KR102194740B1 - Methods for preparing recombinant Acremonium chrysogenum producing deacetoxycephalosporin C with high concentration and Acremonium chrysogenum prepared thereby as bioprocess for 7-ADCA preparation - Google Patents

Abstract

The present invention relates to a method for producing a recombinant Acremonium chrysogenum strain for producing deacetoxycephalosporin C (C(DAOC)) with high concentration and a strain produced thereby and, more specifically, to a method for producing a high DAOC-productive recombinant Acremonium chrysogenum strain, a recombinant Acremonium chrysogenum strain produced thereby, a DAOC production method for culturing the strain under conditions capable of producing DAOC and collecting the same, high DAOC-expression polynucleotide in the Acremonium chrysogenum strain used in the production method, a high DAOC-expression expression vector in the Acremonium chrysogenum strain, and a method for treating the produced DAOC with CPC acylase to produce 7-aminodeacetoxycephalosporanic acid (7-ADCA). According to the present invention, an Acremonium chrysogenum strain having high DAOC production ability can be produced, and DAOC produced by the strain is treated with CPC acylase, and thus 7-ADCA can be produced in an environmentally friendly and efficient manner without toxic by-products.

Description

Methods for preparing recombinant Acremonium chrysogenum producing deacetoxycephalosporin C with high concentration and Acremonium chrysogenum strain for producing 7-ADCA with high concentration of deacetoxycephalosporin C prepared thereby as bioprocess for 7-ADCA preparation}

The present invention relates to a method for producing a recombinant acremonium chrysogenum strain producing a high concentration of deacetoxycephalosporin C (DAOC), and to a strain produced by this method, and more particularly, to a high-productivity acremonium chrysose of DAOC A method for producing a num strain, a recombinant acremonium chrysogenum strain produced by this method, a DAOC production method in which the strain is cultivated under conditions capable of producing DAOC and recovered, acremonium chrysose used in the production method The present invention relates to a polynucleotide for high DAOC expression in num strain, an expression vector for high DAOC expression in acremonium chrysogenum strain, and a method for producing 7-ADCA by treating the produced DAOC with CPC acylase.

Cephalosporin C (hereinafter abbreviated as "CPC") is a beta-lactam antibiotic and is produced by some microorganisms such as the filamentous fungus Acremonium chrysogenum. . CPC exhibits anti-bioactivity against Gram-negative bacteria by inhibiting cell wall synthesis, but its degree is very weak, so it is mainly used for semi-synthetic cephalosporin antibiotics (hereinafter abbreviated as "cephalosporin antibiotics"). It is being used to manufacture raw materials. The raw materials of cephalosporin antibiotics are mainly 7-aminocephalosporanic acid (7-aminocephalosporanic acid, hereinafter abbreviated as "7-ACA"), deacetylglutaryl 7-aminocephalosporanic acid (Deacetylglutaryl). 7-aminocephalosporanic acid, hereinafter abbreviated as “D-7-ACA”), and 7-aminodeacetoxycephalosporanic acid (hereinafter abbreviated as “7-ADCA”).

As an industrial production method, 7-ADCA is known to be made through a chemical conversion method that expands the penam ring, a beta-lactam ring of penicillin, into a cephalos-based cephem ring. In particular, in the production of 7-ADCA, since an organic solvent such as toluene is used in the chemical conversion method involved in the expansion to the Sepham ring, a large amount of toxic waste is generated, resulting in a large disadvantage of increasing environmental pollution and wastewater treatment costs. .

