt agcaccgaac atctgaacaa aattcgtctg acgtatggtg atcacaatcg 300

360

caacgcccgt ggtctgtata ttgcacgcat ggatgctgat gacatttcta tccgggaacg 420

cctggaaaaa caactgaata cgctggaaca gaacaagaaa attggcgtcg tgagctctgg 480

tgtggacttt attgatgaaa atgaccaggc gatccgtgag ggtaaattcc cggaactgac 540

cgacaaagat catcgtcgcc tgctgccgct gatttgctgt atcgcccacc cgacggttat 600

ggtccgcaaa gaaattatca acaaactggg cggttatagt tttggtagtt tctccgaaga 660

ctacgatctg tggctgcgta ttatgcgcga actgccggaa gttgaatttt atcgtatccc 720

ggaatccctg ctgaaatacc gtcgccatgg caatcaggcc accagttcca aaaacattaa 780

gaaaattcgc gcgtacaact cagccctgaa aattcgtgaa ctgtttctgt cgcgcaaact 840

gaaattcatt atcggtatta tcctgccggc acgtatggtg accctgtggc gcaaatgaga 900

attcgagctc ggcgcgcctg caggtcgaca agcttgcggc cgcataatgc ttaagtcgaa 960

cagaaagtaa tcgtattgta cacggccgca taatcgaaat taatacgact cactataggg 1020

gaattgtgag cggataacaa ttccccatct tagtatatta gttaagtata agaaggagat 1080

atacatatgg cagatctcaa ttggatatcg gccggccacg cgatcgctga cgtcggtacc 1140

ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct 1200

actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc 1260

cttggggcct ctaaacggggt cttgaggggt ttttgctga aacctcaggc atttgagaag 1320

cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg 1380

gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc 1440

cattcatccg cttattatca cttattcagg cgtagcacca ggcgtttaag ggcaccaata 1500

actgccttaa aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 1560

aagcattctg ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg 1620

catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg gggcgaagaa 1680

gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 1740

gacgaaaaac atattctcaa taaaccctttt agggaaatag gccaggtttt caccgtaaca 1800

cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 1860

gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 1920

ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag cattcatcag 1980

gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 2040

taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 2100

2160

gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 2220

gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 2280

gtctcatttt cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt 2340

tatttattct gcgaagtgat cttccgtcac aggtatttat tcggcgcaaa gtgcgtcggg 2400

tgatgctgcc aacttactga tttagtgtat gatggtgttt ttgaggtgct ccagtggctt 2460

ctgtttctat cagctgtccc tcctgttcag ctactgacgg ggtggtgcgt aacggcaaaa 2520

gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg gcactgatga 2580

gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag 2640

aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt 2700

cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg 2760

aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc 2820

cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac 2880

tataaagata ccaggcgttt cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt 2940

tcggtttacc ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac 3000

tcagttccgg gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt 3060

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa 3120

aagcaccact ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc 3180

gccggttaag gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta 3240

ccctcggttca aaggttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt 3300

3360

attaatcaga taaaatattt ctagatttca gtgcaattta tctcttcaaa tgtagcacct 3420

gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc cgcgcccacc 3480

ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat 3540

gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 3600

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 3660

ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc 3720

gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa 3780

3840

cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg 3900

cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc 3960

atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc 4020

ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag 4080

acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc 4140

tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg 4200

tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca 4260

tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg 4320

tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg 4380

gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg 4440

gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc 4500

acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc 4560

gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca 4620

tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct 4680

tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc 4740

tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca ctata 4795

<210> 20

<211> 6383

<212> DNA

<213> Artificial sequence

<220>

<223> construct pET-PmnagT

<400> 20

ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag 60

gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag 120

ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag 180

taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 240

gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 300

360

aagaatgtat taatgcagtg attaatcaga catataagaa cttagaaatt attattgtga 420

atgatggttc ttctgataat acttattttc ttttaaaaaa gttagctgaa aaagataatc 480

gtataaaaat attaaatttc aataatcata ttggaataat ttctgcttta aatgaaggtt 540

taaaagagat agctggagaa tatattgctc gaacagattc tgatgatata actaagccag 600

attggattga gaaaatatta acttgtatgc aaaatgatcc taaaatcatc gctatgggat 660

cttatcttac tgtcttgtca gaagaaaata atggtagtgt gcttgctaat catcataaaa 720

ataaagttga atggaaaaat ccattagagc acaaagatat tgttgagaaa atgttatttg 780

gtaatcctat tcataataat tcaatggtta tgagaagtga gatatataca aagtatcact 840

taatttatga tccagattat cattatgctg aagattataa attttggctg gaagttagtc 900

gaattgggaa attagcaaat tatcctgagt cactcgtata ttatagactt caccgaaatc 960

aaatcttc tattcataat agccaacaag aaataaatgg taaaaaatta cgtttacaag 1020

ctcttaatta ttatttaaaa gatcttggta ttgattatca gttacctgaa aaatttttat 1080

1140

1200

ttttattattt tttaataaat aaaaataact ttctttctat aaaagacaaa tttaaaataa 1260

taaaaaaata tcttcgtcct gataaatatt catctactta ttaggacgtc ggtaccctcg 1320

agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta 1380

gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg 1440

gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg 1500

cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 1560

cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 1620

tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 1680

ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 1740

tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 1800

taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 1860

tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 1920

aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttctggcg gcacgatggc 1980

2040

tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 2100

2160 gcacctatct cagcgatctg tctatttcgt tcatccatag

tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 2220

gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 2280

cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 2340

gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 2400

atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 2460

aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 2520

atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 2580

aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 2640

aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 2700

gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 2760

gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 2820

gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 2880

ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 2940

ctcttccttt ttcaatcatg attgaagcat ttatcagggt tattgtctca tgagcggata 3000

catatttgaa tgtatttaga aaaataaaca aataggtcat gaccaaaatc ccttaacgtg 3060

3120

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3180

tttgtttgcc ggatcaagag ctaccaactc ttttccgaa ggtaactggc ttcagcagag 3240

cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 3300

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3360

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3420

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3480

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3540

3600

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3660

gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3720

ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3780

ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3840

3900

ttctccttac gcatctgtgc ggtatttcac accgcatata tggtgcactc tcagtacaat 3960

ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc 4020

atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 4080

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 4140

tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga 4200

agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc 4260

gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc 4320

actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga 4380

gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt 4440

gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa 4500

tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg 4560

atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa 4620

acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag 4680

tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc 4740

cagcctagcc gggtcctcaa cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac 4800

cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa 4860

tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 4920

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 4980

gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac 5040

cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa 5100

atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta 5160

tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc 5220

gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag 5280

catttgcatg gttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat 5340

cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga 5400

gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg 5460

ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg 5520

gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc 5580

atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt 5640

gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct 5700

ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag 5760

ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc 5820

cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt 5880

cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc 5940

atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc 6000

6060

ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc 6120

cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc 6180

cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc 6240

gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg 6300

6360

cgaaattaat acgactcact ata 6383

<210> 21

<211> 6711

<212> DNA

<213> Artificial sequence

<220>

<223> construction pINT-yjhB

<400> 21

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tggcaacagc 1500

atggtataaa caagttaatc caccacaacg gaaagctctt ttttccgcat ggcttggata 1560

tgtatttgat ggctttgatt ttatgatgat attttacatt cttcatatta taaaagcaga 1620

tcttggcatt acggatattc aggctacttt aatagggaca gtggccttca tagccagacc 1680

tattggaggt ggtttttttg gtgccatggc tgataaatat ggtcgtaagc caatgatgat 1740

gtgggcaatt ttcatttact cagtcggaac aggccttagc ggtattgcta caaacttata 1800

1860

ttcaacttat gcggtagaaa gttggcctaa aaatcttcaa tctaaagcta gtgctttttt 1920

ggtaagtggt ttttctgttg gaaatattat tgcggcacaa ataatccctc agtttgctga 1980

agtatatgga tggagaaact ctttttttat aggcctgtta ccagttttac tagttctttg 2040

gatcagaaaa agtgctccag aaagtcagga gtggattgaa gataaatata aggataaatc 2100

2160

ctgtttttgt ctatttggtg caaactggcc gataaacgga ctacttcctt cctacctggc 2220

agataatgga gttaatacag tggtcatttc aactctgatg acaatagcag gtttaggaac 2280

actgacaggt acaatatttt ttggttttgt tggtgataag attggtgtaa aaaaagcctt 2340

tgtagtcggt ctaataactt catttatttt cctttgtcct ctttttttta tttctgtgaa 2400

aaactcttct cttataggat tatgtctctt tggattaatg tttacaaatt taggtattgc 2460

agggttggtt ccaaaattta tatatgatta ctttccaaca aaattaagag gattagggac 2520

cggtcttatt tataacttag gggcaactgg aggaatggcc gcacctgtat tagctacata 2580

catttcagga tattatggct taggtgtttc attattcatt gttacggttg cattctctgc 2640

cttattaatt ttgttagttg gttttgatat tccaggtaaa atttataaac tatccgtggc 2700

taaatgataa atcgatacta gcataacccc ttggggcctc taaacgcgtc gacacgcaaa 2760

aaggccatcc gtcaggatgg ccttctgctt aatttgatgc ctggcagttt atggcgggcg 2820

tcctgcccgc caccctccgg gccgttgctt cgcaacgttc aaatccgctc ccggcggatt 2880

tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt 2940

cgactgagcc tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagacc 3000

ccacactacc atcatgtatg aatatcctcc ttagttccta ttccgaagtt cctattctct 3060

agaaagtata ggaacttcgg cgcgtcctac ctgtgacgga agatcacttc gcagaataaa 3120

taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag 3180

acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg 3240

3300

3360

ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt 3420

taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc 3480

gcctgatgaa tgctcatccg gaattacgta tggcaatgaa agacggtgag ctggtgatat 3540

gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc 3600

3660

3720

tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca 3780

acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga 3840

tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagatgct 3900

taatgaatac aacagtactg cgatgagtgg cagggcgggg cgtaaggcgc gccatttaaa 3960

tgaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcg aagcagctcc 4020

agcctacaca atcgctcaag acgtgtaatg ctgcaatctg catgcaagct tggcactggc 4080

4140

ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttcg caacgttcaa atccgctccc 4200

ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc 4260

cagtctttcg actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgcatg 4320

gggagacccc acactaccat cggggggcca tcgatgcagg tggcactttt cggggaaatg 4380

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 4440

gacaataacc ctgctgcaga ggcctgcatg caagcttggc gtaatcatgg tcatagctgt 4500

ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4560

4620 agtgtaaagc ctggggtgcc taatgagtga gctaactcac

tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4680

cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 4740

gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4800

ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4860

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 4920

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 4980

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5040

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5100

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5160

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5220

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5280

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 5340

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5400

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5460

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5520

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5580

agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5640

ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5700

gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5760

catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 5820

cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 5880

cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 5940

gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6000

tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6060

gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6120

tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6180

gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6240

gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6300

taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6360

tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6420

ctttcaccag cgtttctgggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6480

taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6540

tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6600

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 6660

ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt c 6711

<210> 22

<211> 6867

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-yebQ

<400> 22

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgccaaaagt 1500

tcaggccgac ggcctgccat tgccccagcg atacggtgcg atattaacca ttgtgattgg 1560

tatttcgatg gccgtccttg acggcgcaat cgccaacgtc gccctgccaa caatcgccac 1620

ggaccttcat gccacgccag ccagttccat ctgggtagtg aacgcctatc aaatcgccat 1680

tgtcatctcc ctgctctcgt tttcgtttct gggcgatatg tttggctatc gacgtattta 1740

taaatgcggt ctggtcgttt ttctgttgtc ttcactgttc tgcgcccttt ctgattcgct 1800

gcaaatgctc acccttgcgc gtgtcataca aggtttcggc ggtgcagcgt tgatgagcgt 1860

taataccgca cttatccgcc tgatctatcc acaacgtttt ctgggtagag ggatgggcat 1920

aaactcgttt attgttgccg tctcttctgc tgccgggccg acaattgctg cagcaatcct 1980

2040

gcttctggcg atgcgttttc tgccacccaa tggttctcgc gccagtaaac cccgtttcga 2100

cctgcccagc gccgtgatga acgcgttaac cttcggcctg cttatcactg cgttgagtgg 2160

tttcgctcag gggcaatcgc tgacgttaat tgctgcggaa ctggtggtaa tggttgttgt 2220

tggtattttc tttattcgcc gccagctttc tcttcccgta ccgctgctac cggtggattt 2280

actgcgtatc ccgctgtttt cactttctat ttgcacatct gtttgctctt tctgcgcaca 2340

aatgctggca atggtttccc tgccctttta cctgcaaacc gtgctcgggc gtagtgaagt 2400

cgaaacaggt ttacttctga caccgtggcc gttagcaacg atggtgatgg ctccgctggc 2460

aggctatttg attgaacgcg tacatgcagg attgctgggg gctttagggt tgttcatcat 2520

ggctgcgggg cttttttccc tggttctgct gcccgcgtca cctgcggata tcaatattat 2580

ctggccgatg atcttatgtg gtgctggatt tggcttattc cagtcaccca ataaccacac 2640

cattattacc tccgcgcctc gcgaacgtag cggtggagcc agtggcatgt taggaacggc 2700

tcgtctactg ggtcagagta gcggcgcggc gctggtggcg ctgatgctaa atcagtttgg 2760

agataatggt acacacgtct cgctgatggc tgcggctatt ctggcagtga ttgctgcctg 2820

2880

aaccccttgg ggcctctaaa cgcgtcgaca cgcaaaaagg ccatccgtca ggatggcctt 2940

ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg 3000

ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac 3060

3120

atgcctggca gttccctact ctcgcatggg gagaccccac actaccatca tgtatgaata 3180

tcctccttag ttcctattcc gaagttccta ttctctagaa agtataggaa cttcggcgcg 3240

3300

ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt 3360

ccaactttca ccataatgaa ataagatcac taccgggcgt

tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata 3480

tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct 3540

ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc 3600

3660

tacgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca 3720

ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt 3780

tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct 3840

3900

3960

tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc 4020

atgccgtttg tgatggcttc catgtcggca gatgcttaat gaatacaaca gtactgcgat 4080

gagtggcagg gcggggcgta aggcgcgcca tttaaatgaa gttcctattc cgaagttcct 4140

attctctaga aagtatagga acttcgaagc agctccagcc tacacaatcg ctcaagacgt 4200

gtaatgctgc aatctgcatg caagcttggc actggccacg caaaaaggcc atccgtcagg 4260

atggccttct gcttaatttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct 4320

ccgggccgtt gcttcgcaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga 4380

4440

tttatttgat gcctggcagt tccctactct cgcatgggga gaccccacac taccatcggg 4500

gggccatcga tgcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 4560

ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctgc tgcagaggcc 4620

tgcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 4680

tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 4740

gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 4800

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4860

ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4920

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4980

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 5040

tggcgtttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 5100

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 5160

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 5220

cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 5280

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5340

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5400

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5460

ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 5520

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5580

gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5640

atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5700

ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 5760

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 5820

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 5880

ccgtcgtgta gtaactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 5940

6000

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 6060

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 6120

ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 6180

aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 6240

gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 6300

cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 6360

actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 6420

caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 6480

gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 6540

ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 6600

6660

tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 6720

gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 6780

cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 6840

ataggcgtat cacgaggccc tttcgtc 6867

<210> 23

<211> 6768

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-proP

<400> 23

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgccgaacaa 1500

agctgaaacc tccccggcga aactgcgtct gaaagccttc ctgaaacgta tcaagattat 1560

gaacaccacc gaaaacagca aacagaagcc ggttaacgtg gttgcatttg ctttcctgct 1620

1680

ggcgcaggaa attcaagtct ctccgtttat ggtgggcatg ttctatacct caaatgcagt 1740

gctgggcatc gttctgtcgc agattctggc taaatacagt gattcccaag atgaccgtcg 1800

caagattatc attttctgca gtctgctggc gatcggcggt tgtatcacct tcgcctacaa 1860

ccgtaactac tacgtgctga tgtttttcgc gacgttcctg ctgtccctgg gtagttccgc 1920

aaacccgcag gcatttgcac tggcacgtga atatgcagac tacaccaaac gcgaagctat 1980

catgtttacc acgattatgc gcacgcagat cagcctggca tggattgttg gcccgccgct 2040

gtcattctcg attgcgctgg gctggggttt tgaatatatg tacatggtcg cggcctcagc 2100

ttgctaaagc gctgctgccg tatgtgccgc gtaaagccgt 2160

cgtgccgctg accaagccgg atgaagttgc gggtctgccg gccaaaaata aaaagcagag 2220

2280

catgtatctg atctccatgc cgctgcatgt tattaatgaa ctgcacctga gtgaacgtct 2340

ggcgggcatt ctgatgggta ccgcagctgg cctggaaatc ccggtgatgc tgattgccgg 2400

ctatctgacc aaatacctga cgaaaaagtc tctgatcctg accgccctgt tcatgggtct 2460

gtttttctat attggcatgc tgtttgcaga acagacgtgg caactggtcg ccctgcaggc 2520

atttaacgct atcttcattg gtatcattgc gaccctgggc atggtgtact ttcaagatct 2580

gatgccgggc aaaatgggtt cagccaccac gctgttctcg aacgcggcca aatcatcgtg 2640

gatcgttgca ggtccgtttg tcggcatcat tgctcagatt tggaattata gctctgtgtt 2700

ctacatcagc attgttctgg tcgcggtgtc tctgtttagc atgtctaaag ttaagagcgt 2760

ctaataaatc gatactagca taaccccttg gggcctctaa acgcgtcgac acgcaaaaag 2820

gccatccgtc aggatggcct tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc 2880

tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt 2940

cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga 3000

ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg ggagacccca 3060

cactaccatc atgtatgaat atcctcctta gttcctattc cgaagttcct attctctaga 3120

aagtatagga acttcggcgc gtcctacctg tgacggaaga tcacttcgca gaataaataa 3180

atcctggtgt ccctgttgat accgggaagc cctggggccaa cttttggcga aaatgagacg 3240

ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg 3300

tattttttga gttgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca 3360

ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc 3420

agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa 3480

agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc 3540

tgatgaatgc tcatccggaa ttacgtatgg caatgaaaga cggtgagctg gtgatatggg 3600

atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct 3660

ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt 3720

3780

cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact 3840

tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc 3900

cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agatgcttaa 3960

tgaatacaac agtactgcga tgagtggcag ggcggggcgt aaggcgcgcc atttaaatga 4020

agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcgaag cagctccagc 4080

ctacacaatc gctcaagacg tgtaatgctg caatctgcat gcaagcttgg cactggccac 4140

gcaaaaaggc catccgtcag gatggccttc tgcttaattt gatgcctggc agtttatggc 4200

gggcgtcctg cccgccaccc tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc 4260

ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag 4320

tctttcgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg 4380

agaccccaca ctaccatcgg ggggccatcg atgcaggtgg cacttttcgg ggaaatgtgc 4440

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 4500

aataaccctg ctgcagaggc ctgcatgcaa gcttggcgta atcatggtca tagctgtttc 4560

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 4620

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 4680

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 4740

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 4800

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 4860

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 4920

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 4980

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 5040

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 5100

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 5160

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 5220

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 5280

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 5340

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 5400

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 5460

gcaaacaaac caccgctggt agcggtggtt ttttgtttg caagcagcag attacgcgca 5520

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 5580

5640

5700

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 5760

catccatagt tgcctgactc cccgtcgtgt agataactac gatacggggag ggcttaccat 5820

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gattatcag 5880

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 5940

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 6000

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 6060

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 6120

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 6180

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 6240

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 6300

6360

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 6420

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 6480

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 6540

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 6600

6660

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 6720

tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 6768

<210> 24

<211> 6672

<212> DNA

<213> Artificial sequence

<220>

<223> construction pINT-Cn-setA

<400> 24

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgctgtggtt 1500

1560

gattggtgtg gccggtgcac tgcaggcacc gaccctgtct ctgtttctga cgcgtgaagt 1620

tgaagtccgc ccgtttgggg ttggtctgtt ctacacggtc aacgcaattg ctggcatcgg 1680

tgtgagtctg ctgctggcca aacgtagtga ttcccaaggc gaccgtcgca aactgattat 1740

ggtgtgctgt gttatggcgg tcgccaactg cgtcctgttt gcattcaatc gccattatct 1800

gaccctgatc acgctgggtg tgatgtttgc aagcattgct aataccgcga tgccgcagat 1860

cttcgcactg gctcgtgaat acgccgatcg ttctgcacgc gaagtggtta tgtttagctc 1920

tattatgcgc gcccaactga gtctggcatg ggttattggc ccgccgctgt ccttcatgct 1980

ggccctgaaa tatggtttta ccacgatgtt cctgattgca gctggcattt ttgtgatctc 2040

actggctctg attachtcttcg cgctgccgtc ggtgccgcgt gttgaacagc cggccgaagt 2100

ggcaattacc caagttagcg gttggaaaga ttctaacgtt cgcatgctgt ttatcgcctc 2160

aatgctgatg tggacctgta atacgatgta tattatcgac atgccgctgt ggatttcgca 2220

ggatctgggt ctgccggatg aactggccgg tctgctgatg ggtaccgccg caggcattga 2280

aatcccggct atgatcctgg cgggttatta cgtgaaacgt tttggcaaac gcaacatgat 2340

ggtcgcagct gtggcggccg gtattctgtt ttacgttggc ctgatcctgt tccatagcaa 2400

aacggcgctg gtcgtgctgc agctgtttaa tgccgtcttc attggtatta tcgcaggcat 2460

cggtatgctg tggtttcaag atctgatgcc gggtcgtccg ggtagcgcaa ccaccctgtt 2520

caccaactca atttcgacgg gcgtgattct ggccggtatt ctgcagggtg ccctggcaga 2580

aggttttggt cactatagtg tgtactggct gatggcagct ctggctgtta tcgcgctgtt 2640

cctgaccagc cgcgttaaaa acgtctaata aatcgatact agcataaccc cttggggcct 2700

ctaaacgcgt cgacacgcaa aaaggccatc cgtcaggatg gccttctgct taatttgatg 2760

ccctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct tcgcaacgtt 2820

2880

aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggcagttcc 2940

ctactctcgc atggggagac cccacactac catcatgtat gaatatcctc cttagttcct 3000

3060

aagatcactt cgcagaataa ataaatcctg gtgtccctgt tgataccgggg aagccctggg 3120

ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata 3180

atgaaataag atcactaccg ggcgtatttt ttgagttgtc gagattttca ggagctaagg 3240

aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc 3300

gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc 3360

agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg 3420

cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattacgt atggcaatga 3480

aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc 3540

aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac 3600

acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt 3660

ttattgagaa tatgtttttc gtctcagcca atccctggggt gagtttcacc agttttgatt 3720

taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata 3780

cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg 3840

gcttccatgt cggcagatgc ttaatgaata caacagtact gcgatgagtg gcagggcggg 3900

3960

taggaacttc gaagcagctc cagcctacac aatcgctcaa gacgtgtaat gctgcaatct 4020

gcatgcaagc ttggcactgg ccacgcaaaa aggccatccg tcaggatggc cttctgctta 4080

atttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 4140

gcaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 4200

caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg 4260

gcagttccct actctcgcat ggggagaccc cacactacca tcggggggcc atcgatgcag 4320

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 4380

caaatatgta tccgctcatg agacaataac cctgctgcag aggcctgcat gcaagcttgg 4440

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 4500

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 4560

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 4620

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 4680

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 4740

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 4800

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 4860

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 4920

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 4980

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 5040

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 5100

gctgtgtgca cgaaccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 5160

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 5220

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 5280

gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 5340

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 5400

tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 5460

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 5520

tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 5580

aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 5640

tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 5700

ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 5760

gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 5820

gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 5880

taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 5940

tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 6000

6060

tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 6120

ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 6180

6240

ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 6300

aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 6360

actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 6420

aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 6480

6540

aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 6600

ctgacgtcta agaaaccattattatcatga cattaaccta taaaaatagg cgtatcacga 6660

ggccctttcg tc 6672

<210> 25

<211> 7074

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-spoVB

<400> 25

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgaatgactc 1500

ggtcggtgct aagcgtcaaa gtgcgtggaa gggtgcgttc gtcctggtcg ttgcgggcat 1560

1620 cgttaccaag atcctgtctg ccgtgtatcg tgttccgttt

gggtttctat atctaccagc aagtttaccc gtttctgggc attgcggtca tgctgagtac 1680

ctccggtttt ccggtgatca tctcgaagct gatgaacgat tacagcgacc ataaacagaa 1740

gattatgaag atcagtgcac tgtatgtgac ggcagcaggt ctggttctgt ttgccctgat 1800

gtacgcaggt gcagctccgc tggcgggctt catgggtgat gaccgtctgg tcatgctgat 1860

tcgcgtggcg gcctttgctt tcatcctgtt tccgttcacc gcggtttttc gcggctattt 1920

ccagggtgtg cacgacatga tgccgtctgc tctgagtcag attacggaac aactgctgcg 1980

tgtggcagtt ctgctgggcc tgtctttttg gctgctgaaa tccggtcgtt cactgtacgc 2040

agctggtgca ggtgcagcat caggttcgat tgcaggtagt ctggcagctc tgtgcgttct 2100

ggcagtcttc tggtataaac gtgaagaaac caaaaaggat ggcggtcata tcgaaacggc 2160

ggttattatc aaaaagctgc tgctgtactc cgtgaccatt tgtatcagct ctgttctgat 2220

gctgctgctg cagctggttg atgcgctgaa cctgtattcg ctgctgagcg acggcaccga 2280

atcacatgcg gccaaacaac tgaagggcat ttacgaccgt ggtcagccgc tgctgcaact 2340

gggtacggtg tttgcggttt ccattgcagc ttcactggtc ccgagcatct ctaaagccgt 2400

gcacgaaaat aagccgttca ttatcaaaga aaaggctacc tctgcggtca aactgtgcct 2460

ggcggtgggc attggtgcta gtgcgggcct gttttgtatt ctggaaccgg ttaacatcat 2520

gctgttccag aattccgaag gtacccagac gctgcaaatc tttagtctgt ccattttctt 2580

tgcctcaatc gcactgaccg cagcagcaat cctgcaaggt gcaggtcata cggtgttccc 2640

ggcagtcagc gtgctggctg gcggtgcgct gaaatgggtc ctgaacgtgt ggctggttcc 2700

gggttggggt attaccggtg ctgcactggc tacggttctg gcatttgcag cagtcgcatg 2760

cctgaacctg cgtcgcatct ggtcgaaagg ttggctgacc aatattggcg gtgtgatcgc 2820

acgtctgtgc tggtgtagcc tgctgatggt gtttttcctg ctggtctata tgaaactgtg 2880

gcagctgttt gttccggtca gccgtgccgg cgcagtttgc gaatcactgt cggccagcgt 2940

gattggcggt ctgctgttca tctactgtat gatccgcatg aagatcttca ccgatgaaga 3000

actgagcggc ctgccgttcg gttctgcgct gagtaaactg aaaaagcgtc gcgaaaagca 3060

cggtcgctaa taaatcgata ctagcataac cccttggggc ctctaaacgc gtcgacacgc 3120

3180

gcgtcctgcc cgccaccctc cgggccgttg cttcgcaacg ttcaaatccg ctcccggcgg 3240

atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc 3300

3360

accccacact accatcatgt atgaatatcc tccttagttc ctattccgaa gttcctattc 3420

tctagaaagt ataggaactt cggcgcgtcc tacctgtgac ggaagatcac ttcgcagaat 3480

aaataaatcc tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat 3540

gagacgttga tcggcacgta agaggttcca actttcacca taatgaaata agatcactac 3600

3660

aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg 3720

catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct 3780

tttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg 3840

cccgcctgat gaatgctcat ccggaattac gtatggcaat gaaagacggt gagctggtga 3900

3960

cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg 4020

tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt 4080

tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg 4140

acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc 4200

tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagat 4260

gcttaatgaa tacaacagta ctgcgatgag tggcagggcg gggcgtaagg cgcgccattt 4320

aaatgaagtt cctattccga agttcctatt ctctagaaag tataggaact tcgaagcagc 4380

tccagcctac acaatcgctc aagacgtgta atgctgcaat ctgcatgcaa gcttggcact 4440

ggccacgcaa aaaggccatc cgtcaggatg gccttctgct taatttgatg cctggcagtt 4500

tatggcgggc gtcctgccg ccaccctccg ggccgttgct tcgcaacgtt caaatccgct 4560

cccggcggat ttgtcctact caggaagcg ttcaccgaca aacaacagat aaaacgaaag 4620

gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc 4680

atggggagac cccacactac catcgggggg ccatcgatgc aggtggcact tttcggggaa 4740

atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 4800

tgagacaata accctgctgc agaggcctgc atgcaagctt ggcgtaatca tggtcatagc 4860

tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 4920

taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 4980

cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 5040

gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 5100

tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 5160

tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 5220

cgggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 5280

agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 5340

accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 5400

ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 5460

gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 5520

ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 5580

gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 5640

taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 5700

tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 5760

gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 5820

cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5880

agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5940

cctagatcct tttaaattaa 6000

cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 6060

ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggaggggct 6120

taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 6180

tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 6240

ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 6300

6360

gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 6420

tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 6480

cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 6540

taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 6600

ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 6660

ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 6720

cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 6780

ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 6840

gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 6900

6960

aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca 7020

ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc 7074

<210> 26

<211> 6699

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-yabM

<400> 26

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgaaggctct 1500

1560

ggttggcatc gcaggtgcgc tgcagtcacc gaccctgtcg ctgtttctga gccgtgaagt 1620

gggtgttcgc ccgttttgggg tgggcctgtt ctatacggtt aacgcagtcg ctggtattat 1680

cgttccctg ctgctggcca aacgttcaga taatcagggc gaccgtcgca tgctgattct 1740

gttctgctgt gttatggcga tcgccaacgc agtcctgttt gccttcaatc gccattatct 1800

gaccctggtc attgcaggtg tgctgctgag ctctatcgct agcgtggcga tgccgcagat 1860

ttttgctctg gcgcgtgaat acgcagatag ttccgcccgc gaagcagtca tgttctcatc 1920

ggtgatgcgt gcccaactgt cgctggcatg ggttatcggt ccgccgctga gctttgccat 1980

tgcactgaac tacggcttta ccgcgatgtt cctggtggcg gccctgctgt ttttcgtctg 2040

cgtggctctg atttggttca ccctgccgag cgttccgcgt gcagaaaaca cggcagctga 2100

accgctgagt gatatctccg gttggaaaca ccgtgacgtg cgcatgctgt ttattgcctc 2160

tgttttcatg tggacctgta atacgatgta tgttatcgat atgccgctgt acattagtat 2220

2280

aattccggtc atgctgctgg ctggtcatta tgtgaaacgt tttggcaagc gcccgatgat 2340

gctgctggcg gttggctgcg gtgtcctgtt ttacctgggt ctggtgctgt tccacggccg 2400

tacggaactg atgctgctgc agctgctgaa cgctctgttt atcggcatta tcgcgggcat 2460

tggtatgatc tggttccaag atctgatgcc gggtcgtccg ggttctgcaa ccacgctgtt 2520

taccaatagc atttctacgg gtgtgatcct ggcaggtgtg ctgcaggggcg ttatggccga 2580

aacctttggc catcacgcag tctattggct ggcttccctg ctggcgctga tttctttcgc 2640

tctgagttgg caagttcgtg aagcgcgcac ggtgaagagt gttccgctgg cctaataaat 2700

2760

caggatggcc ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca 2820

ccctccgggc cgttgcttcg caacgttcaa atccgctccc ggcggatttg tcctactcag 2880

gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt 2940

tcgttttatt tgatgcctgg cagttcccta ctctcgcatg gggagacccc acactaccat 3000

3060

aacttcggcg cgtcctacct gtgacggaag atcacttcgc agaataaata aatcctggtg 3120

tccctgttga taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc 3180

acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg 3240

agttgtcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata 3300

3360

ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa 3420

agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg 3480

ctcatccgga attacgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc 3540

acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat 3600

3660

aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc 3720

cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc 3780

ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga 3840

ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagatgctta atgaatacaa 3900

cagtactgcg atgagtggca gggcggggcg taaggcgcgc catttaaatg aagttcctat 3960

tccgaagttc ctattctcta gaaagtatag gaacttcgaa gcagctccag cctacacaat 4020

cgctcaagac gtgtaatgct gcaatctgca tgcaagcttg gcactggcca cgcaaaaagg 4080

ccatccgtca ggatggcctt ctgcttaatt tgatgcctgg cagtttatgg cgggcgtcct 4140

gcccgccacc ctcggggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc 4200

ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac 4260

tgagcctttc gttttatttg atgcctggca gttccctact ctcgcatggg gagaccccac 4320

actaccatcg gggggccatc gatgcaggtg gcacttttcg gggaaatgtg cgcggaaccc 4380

ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 4440

gctgcagagg cctgcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 4500

attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 4560

ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 4620

agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 4680

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 4740

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 4800

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 4860

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 4920

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 4980

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccggga tacctgtccg 5040

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 5100

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 5160

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 5220

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 5280

agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 5340

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 5400

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 5460

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 5520

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 5580

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 5640

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 5700

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 5760

gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 5820

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 5880

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 5940

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 6000

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 6060

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 6120

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 6180

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 6240

cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 6300

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 6360

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 6420

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 6480

6540

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 6600

cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 6660

taacctataa aaataggcgt atcacgaggc cctttcgtc 6699

<210> 27

<211> 6684

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-ydeA

<400> 27

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgacaacaaa 1500

cactgtttcc cgcaaagtgg cgtggctacg ggtcgttacg ctggcagtcg ccgccttcat 1560

cttcaacacc accgaatttg tccctgttgg cctgctctct gacattgcgc aaagttttca 1620

catgcaaacc gctcaggtcg gcatcatgtt gaccattttac gcatgggtag tagcgctaat 1680

gtcattgcct tttatgttaa tgaccagtca ggttgaacgg cgcaaattac tgatctgcct 1740

1800

ggtgatcagt cgcattggtg tggcttttgc acatgcgatt ttctggtcga ttacggcgtc 1860

tctggcgatc cgtatggctc cggccgggaa gcgagcacag gcattgagtt taattgccac 1920

cggtacagca ctggcgatgg tcttaggttt acctctcggg cgcattgtgg gccagtattt 1980

cggttggcga atgaccttct tcgcgattgg tattggggcg cttatcaccc ttttgtgcct 2040

gattaagtta cttcccttac tgcccagtga gcattccggt tcactgaaaa gcctcccgct 2100

attgttccgc cgcccggcat tgatgagcat ttatttgtta actgtggtgg ttgtcaccgc 2160

ccattacacg gcatacagct atatcgagcc ttttgtacaa aacattgcgg gattcagcgc 2220

caactttgcc acggcattac tgttattact cggtggtgcg ggcattattg gcagcgtgat 2280

2340

gctggtgtgc ctggcattgc tgttacctgc ggcgaacagt gaaatacacc tcggggtgct 2400

gagtattttc tgggggatcg cgatgatgat catcgggctt ggtatgcagg ttaaagtgct 2460

ggcgctggca ccagatgcta ccgacgtcgc gatggcgcta ttctccggca tatttaatat 2520

tggaatcggg gcgggtgcgt tggtaggtaa tcaggtgagt ttgcactggt caatgtcgat 2580

gattggttat gtgggcgcgg tgcctgcttt tgccgcgtta atttggtcaa tcattatatt 2640

tcgccgctgg ccagtgacac tcgaagaaca gacgcaatag taaatcgata ctagcataac 2700

cccttggggc ctctaaacgc gtcgacacgc aaaaaggcca tccgtcagga tggccttctg 2760

cttaatttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg 2820

cttcgcaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga 2880

2940

ccctggcagtt ccctactctc gcatggggag accccacact accatcatgt atgaatatcc 3000

tccttagttc ctattccgaa gttcctattc tctagaaagt ataggaactt cggcgcgtcc 3060

tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg 3120

3180

3240

caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat 3300

cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata 3360

accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca 3420

agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattac 3480

3540

ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc 3600

ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt 3660

tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca 3720

ccagttttga tttaaacgtg gccaatatgg acaacttctt cgccccccgtt ttcaccatgg 3780

gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg 3840

ccgtttgtga tggcttccat gtcggcagat gcttaatgaa tacaacagta ctgcgatgag 3900

3960

ctctagaaag tataggaact tcgaagcagc tccagcctac acaatcgctc aagacgtgta 4020

atgctgcaat ctgcatgcaa gcttggcact ggccacgcaa aaaggccatc cgtcaggatg 4080

4140

ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact caggaagcg 4200

ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt 4260

atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcgggggg 4320

ccatcgatgc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 4380

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgctgc agaggcctgc 4440

atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4500

caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 4560

tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 4620

cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 4680

gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 4740

tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 4800

agaacatgtg agcaaaaggc cagcaaaagg cgggaaccg taaaaaggcc gcgttgctgg 4860

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 4920

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 4980

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5040

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5100

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5160

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5220

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5280

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 5340

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5400

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaagggatct caagaagatc 5460

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5520

tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 5580

ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 5640

gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 5700

tcgtgtagat aactacgata cgggaggggct taccatctgg ccccagtgct gcaatgatac 5760

cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 5820

ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 5880

gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 5940

caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 6000

6060 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa

ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 6120

tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 6180

caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 6240

tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 6300

cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6360

ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 6420

aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6480

tcatactctt ccttttttcaa tattattgaa gcatttatca gggtttattgt ctcatgagcg 6540

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 6600

acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 6660

ggcgtatcac gaggcccttt cgtc 6684

<210> 28

<211> 6738

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-propP2

<400> 28

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgaatgaatc 1500

cgccgtgaaa atcaaccgca ccttttatctc ccactatgct ctgctgctga aactgatgac 1560

catgttcgtc caacaacaga acagtacccc gtccaatatt gtggcgttta acttcctgct 1620

1680

gtcgcaagaa atcaatgtta gtccgttttt cgttggtctg ttttactccg ttaacgcgat 1740

tatcggcatt atcctgagcc agattctggc caaatattct gataagcaag atgaccgtcg 1800

caaagtcatg attgtgtgct gtctgatcgc agtgctgggt tgcctgatct ttgcttacag 1860

ccgtaattat tacgttctga ttatcattgg caccacgctg ctgggcctgg gtagctctgc 1920

aaacccgcag tcatttgcac tggctcgtga atatgcagaa agttcccatc gcgaagctgt 1980

tatgttcacc acgattatgc gcacccagat cagtctggca tggattgtcg gtccgccgct 2040

gtcctttttc attgctctga attggggctt tgattatatg tacctggtcg caggttcagc 2100

tttcctgctg tgcgccggcg tgtcgaaact gctgccgaag atcccgcgtc agtctgcagt 2160

2220

cgccaatctg ctgctgtgga cgtgtaattc catgtacctg atcaacatgc cgctgttcgt 2280

gattaacgaa ctgcacctgg gtaaagaact ggcaggtacc ctgatgggta cggcagcagg 2340

cctggaaatt ccggtgatga tctttgccgg ctatctgacc aaatacttct caaaaaagcg 2400

cctgatgatg attgcactgg tttcgggtct ggctttttat tcatcgctgc tgttcagcga 2460

tcagacctgg caactgatcg gcctgcagat gctgaacgcg atctttattg gtatcaccgc 2520

cacgattggc atggtttatt tccaagacct gatgccgacc aaaatgggta cggcgaccac 2580

gctgtttagt aatgcagcta agagctcttg gatcattggc ggtccgatcg cgggcatcat 2640

tgccgaaatc tggcattaca actctgtgtt ttatgtggcg gttgccctga ttttcatcag 2700

cgtcggctgt atgtggaagg ttaagtctgt ctaataaatc gatactagca taaccccttg 2760

gggcctctaa acgcgtcgac acgcaaaaag gccatccgtc aggatggcct tctgcttaat 2820

ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac cctccgggcc gttgcttcgc 2880