Some companies are known to apply a two-step enzymatic method to overcome this problem.In detail, the expansion enzyme (expandase) that extends from the pan-am ring to the sepham ring is improved to increase substrate affinity and activity for penicillin G, etc. It is known that it is possible to produce 7-ADCA using two enzymes, extension enzyme and acylase, by improving the strain to produce adipoyl-6-APA, which can function well instead of penicillin G, or by developing a fermentation method. To do this, adipoyl-6-APA is produced and purified by fermentation of Penicillium (Penicillium chrysogenum), and the enzyme is treated with the enzyme in two stages of an expansion enzyme and an acylase (Crawford L et al., 1995 Production of cephalosporin intermediates by). feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity, Biotechnology (NY). 1995 Jan;13(1):58-62, International Patent WO2010015624A1 Adipoyl-7-adca producing strains). This may have an advantage in terms of the environment, but the cost used for purification and immobilization of the two enzymes is high, and thus the efficiency may decrease in terms of manufacturing cost.

Therefore, there is an increasing demand for a method capable of producing 7-ADCA in an economical manner without environmental pollution.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

As a result of continuous research to develop an eco-friendly and efficient production method of 7-ADCA, the inventors of the present invention deficient CefEF and CefG genes in acremonium chrysogenum strain, When the microorganism was transformed with an expression vector containing the CefE gene, it was found that DAOC, a precursor of 7-ADCA, can be obtained with high productivity, and the present invention was completed. In addition, it was discovered that 7-ADCA can be produced on an industrial scale by treating the DAOC obtained by this method with CPC acylase, and the present invention was completed.

Accordingly, an object of the present invention is (a) preparing a CPC (cephalosporin C) highly productive Acremonium chrysogenum strain; (b) deleting the CefEF and CefG genes of the strain; And (c) Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydrocarbonoxydans , I-shi seutiseu ekse dense (Nannocystis exedens), also Pseudomonas sealing it (Pseudomonas synringae), Streptomyces Cloud disadvantage claim Russ (Streptomyces clavuligerus) and Sphingomonas islets norbornene cis CefE genes of selected microorganisms from the group consisting of (Sphingomonas dokdonensis) It is to provide a method for producing a high-productivity acremonium chrysogenum strain including the step of transforming the strain of step (b) with an expression vector comprising a nucleotide sequence.

Another object of the present invention is (a1) preparing a CPC (cephalosporin C) highly productive acremonium chrysogenum strain; (b1) deleting the CefEF and CefG genes of the strain; (c1) Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydrocarbonoxydans , I shi seutiseu ekse dense (Nannocystis exedens), also Pseudomonas sealing it (Pseudomonas synringae), Streptomyces Cloud disadvantage claim Russ (Streptomyces clavuligerus) and Sphingomonas islets norbornene sheath of the second microorganism or more member selected from the group consisting of (Sphingomonas dokdonensis) Transforming the strain of step (b1) with each expression vector including the CefE gene sequence; And (d1) protoplast fusion of each of the transformed strains. It is to provide a method for producing a high-productivity acremonium chrysogenum strain, including DAOC (Deacetoxycephalosporin C).

Another object of the present invention is to provide an acremonium chrysogenum strain prepared according to the above method.

Another object of the present invention is (i) culturing the acremonium chrysogenum strain under conditions capable of producing DAOC; And (ii) to provide a method for producing DAOC comprising the step of recovering the DAOC from the medium.

Another object of the present invention is (I) culturing the acremonium chrysogenum strain under conditions capable of producing DAOC; (II) recovering DAOC from the medium; And (III) to provide a method for producing 7-ADCA (7-aminodeacetoxycephalosporanic acid) comprising the step of treating CPC (Cephalosporin C) acylase on the recovered DAOC.

In order to achieve the object of the present invention described above, the present invention comprises the steps of: (a) preparing a CPC (cephalosporin C) high-productivity Acremonium chrysogenum strain; (b) deleting the CefEF and CefG genes of the strain; And (c) Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydrocarbonoxydans , I-shi seutiseu ekse dense (Nannocystis exedens), also Pseudomonas sealing it (Pseudomonas synringae), Streptomyces Cloud disadvantage claim Russ (Streptomyces clavuligerus) and Sphingomonas islets norbornene cis CefE genes of selected microorganisms from the group consisting of (Sphingomonas dokdonensis) It provides a method for producing a high-productivity acremonium chrysogenum strain including the step of transforming the strain of step (b) with an expression vector comprising a nucleotide sequence.

In order to achieve the object of the present invention described above, the present invention comprises the steps of: (a1) preparing a CPC (cephalosporin C) highly productive acremonium chrysogenum strain; (b1) deleting the CefEF and CefG genes of the strain; (c1) Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydrocarbonoxydans , I shi seutiseu ekse dense (Nannocystis exedens), also Pseudomonas sealing it (Pseudomonas synringae), Streptomyces Cloud disadvantage claim Russ (Streptomyces clavuligerus) and Sphingomonas islets norbornene sheath of the second microorganism or more member selected from the group consisting of (Sphingomonas dokdonensis) Transforming the strain of step (b1) with each expression vector including the CefE gene sequence; And (d1) protoplast fusion of each of the transformed strains. It provides a method for producing a high-productivity acremonium chrysogenum strain, including DAOC (Deacetoxycephalosporin C).

In order to achieve another object of the present invention, the present invention provides an acremonium chrysogenum strain prepared according to the above method.

In order to achieve another object of the present invention, the present invention comprises the steps of: (i) culturing the acremonium chrysogenum strain under conditions capable of producing DAOC; And (ii) it provides a method of producing DAOC comprising the step of recovering the DAOC from the medium.

In order to achieve another object of the present invention, the present invention comprises the steps of: (I) culturing the acremonium chrysogenum strain under conditions capable of producing DAOC; (II) recovering DAOC from the medium; And (III) it provides a method for producing 7-ADCA (7-aminodeacetoxycephalosporanic acid) comprising the step of treating CPC (Cephalosporin C) acylase on the recovered DAOC.

Hereinafter, the present invention will be described in more detail.

The present invention (a) CPC (cephalosporin C) high productivity acremonium chrysogenum ( Acremonium chrysogenum ) preparing a strain; (b) deleting the CefEF and CefG genes of the strain; And (c) Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydrocarbonoxydans , I-shi seutiseu ekse dense (Nannocystis exedens), also Pseudomonas sealing it (Pseudomonas synringae), Streptomyces Cloud disadvantage claim Russ (Streptomyces clavuligerus) and Sphingomonas islets norbornene cis CefE genes of selected microorganisms from the group consisting of (Sphingomonas dokdonensis) It provides a method for producing a high-productivity acremonium chrysogenum strain including the step of transforming the strain of step (b) with an expression vector comprising a nucleotide sequence.

Acremonium chrysogenum has a pathway for synthesizing cephalosporin, and the final product is CPC. CPC is converted from precursors L-2-aminoadipate, L-cysteine, and L-valine to isopenicillin N and penicillin N through N-[(5S)-5-amino-5-carboxylpentanoyl]-L-cysteinyl-D-valine. After that, the penam ring is converted into a cephem ring by receiving the catalyst of an expandase (or DAOC synthase) encoded by the CefEF gene to become a cephalosporin-based precursor DAOC. The enzyme encoded by the CefEF gene is a bifuntional enzyme that has the activity of hydroxylase (hydorxylase) that removes the hydroxyl group of DAOC in addition to ring expansion, and is a deacetylcephalosporin C (DAC). ). DAC is converted into the final product, CPC, by acetyltransferase, which is encoded by the CefG gene.

Some of these pathways for the synthesis of cephalosporins are also present in some actinomycetes that produce cephalosporins, but in actinomycetes, CefEF-encoded expandases and hydroxylases are commonly used to perform dual functions. Separately exist and act as CefE encoding expandase and CefF encoding hydroxylase.