aacgttcaaa tccgctcccg gcggatttgt cctactcagg agagcgttca ccgacaaaca 2940

acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc 3000

agttccctac tctcgcatgg ggagacccca cactaccatc atgtatgaat atcctcctta 3060

gttcctattc cgaagttcct attctctaga aagtatagga acttcggcgc gtcctacctg 3120

tgacggaaga tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc 3180

cctgggccaa cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc 3240

accataatga aataagatca ctaccgggcg tattttttga gttgtcgaga ttttcaggag 3300

3360

ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga 3420

3480

atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttacgtatgg 3540

caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc 3600

3660

ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta 3720

3780

ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat 3840

attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt 3900

3960

ggcggggcgt aaggcgcgcc atttaaatga agttcctatt ccgaagttcc tattctctag 4020

aaagtatagg aacttcgaag cagctccagc ctacacaatc gctcaagacg tgtaatgctg 4080

caatctgcat gcaagcttgg cactggccac gcaaaaaggc catccgtcag gatggccttc 4140

tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt 4200

tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc 4260

gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga 4320

tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg ggggccatcg 4380

atgcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 4440

tacattcaaa tatgtatccg ctcatgagac aataaccctg ctgcagaggc ctgcatgcaa 4500

gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 4560

cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 4620

aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 4680

agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 4740

ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 4800

ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 4860

tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 4920

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 4980

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5040

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5100

tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5160

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5220

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5280

acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 5340

actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 5400

tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 5460

tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 5520

tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 5580

tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 5640

caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 5700

cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 5760

agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 5820

acccacgctc accggctcca gattatcag caataaacca gccagccgga agggccgagc 5880

gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 5940

ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 6000

tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 6060

ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 6120

tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 6180

attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 6240

agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 6300

ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 6360

ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 6420

cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 6480

gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 6540

tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 6600

6660

tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 6720

tcacgaggcc ctttcgtc 6738

<210> 29

<211> 6513

<212> DNA

<213> Artificial sequence

<220>

<223> construction pINT-Pc-setA

<400> 29

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgttctacac 1500

gggctcggca gttattggta tcgtcgtctc gcagatgctg gctacgcgct cggatcgtca 1560

gggtgaccgc aagtcgctga tcttcgtttg ctgtctgctg ggtgcgctgg cctgcatgct 1620

gtttgcgtgg aaccgcaatt atttcatcct gctgtttatt ggtgtgctgc tgagctcttt 1680

cggcagtacc gccaacccgc agctgtttgc actggctcgc gaacatgcag ataaaacggg 1740

tcgtgaagcg gccatgttca gttccatcct gcgtgcccaa atttccctgg catgggtggt 1800

tggtccgccg attgcgtttg ccctggcact gggcttcggt tttaccacga tgtacctgac 1860

cgcagctgtc gtgttcatcc tgtgtggtat tctggtgaag ctgtttctgc cgagcatgcc 1920

gaaagccgtt gaaaagacca cgagcaccct ggaatctccg cgtcgcaatc gtcgcgacac 1980

gctgctgctg tttgttgcgt gcaccctgat gtggacgtgt aacggcatct atctgattaa 2040

tatgccgctg tacctggttc atgaactgca cctgccggaa aaactggcag gtatcatgat 2100

gggtgtcgca gcaggtctgg aaatcccggt tatgctgatt gccggttatg tcgcaaaacg 2160

tttcggcaag cgctttctga tgcgtctggc tgtcgcgagc ggtctgctgt ttttcggcgg 2220

tctgctggtg ctggatggcg aaatcgccct gctggcactg caggctctga acgcgatttt 2280

catcggcatt ctggctggca ttggtatgct gtactttcag gacctgatgc cgggccaagc 2340

aggtgcagct accacgctgt ttaccaacac cacgcgcgtg ggttggatta tctcaggttc 2400

gctggctggc atcgtggcgg aaatttggaa ttatcacgct gtgtttttct ttgcgctgct 2460

gatgatcgtc ggctctattt actgcatgtg gcgtattaaa gatgcgtaat aaatcgatac 2520

tagcataacc ccttggggcc tctaaacgcg tcgacacgca aaaaggccat ccgtcaggat 2580

ggccttctgc ttaatttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc 2640

gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc 2700

2760

tatttgatgc ctggcagttc cctactctcg catggggaga ccccacacta ccatcatgta 2820

2880

ggcgcgtcct acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg 2940

ttgataccgg gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcacgtaa 3000

3060

cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg 3120

ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat 3180

gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa 3240

ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc 3300

3360

3420

acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc 3480

tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg 3540

3600

tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 3660

ttcatcatgc cgtttgtgat ggcttccatg tcggcagatg cttaatgaat acaacagtac 3720

tgcgatgagt ggcagggcgg ggcgtaaggc gcgccattta aatgaagttc ctattccgaa 3780

gttcctattc tctagaaagt ataggaactt cgaagcagct ccagcctaca caatcgctca 3840

agacgtgtaa tgctgcaatc tgcatgcaag cttggcactg gccacgcaaa aaggccatcc 3900

gtcaggatgg ccttctgctt aatttgatgc ctggcagttt atggcgggcg tcctgcccgc 3960

caccctccgg gccgttgctt cgcaacgttc aaatccgctc ccggcggatt tgtcctactc 4020

aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc 4080

tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagacc cccacactacc 4140

atcggggggc catcgatgca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4200

gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgctgca 4260

gaggcctgca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 4320

atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 4380

cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 4440

gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4500

gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 4560

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 4620

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 4680

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 4740

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 4800

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 4860

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 4920

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 4980

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 5040

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 5100

tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 5160

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 5220

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 5280

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 5340

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 5400

aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 5460

gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 5520

gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 5580

caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 5640

ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 5700

attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 5760

ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 5820

gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 5880

ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 5940

tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 6000

gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 6060

cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 6120

gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 6180

tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 6240

ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 6300

gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 6360

tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 6420

catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 6480

ataaaaatag gcgtatcacg aggccctttc gtc 6513

<210> 30

<211> 6810

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-fucP

<400> 30

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgggaaacac 1500

atcaatacaa acgcagagtt accgtgcggt agataaagat gcagggcaaa gcagaagtta 1560

cattattcca ttcgcgctgc tgtgctcact gttttttctt tgggcggtag ccaataacct 1620

taacgacatt ttattacctc aattccagca ggcttttacg ctgacaaatt tccaggctgg 1680

cctgatccaa tcggcctttt actttggtta tttcattatc ccaatccctg ctgggatatt 1740

gatgaaaaaa ctcagttata aagcagggat tattaccggg ttatttttat atgccttggg 1800

tgctgcatta ttctggccg ccgcagaaat aatgaactac accttgtttt tagttggcct 1860

atttattatt gcagccggat taggttgtct ggaaactgcc gcaaaccctt ttgttacggt 1920

attagggccg gaaagtagtg gtcacttccg cttaaatctt gcgcaaacat ttaactcgtt 1980

tggcgcaatt atcgcggttg tctttgggca aagtcttatt ttgtctaacg tgccacatca 2040

atcgcaagac gttctcgata aaatgtctcc agagcaattg agtgcgtata aacacagcct 2100

ggtattatcg gtacagacac cttatatgat catcgtggct atcgtgttac tggtcgccct 2160

gctgatcatg ctgacgaaat tcccggcatt gcagagtgat aatcacagtg acgccaaaca 2220

aggatcgttc tccgcatcgc tttctcgcct ggcgcgtatt cgccactggc gctgggcggt 2280

attagcgcaa ttctgctatg tcggcgcaca aacggcctgc tggagctatt tgattcgcta 2340

cgctgtagaa gaaattccag gtatgactgc aggctttgcc gctaactatt taaccggaac 2400

catggtgtgc ttctttattg gtcgtttcac cggtacctgg ctcatcagtc gcttcgcacc 2460

acacaaagtc ctggccgcct acgcattaat cgctatggca ctgtgcctga tctcagcctt 2520

cgctggcggt catgtgggct taatagccct gactttatgc agcgccttta tgtcgattca 2580

gtacccaaca atcttctcgc tgggcattaa gaatctcggc caggacacca aatatggttc 2640

gtccttcatc gttatgacca ttattggcgg cggtattgtc actccggtca tgggttttgt 2700

cagtgacgcg gcgggcaaca tccccactgc tgaactgatc cccgcactct gcttcgcggt 2760

catctttatc tttgcccgtt tccgttctca aacggcaact aactgataaa tcgatactag 2820

cataacccct tggggcctct aaacgcgtcg acacgcaaaa aggccatccg tcaggatggc 2880

cttctgctta atttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccgggg 2940

ccgttgcttc gcaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt 3000

3060

ttgatgcctg gcagttccct actctcgcat ggggagaccc cacactacca tcatgtatga 3120

atatcctcct tagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcggc 3180

gcgtcctacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg 3240

ataccgggaa gccctggggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag 3300

3360

gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat acccgttg 3420

atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta 3480

aaagaccgta aagaaaaata 3540

agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg 3600

3660

acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg 3720

atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg 3780

3840

gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca 3900

ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc 3960

atcatgccgt ttgtgatggc ttccatgtcg gcagatgctt aatgaataca acagtactgc 4020

gatgagtggc agggcggggc gtaaggcgcg ccatttaaat gaagttccta ttccgaagtt 4080

4140

cgtgtaatgc tgcaatctgc atgcaagctt ggcactggcc acgcaaaaag gccatccgtc 4200

aggatggcct tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc tgcccgccac 4260

cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt cctactcagg 4320

agagcgttca ccgacaaaca acagataaaa cgaaaaggccc agtctttcga ctgagccttt 4380

cgttttattt gatgcctggc agttccctac tctcgcatgg ggagacccca cactaccatc 4440

ggggggccat cgatgcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4500

tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgctgcagag 4560

gcctgcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 4620

cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 4680

aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 4740

acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 4800

ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 4860

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 4920

catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 4980

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 5040

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 5100

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 5160

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 5220

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5280

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5340

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5400

agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 5460

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 5520

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 5580

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 5640

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 5700

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 5760

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 5820

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 5880

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 5940

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 6000

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 6060

ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 6120

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6180

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 6240

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 6300

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 6360

cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 6420

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 6480

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 6540

6600

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcaggggt tattgtctca 6660

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 6720

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 6780

aaaataggcg tatcacgagg ccctttcgtc 6810

<210> 31

<211> 6933

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-mdeA

<400> 31

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgtccaaaaa 1500

acaaaaactg acgatgatta ttacgatgct gatgggtggc ttcttcggtc tgctgaatga 1560

aacgctgctg gtgacggcac tgccgagcat catgaaagac ttcgaaattt cttatacgca 1620

ggttcaatgg ctgaccacgg catttctgct gaccaacggc atcgttattc cgctgtcagc 1680

tctggtcatt cagcgttaca ccacgcgcca agttttcctg gtcggtatct ctattttctt 1740

tctgggcacg ctgctgtcag gtctgtcgcc gcattttgcg accctgctgg ttgcgcgtat 1800

tatccaggca ctgggcgctg gtatcatgat gccgctgatg atgaccacga ttctggatgt 1860

cttccaaccg cacgaacgcg gcaaatatat gggcattttt ggtctggtga tcggtctggc 1920

accggcaatc ggtccgaccc tgagtggtta tctggttgaa tacttcaact ggcgttccct 1980

gtttcatgtg gttgcgccga tcgcggccgt tacctttctg attggcttca aaacgatcaa 2040

aaatgtgggt accacgatta aagttccgat cgactttatt tcagtcatct tctcggtgct 2100

gggctttggc ggtctgctgt atggtaccag ctctatttca gaaaaaggct tcgataatcc 2160

gatcgtcctg gtgtcgatga ttggcggtgt cgtgctggtt gcactgtttg tcctgcgtca 2220

gtaccgcctg agcaccccgc tgctgaactt cgctgtgttc aaaaacaaac aattcaccgt 2280

tggcattatc attatgggtg tgacgatggt tagcatgatc ggctctgaaa ccattctgcc 2340

gatctttgtt cagaacctgc tgcatcgtag tgcactggac tccggtctga cgctgctgcc 2400

gggtgcaatt gtgatggcct tcatgagcat gacctctggc gccctgtatg aaaaatttgg 2460

2520

ggttatggat gaacagacca gtacgattat gctggcaacc gtctacgcta ttcgcatggt 2580

gggcatcgcg ctgggtctga ttccggttat gacccatacg atgaaccagc tgaaaccggga 2640

aatgaatgcg cacggcagtt ccatgaccaa cacggtgcag caaattgccg gcagcatcgg 2700

taccgcagct ctgatcacga ttctgagtca cgcctccaaa aacttttcac cgaccatgtc 2760

ggattacaac ggtatgaaca aaatcgacat gatgaaccag atcaaagtcg ataccatgct 2820

gcatggctac cacgcgggtt ttctgttcgc cctgctgatt accgtggtgt cgttcttctg 2880

ttcatttatg ctgcaaggca aaaagaaaga agtggattcc cgccagtaat aaatcgatac 2940

tagcataacc ccttggggcc tctaaacgcg tcgacacgca aaaaggccat ccgtcaggat 3000

ggccttctgc ttaatttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc 3060

gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc 3120

3180

tatttgatgc ctggcagttc cctactctcg catggggaga ccccacacta ccatcatgta 3240

tgaatatcct ccttagttcc tattccgaag ttcctattct ctagaaagta taggaacttc 3300

ggcgcgtcct acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg 3360

ttgataccgg gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcacgtaa 3420

3480

cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg 3540

ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat 3600

gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa 3660

ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc 3720

cggaattacg tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt 3780

3840

acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc 3900

tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg 3960

tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gccccccgttt 4020

tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg 4080

ttcatcatgc cgtttgtgat ggcttccatg tcggcagatg cttaatgaat acaacagtac 4140

tgcgatgagt ggcagggcgg ggcgtaaggc gcgccattta aatgaagttc ctattccgaa 4200

gttcctattc tctagaaagt ataggaactt cgaagcagct ccagcctaca caatcgctca 4260

agacgtgtaa tgctgcaatc tgcatgcaag cttggcactg gccacgcaaa aaggccatcc 4320

gtcaggatgg ccttctgctt aatttgatgc ctggcagttt atggcgggcg tcctgcccgc 4380

caccctccgg gccgttgctt cgcaacgttc aaatccgctc ccggcggatt tgtcctactc 4440

aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc 4500

tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagacc cccacactacc 4560

atcggggggc catcgatgca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4620

gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgctgca 4680

gaggcctgca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 4740

atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 4800

cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 4860

gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4920

gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 4980

ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 5040

acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 5100

cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 5160

caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 5220

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 5280

tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 5340

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 5400

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 5460

cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 5520

tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtc tgcgctctgc 5580

tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 5640

ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 5700

aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 5760

aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 5820

aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 5880

gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 5940

gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 6000

caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 6060

6120

attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 6180

ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 6240

gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 6300

6360

tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 6420

gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 6480

cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 6540

gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 6600

tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 6660

ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 6720

gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 6780

tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 6840

catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 6900

ataaaaatag gcgtatcacg aggccctttc gtc 6933

<210> 32

<211> 7248

<212> DNA

<213> Artificial sequence

<220>

<223> construct pINT-lmrA

<400> 32

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tggcgaaccg 1500

tatcgaaggc aaagctgtgg acaaaacctc aatcaaacat ttcattaaac tgatccgtgc 1560

1620

gattcagctg caagtcccga aaatggtgca gccgctggtt aactcttttg gtcatggcgt 1680

taatggcggt aaagttgccc tggtcattgc actgtatatc ggtagtgcag cagtctccgc 1740

aattgcagct atcgtgctgg gtatctttgg cgaaagcgtg gttaaaaacc tgcgtacgcg 1800

cgtttgggat aaaatgattc acctgccggt gaaatacttc gacgaagtta aaaccggtga 1860

aatgagctct cgtctggcga atgataccac gcaagtgaaa aacctgattg caaatagcat 1920

cccgcaggct tttacgtcta ttctgctgct ggtcggcagt atcgtgttca tgctgcagat 1980

gcaatggcgc ctgaccctgg ctatgattat cgcggttccg gtcgtgatgc tgattatgtt 2040

tccgatcatg acgttcggtc agaaaattgg ccgtacccgc caagatagcc tggcgaactt 2100

tcagggtatt gcctcagaat cgctgagcga aatccgtctg gtgaaaagtt ccaatgccga 2160

2220

agaagcgatc tttgatggcc tgatgagtcc ggtcatgatg ctgtccatga tgctgatgat 2280

cttcggtctg ctggcctatg gcatttacct gatcagcacg ggtgtgatgt ctctgggtac 2340

cctgctgggc atgatgatgt acctgatgaa cctgattggc gcggtgccga ccgttgccac 2400

gtttttcacc gaactggcga aagcctctgg tagtacgggc cgtctgaccg aactgctgga 2460

tgaagaacag gaagttctgc atcagggtga atcgctggat ctggaaggca aaaccctgag 2520

cgcacgtcac gtcgactttg cttatgatga ctctgaacaa attctgcgcg atatctcctt 2580

tgaagcgcag ccgaattcaa ttatcgcatt cgctggcccg agtggcggtg gcaaatcaac 2640

catcttttcg ctgctggaac gcttctacca accgacggcc ggtgaaatta ccatcgatgg 2700

ccagccgatt gacaacatct cactggaaaa ttggcgttcg cagattggtt tcgttagcca 2760

agactctgct attatggcgg gcacgatccg cgaaaacctg acctatggtc tggaaggcga 2820

ttacacggat gaagacctgt ggcaggtcct ggacctggcg tttgcccgtt cattcgtgga 2880

aaacatgccg gatcagctga ataccgaagt tggtgaacgc ggcgtcaaaa tttcgggtgg 2940

ccagcgtcaa cgcctggcaa tcgctcgtgc gtttctgcgc aatccgaaaa ttctgatgct 3000

ggatgaagcc accgcatctc tggactccga atcagaatcg atggtgcaga aagcgctgga 3060

tagtctgatg aaaggtcgta ccacgctggt gattgcccat cgcctgtcca cgatcgttga 3120

tgcagacaaa atctacttca tcgaaaaagg ccagatcacc ggtagcggca aacacaacga 3180

actggtcgca acccacccgc tgtacgcaaa atatgtctcg gaacaactga cggtcggcca 3240

ataataaatc gatactagca taaccccttg gggcctctaa acgcgtcgac acgcaaaaag 3300

gccatccgtc aggatggcct tctgcttaat ttgatgcctg gcagtttatg gcgggcgtcc 3360

tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt 3420

cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga 3480

ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcatgg ggagacccca 3540

cactaccatc atgtatgaat atcctcctta gttcctattc cgaagttcct attctctaga 3600

aagtatagga acttcggcgc gtcctacctg tgacggaaga tcacttcgca gaataaataa3660

atcctggtgt ccctgttgat accgggaagc cctggggccaa cttttggcga aaatgagacg 3720

ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg 3780

tattttttga gttgtcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca 3840

ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc 3900

agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa 3960

agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc 4020

tgatgaatgc tcatccggaa ttacgtatgg caatgaaaga cggtgagctg gtgatatggg 4080

atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct 4140

ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt 4200

gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg ttttcgtct 4260

cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact 4320

tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc 4380

cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agatgcttaa 4440

tgaatacaac agtactgcga tgagtggcag ggcggggcgt aaggcgcgcc atttaaatga 4500

agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcgaag cagctccagc 4560

ctacacaatc gctcaagacg tgtaatgctg caatctgcat gcaagcttgg cactggccac 4620

gcaaaaaggc catccgtcag gatggccttc tgcttaattt gatgcctggc agtttatggc 4680

gggcgtcctg cccgccaccc tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc 4740

ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg aaaggcccag 4800

tctttcgact gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg 4860

agaccccaca ctaccatcgg ggggccatcg atgcaggtgg cacttttcgg ggaaatgtgc 4920

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 4980

aataaccctg ctgcagaggc ctgcatgcaa gcttggcgta atcatggtca tagctgtttc 5040

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 5100

gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 5160

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 5220

ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 5280

cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 5340

cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 5400

accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 5460

acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 5520

cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 5580

acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 5640

atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 5700

agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 5760

acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 5820

gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 5880

gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 5940

gcaaacaaac caccgctggt agcggtggtt ttttgtttg caagcagcag attacgcgca 6000

gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 6060

6120

6180

ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 6240

catccatagt tgcctgactc cccgtcgtgt agataactac gatacggggag ggcttaccat 6300

ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gattatcag 6360

caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 6420

ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 6480

tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 6540

cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 6600

aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 6660

tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 6720

gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 6780

6840

aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 6900

tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 6960

tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 7020

gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 7080

7140

taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 7200

tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 7248

<210> 33

<211> 6684

<212> DNA

<213> Artificial sequence

<220>

<223> construction pINT-Ps-setA

<400> 33

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgaacgaaag 1500

cggagctct catggcggtt catggctgtc ggttattgcg ctggccctgg cggcctttat 1560

cttcaatacc acggaattcg ttccggtcgc gctgctgtca gatattggcc gttcgtttga 1620

catgccgcca tcacaagtgg gtctgatgct gaccatctat gcgtgggtgg ttgccctgat 1680

gtcgctgccg atgatgctgc tgacccgcaa cgtcgaacgt cgcacgctgc tgatttttgt 1740

gttcgtcgtg ttcatcggca gtcatctggt gagttccgtg gcgtcatcgt ttagcatgct 1800

gatgatttct cgtattggta tcgcactgtc ccacgctgtg ttttggagta tcaccgcatc 1860

cctggctgtg cgtgttgcac cggctggtaa acaggcccag gcactgggtc tgctggcaac 1920

cggttcagca ctggctatgg tcctgggtat tccgctgggc cgtgttgtcg gtgaactgct 1980

ggattggcgc accacgttcc tgagcattgc catcgtggca gctctggtgg ttctgtgtct 2040

ggcacgtacc ctgccgctgc tgccgagtca gaatagtggt tccctgcgtt ccctgccgat 2100

gctgtttaaa cgtccggcgc tggttgcggc atatgttctg accgccctgg ttattacggc 2160

gcagtttacc gcctatacgt acattgaacc gttcgcacaa accatcgctc atctgtctgg 2220

caacatgacc acggcactgc tgctgctgtt tggcggtgct ggtattctgg gcacggtgct 2280

gttcagccgt tattctaatc gctacccgaa aggttttctg atcgcagcta ttagtatcat 2340

ggcaatgtgt ctgctgctgc tgctgccggc ctcccgcgat agctctctgc tggccgccct 2400

ggtcgtggtt tggggtattg cgggcatgtg tttcggcctg gcgctgcagg ccaaagttct 2460

gaacctggca agcgatgcta ccgacgtcgc gatggccctg ttttctggca tttataatgt 2520

tggtatcggc ggtggcgcc tgctgggttc actggttacg gcacacctgg gcctgtcgga 2580

cgttggtatt gtcggtggcc tgctggccct gagcggcgtc gtgctgtgct gttttgccac 2640

ctatcgcttt gcacgtccgg tgggttctgc agctctgtaa taaatcgata ctagcataac 2700

cccttggggc ctctaaacgc gtcgacacgc aaaaaggcca tccgtcagga tggccttctg 2760

cttaatttga tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg 2820

cttcgcaacg ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga 2880

2940

ccctggcagtt ccctactctc gcatggggag accccacact accatcatgt atgaatatcc 3000

tccttagttc ctattccgaa gttcctattc tctagaaagt ataggaactt cggcgcgtcc 3060

tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg 3120

3180

3240

caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat 3300

cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata 3360

accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca 3420

agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattac 3480

3540

ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc 3600

ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt 3660

tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca 3720

ccagttttga tttaaacgtg gccaatatgg acaacttctt cgccccccgtt ttcaccatgg 3780

gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg 3840

ccgtttgtga tggcttccat gtcggcagat gcttaatgaa tacaacagta ctgcgatgag 3900

3960

ctctagaaag tataggaact tcgaagcagc tccagcctac acaatcgctc aagacgtgta 4020

atgctgcaat ctgcatgcaa gcttggcact ggccacgcaa aaaggccatc cgtcaggatg 4080

4140

ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact caggaagcg 4200

ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt 4260

atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcgggggg 4320

ccatcgatgc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 4380

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgctgc agaggcctgc 4440

atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4500

caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 4560

tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 4620

cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 4680

gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 4740

tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 4800

agaacatgtg agcaaaaggc cagcaaaagg cgggaaccg taaaaaggcc gcgttgctgg 4860

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 4920

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 4980

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5040

gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5100

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5160

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5220

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5280

ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag 5340

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5400

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaagggatct caagaagatc 5460

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 5520

tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 5580

ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 5640

gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 5700

tcgtgtagat aactacgata cgggaggggct taccatctgg ccccagtgct gcaatgatac 5760

cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 5820

ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 5880

gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 5940

caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 6000

6060 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa

ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 6120

tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 6180

caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 6240

tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 6300

cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 6360

ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 6420

aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 6480

tcatactctt ccttttttcaa tattattgaa gcatttatca gggtttattgt ctcatgagcg 6540

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 6600

acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 6660

ggcgtatcac gaggcccttt cgtc 6684

<210> 34

<211> 6692

<212> DNA

<213> Artificial sequence

<220>

<223> construction pINT-Bb-setA

<400> 34

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaagatcct ttgatctttt 420

ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 480

tatcaaaaag gatcttcacc tagatccttt taaactagtg aagttaccat cacggaaaaa 540

ggttatgctg cttttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 600

tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 660

cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 720

cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccact gcgctgagtg 780

catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 840

gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 900

gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 960

ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1020

cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1080

tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1140

cgctgttaat cactttactt ttatctaaac gagacatact cttccttttt caatattatt 1200

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 1260

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa attggccaga 1320

tgattaattc ctaatttttg ttgacactct atcattgata gagttatttt accactccct 1380

atcagtgata gagaaaagtg aaatgaatag ttcgacaaaa atctagaaat aattttgttt 1440

aactttaaga aggagatata caagagctcg agtcgaagga gatagaacca tgaccattgc 1500

1560

1620

catggaaacc gcccaagtgg gtattatgct gacgatctat gcctgggttg tggcactgat 1680

gagcctgccg tttatgctga tgacctctca ggtggaacgt cgccgtctgc tgattagcat 1740

ctttctgctg ttcatcgcaa gtcatgttct gtcctttctg gcgtggaatt tcaccgttct 1800

ggtcatttct cgcattggta tcgcgtttgc ccacgcaatt ttctggtcaa tcacggcttc 1860

gctggcgatt cgtatggctc cggcgggcaa gaaagcgcag gcactgagtc tgctggcgac 1920

cggtacggct ctggcgatgg ttctgggtct gccgatcggc cgcattgtcg gtcaatactt 1980

tggctggcgt accacgtttt tcgtgattgg cgttgtcgca gctatcaccc tgttctgcct 2040

gattaaactg ctgccgaaac tgccgagcga acatagtggt tccctgagct ctgtgccgaa 2100

actgtttcgc cgtccggcgc tggttaacat ctatgccctg attgcaatcg tggttaccgc 2160

acactacacg gcttatagtt acatcgaacc gttcgtgcag caaattgccg gcctgtccgc 2220

taactttgcg accctgctgc tgctgctgtt tggcggtgcg ggtattatcg gctctgttct 2280

gtttggtaaa tggggcaata aacatgccag cggtctggtc tctggcgcca ttgcactgat 2340

ggccgcatgt ctggtgctgc tgctgccggc agctcagggt gaactgaccc tggccggcct 2400

2460

ggctctggcc ccggatgcca ccgatgttgc catgagcctg ttttctggca tcttcaacat 2520

cggcattggt gccggcgcac tgctgggtaa tcaggtgtca ctgcacattt caatgtcgga 2580

catcggtttt attggcgcca tcccggcaat tatcgctctg gtctggtcga ttctggtgtc 2640

cgccgttggc cggttgccct ggaagaacat ccgcaggcaa cccactaata aatcgatact 2700

agcataaccc cttggggcct ctaaacgcgt cgacacgcaa aaaggccatc cgtcaggatg 2760

2820

ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact caggaagcg 2880

ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt 2940

atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcatgtat 3000

gaatatcctc cttagttcct attccgaagt tcctattctc tagaaagtat aggaacttcg 3060

gcgcgtccta cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt 3120

3180

aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttgtc 3240

3300

tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg 3360

tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa 3420

taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc 3480

ggaattacgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg 3540

ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga 3600

cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct 3660

3720

3780

caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt 3840

tcatcatgcc gtttgtgatg gcttccatgt cggcagatgc ttaatgaata caacagtact 3900

gcgatgagtg gcagggcggg gcgtaaggcg cgccatttaa atgaagttcc tattccgaag 3960

ttcctattct ctagaaagta taggaacttc gaagcagctc cagcctacac aatcgctcaa 4020

gacgtgtaat gctgcaatct gcatgcaagc ttggcactgg ccacgcaaaa aggccatccg 4080

tcaggatggc cttctgctta atttgatgcc tggcagttta tggcgggcgt cctgcccgcc 4140

accctccggg ccgttgcttc gcaacgttca aatccgctcc cggcggattt gtcctactca 4200

ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct 4260

ttcgttttat ttgatgcctg gcagttccct actctcgcat ggggagaccc cacactacca 4320

tcggggggcc atcgatgcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4380

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgctgcag 4440

aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 4500

tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 4560

ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 4620

aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 4680

tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 4740

gcgagcggta tcagctcact caaaaggcggt aatacggtta tccacagaat caggggataa 4800

cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 4860

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 4920

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 4980

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 5040

cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 5100

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 5160

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 5220

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 5280

gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 5340

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 5400

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 5460

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 5520

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 5580

atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 5640

cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 5700

actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 5760

aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 5820

cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 5880

ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 5940

cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 6000

ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 6060

cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 6120

ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 6180

tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 6240

ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 6300

aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 6360

gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 6420

6480

6540

catgagcggga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 6600

atttccccga aaagtgccac ctgacgtcta agaaaccatt

taaaaatagg cgtatcacga ggccctttcg tc 6692

<210> 35

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 35

ttactcagca ataaactgat attccgtcag gctgg 35

<210> 36

<211> 83

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 36

ttgtaatctc gcgctcttca catcagactt tccatataga gcgtaatttc cgttaacgtc 60

ggtagtgctg accttgccgg agg 83

<210> 37

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 37

ctgtctctta tcacatctcc tgaaatggcc agatgtaatt cctaattttt gtt 53

<210> 38

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 38

ctgtctctta tcacatctca cattacatct gagcgattgt tagg 44

<210> 39

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 39

gatcacatat gagagttctg gttaccggtg 30

<210> 40

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 40

gatcactcga gtcattaatc gggatatccc tgtggatggc 40

<210> 41

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 41

gtcgatgaag ccctgaaaga cgcgcagact atgcacttca ttgaaaacaa aaacttcgtc 60

<210> 42

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 42

gatggccttt ttgcgtgtcg acgcggccgc ctagataaac aggatgatat ttttgccttg 60

<210> 43

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 43

caaggcaaaa atatcatcct gtttatctag gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 44

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 44

gacgaagttt ttgttttcaa tgaagtgcat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 45

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 45

atggcctttt tgcgtgtcga cgcggccgct taattcgagc gggtaaagat cttcatcagg 60

<210> 46

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 46

atggcctttt tgcgtgtcga cgcggccgct taattcgagc gggtaaagat cttcatcagg 60

<210> 47

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 47

ctgatgaaga tctttacccg ctcgaattaa gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 48

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 48

cggggtacgc ttaatcaggt tatcaatcat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 49

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 49

gtcgatgaag ccctgaaaga cgcgcagact atgagcggtg aacactatgt cattagcctg 60

<210> 50

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 50

gatggccttt ttgcgtgtcg acgcggccgc tcatttaaat tcgatgatca tcttgtcgtt

<210> 51

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 51

aacgacaaga tgatcatcga atttaaatga gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 52

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 52

caggctaatg acatagtgtt caccgctcat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 53

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 53

gtcgatgaag ccctgaaaga cgcgcagact atggatgaaa tcaaactgtc ggtggttatg 60

<210> 54

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 54

gatggccttt ttgcgtgtcg acgcggccgc tcattggcga cgccaatcga acgcaacgcg 60

<210> 55

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 55

cgcgttgcgt tcgattggcg tcgccaatga gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 56

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 56

cataaccacc gacagtttga tttcatccat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 57

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 57

gtcgatgaag ccctgaaaga cgcgcagact atggaaaact atgtcgtctc tatccgcacc 60

<210> 58

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 58

gatggccttt ttgcgtgtcg acgcggccgc tcatttgaac ggaacaatct ttttgtcatc 60

<210> 59

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 59

gatgacaaaa agattgttcc gttcaaatga gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 60

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 60

ggtgcggata gagacgacat agttttccat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 61

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 61

gtcgatgaag ccctgaaaga cgcgcagact atgtcctcag ctttccatta cgtcattagc 60

<210> 62

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 62

gatggccttt ttgcgtgtcg acgcggccgc tcattcaaat tcgataatca tggtgatttt 60

<210> 63

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 63

aaaatcacca tgattatcga atttgaatga gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 64

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 64

gctaatgacg taatggaaag ctgaggacat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 65

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 65

gtcgatgaag ccctgaaaga cgcgcagact atgaacgtga ataagccgac caccgaaaag 60

<210> 66

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 66

gatggccttt ttgcgtgtcg acgcggccgc tcagtattct tcaattttgt ccagttgata 60

<210> 67

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 67

tatcaactgg acaaaattga agaatactga gcggccgcgt cgacacgcaa aaaggccatc 60

<210> 68

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 68

cttttcggtg gtcggcttat tcacgttcat agtctgcgcg tctttcaggg cttcatcgac 60

<210> 69

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 69

gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact 60

<210> 70

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 70

gcggccgcgt cgacacgcaa aaaggccatc catccgtcag gatggccttc tgcttaattt 60

<210> 71

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 71

aaattaagca gaaggccatc ctgacggatg gatggccttt ttgcgtgtcg acgcggccgc 60

<210> 72

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 72

agtctgcgcg tctttcaggg cttcatcgac agtctgacga ccgctggcgg cgttgatcac 60

<210> 73

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 73

gtttaacttt aataaggaga tataccatgc tgacggaagt gcgcccggtc tctacgacga 60

aaccgc 66

<210> 74

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 74

cgacctgcag gcgcgccgag ctcgaattca tttgatgtat ttgcaataga acacagaaaa 60

gaccgt 66

<210> 75

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 75

gtgttctatt gcaaatacat caaatgaatt cgagctcggc gcgcctgcag gtcgacaagc 60

ttgcgg 66

<210> 76

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 76

gagaccgggc gcacttccgt cagcatggta tatctcctta ttaaagttaa acaaaattat 60

ttctacagg 69

<210> 77

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 77

gtatggtgac cctgtggcgc aaatgagaat tcgagctcgg cgcgcctgca ggtcgacaag 60

ct62

<210> 78

<211> 64

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 78

gcgctgccct gtttgatttt atccatggta tatctcctta ttaaagttaa acaaaattat 60

ttct 64

<210> 79

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 79

cctgcaggcg cgccgagctc gaattctcat ttgcgccaca gggtcaccat acgtgccggc 60

agg 63

<210> 80

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 80

gtttaacttt aataaggaga tataccatgg ataaaatcaa acagggcagc gcctctctgg 60

ttgtcg 66

<210> 81

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 81

cagactcgag ggtaccgacg tcctaataag tagatgaata tttatcagga cgaagat 57

<210> 82

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 82

aactaaaggt ttatttttcca tatgtatatc tccttcttat acttaactaa tatac 55

<210> 83

<211> 64

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 83

taaatattca tctacttatt aggacgtcgg taccctcgag tctggtaaag aaaccgctgc 60

tgcg 64

<210> 84

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 84

gtataagaag gagatataca tatggaaaat aaacctttag tttcagtttt gatttgtgc 59

<210> 85

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 85

taactttaag aaggagatat acaagagctc gagtcgaagg agatagaacc atggcaacag 60

catggtataa acaag 75

<210> 86

<211> 82

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 86

gcgtgtcgac gcgtttagag gccccaaggg gttatgctag tatcgattta tcatttagcc 60

acggatagtt tataaattttt ac 82

<210> 87

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 87

ggttctatct ccttcgactc gagctcttgt atatctcctt cttaaagtta aacaaaatta 60

tttctagatt tttgtcgaac 80

<210> 88

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 88

taaatcgata ctagcataac cccttggggc ctctaaacgc gtcgacacgc aaaaaggcca 60

tcc63

<210> 89

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 89

60

gagtcgaagg agatagaacc 80

<210> 90

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 90

ggatggcctt tttgcgtgtc gacgcgttta gaggccccaa ggggttatgc tagtatcgat 60

tta 63

<210> 91

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 91

atatgacgtc tcattagcgg ttttcagga gacg 34

<210> 92

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 92

atatcatatg ccgtccgaag cattccgtcg tcacc 35

<210> 93

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 93

taactttaat aaggagatat accatgacgc aatttaatcc cgttgatcat ccacatcgcc 60

gc62

<210> 94

<211> 67

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 94

attttcgcga atccggagtg taaaagcttg cggccgcata atgcttaagt cgaacagaaa 60

gtaatcg 67

<210> 95

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 95

aagcattatg cggccgcaag cttttacact ccggattcgc gaaaatggat atcgctgact 60

gcgcgcaaac gc 72

<210> 96

<211> 68

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 96

tcaacgggat taaattgcgt catggtatat ctccttatta aagttaaaca aaattatttc 60

tacagggg 68

<210> 97

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 97

atggtgatgg ctgctgccca tttaaaccgc tttgactgcg tcggcaatac ggtgcgc 57

<210> 98

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 98

gtttaacttt aataaggaga tataccatgc tgaacaacgc gatgtctgtt gttatcctgg 60

<210> 99

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 99

cgcagtcaaa gcggtttaaa tgggcagcag ccatcaccat catcaccaca gcc 53

<210> 100

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 100

tcgcgttgtt cagcatggta tatctcctta ttaaagttaa acaaaattat ttctacagg 59

<210> 101

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 101

atatatcata tgtgcggtat cgttggtgct atcgc 35

<210> 102

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 102

atatatgacg tcttattcca cggtcacgga tttcgc 36

<---

Claims (20)

1. A method for the production of lacto- N -triose II by a genetically modified cell of a host microorganism, including the steps in which - receive a genetically modified host microorganism cell, which includes: - at least one recombinant β-1,3- N -acetylglucosaminyltransferase, - increased expression or activity of at least one sugar export protein capable of exporting lacto- N -triose II, wherein said at least one sugar export protein is selected from the group consisting of YjhB from Escherichia coli , ProP from Mannheimia succiniciproducens , SetA from Cedecea neteri and their functional fragments; - cultivating the cell of the host microorganism in the environment under conditions that ensure the production of lacto- N -triose II, resulting in an increased level of export of lacto-N-triose II to the environment compared to the unmodified cell of the host microorganism, and - get lacto- N -triose II from the environment. 2. The method of claim 1 wherein said β-1,3- N -acetylglucosaminyltransferase is selected from the group consisting of IgtA from Neisseria meningitides and PmnagT from Pasteurella multocida . 3. The method according to claim 1, in which inactivation of endogenous β-galactosidase and glucosamine-6-phosphate deaminase genes is carried out in the genetically modified cell of the host microorganism, and at the same time, said genetically modified cell of the host microorganism contains a nucleic acid sequence encoding a functional protein lactose permease. 4. The method of claim 1, wherein said genetically modified host microorganism cell further comprises increased production of UDP- N -acetylglucosamine and UDP-galactose or GDP-fucose or CMP- N -acetylneuraminic acid compared to the non-genetically modified host cell. 5. The method of claim 4 wherein said increased production of UDP- N -acetylglucosamine and UDP-galactose is due to overexpression of one or more genes encoding a protein selected from the group consisting of L-glutamine:D-fructose-6 -phosphate aminotransferase, N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase and glucose-1-phosphate uridylyltransferase. 6. The method of claim 3, wherein said genetically modified host microorganism cell is cultured in the presence of glucose, sucrose, glycerol, or a combination thereof. 7. The method of claim 6, wherein said genetically modified host microorganism cell is not cultured in the presence of N -acetylglucosamine or galactose. 8. A genetically modified host microorganism cell to produce lacto- N -triose II, wherein said host microorganism cell comprises: - at least one recombinant β-1,3- N -acetylglucosaminyltransferase, And - increased expression or activity of at least one sugar export protein capable of exporting lacto- N -triose II, wherein said at least one sugar export protein is selected from the group consisting of YjhB from Escherichia coli , ProP from Mannheimia succiniciproducens , SetA from Cedecea neteri and their functional fragments. 9. The genetically modified host microorganism cell of claim 8, wherein said β-1,3- N -acetylglucosaminyl transferase is selected from the group consisting of IgtA from Neisseria meningitides and PmnagT from Pasteurella multocida . 10. The genetically modified host microorganism cell according to claim 8, wherein in said genetically modified host microorganism cell, endogenous β-galactosidase and glucosamine-6-phosphate deaminase genes have been inactivated, and said genetically modified host microorganism cell contains a nucleic acid a sequence encoding a functional lactose permease protein. 11. The genetically modified host microorganism cell of claim 8, wherein said genetically modified host microorganism cell further comprises increased production of UDP- N -acetylglucosamine and UDP-galactose or GDP-fucose or CMP- N -acetylneuraminic acid compared to genetically non modified host cell. 12. A genetically modified host microorganism cell according to claim 11, wherein said increased production of UDP- N -acetylglucosamine and UDP-galactose is due to overexpression of one or more genes encoding a protein selected from the group consisting of L-glutamine: D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase, phosphoglucosamine mutase, UDP-galactose-4-epimerase, phosphoglucomutase and glucose-1-phosphate uridylyltransferase.

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