Thus, in the present invention, a bacterial-derived homology gene of the actinomycetes CefE gene was used to produce DAOC from acremonium chrysogenum. In more detail, the present invention is a very high DAOC productivity, including the step of introducing CefE genes of various bacteria having homology to the CefE gene of actinomycetes after deficient CefEF and CefG of acremonium chrysogenum. It provides a method of preparing a chrysogenum strain.

The strain produced by the method of the present invention has a characteristic of high DAOC productivity. DAOC production capacity of the DAOC high productivity acremonium chrysogenum strain prepared by the method of the present invention is 25g/L or more, 26g/L or more, 27g/L or more, 28g/L or more, 29g/L or more, 30g /L or more, 31g/L or more, 32g/L or more, 33g/L or more, 34g/L or more, 35g/L or more, preferably 30g/L or more, most preferably 33g/L or more. The upper limit of the production capacity can be adjusted by the growth conditions of the recombinant acremonium chrysogenum, the amount of raw materials in the medium, etc., and can be easily set by those of ordinary skill in the art.

Hereinafter, the method for preparing the strain of the present invention will be described in more detail for each step.

The step (a) is a step of preparing a CPC (cephalosporin C) highly productive Acremonium chrysogenum strain.

In the cephalosporin synthesis pathway possessed by acremonium chrysogenum, DAOC is converted to DAC by the hydroxylase activity of the enzyme encoded by the CefEF gene, and the DAC is converted to CPC by acetyltransferase. Therefore, in order to manufacture acremonium chrysogenum having high DAOC productivity, it is necessary to prepare a strain having high CPC productivity.

In the present invention, the "CPC high productivity" refers to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 g/L or more under appropriate culture conditions, It is preferably 30, 31, 32, 33, 34 or 35 g/L or more, and most preferably 35 g/L or more. The "appropriate culture conditions" means culturing the CPC highly productive acremonium chrysogenum strain in a specific medium composition, culture temperature, culture time, and pH environment to exhibit the CPC production ability. "Proper culture conditions" can be appropriately set by a person skilled in the art according to the following technique.

In the present invention, the CPC high productivity acremonium chrysogenum strain may be a strain in which the wild-type strain itself has the above-defined CPC-producing ability, and may have high CPC productivity by mutation.

The inventors of the present invention induce mutations of acremonium chrysogenum strains by treatment with NTG (N-methyl-N'-nitro-N-nitrosoguanidine) through previous studies, and select and deposit cells with high CPC productivity among them. In the present invention, the CPC highly productive acremonium chrysogenum strain may preferably be acremonium chrysogenum of deposit number KCTC13079BP deposited on August 18, 2016 at the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center. .

The step (b) is a step of deleting the CefEF and CefG genes of the strain.

In the present invention, “deletion” refers to removing the start codon to the stop codon of each of the gene sequences of CefEF and CefG identified in the chromosomal sequence of the strain, or terminating the two gene sequences of adjacent CefEF and CefG at each start codon. It can be defined as removing codons at the same time.

In an embodiment of the present invention, a plasmid vector containing a homologous nucleotide sequence on the outer sides of both ends of the gene to be deleted or the nucleotide sequence containing the gene and having an antibiotic resistance marker therein is prepared, and then the ( Defects of CefEF and CefG genes were induced by a method of transforming acremonium chrysogenum with increased CPC productivity obtained in step a).

More specifically, in one embodiment of the present invention, homologous recombination by linking nucleotide sequences homologous to both sides of a gene to be deleted on both sides of a geneticin resistance marker gene set by a conventional method. CefEF and CefG-deficient strains were prepared by selecting the strain lacking CPC production capability through in vitro culture by inducing the deletion by and analyzing the chromosomal sequence through PCR. The cells that confirmed the defect were named “Acremonium chrysogenum D” and deposited with the relevant institution.

Therefore, preferably, the cell produced by completing the step (b) of the present invention may be a recombinant Acremonium chrysogenum D strain lacking CPC production ability due to the deletion of CefEF and CefG genes, which is the Korea Research Institute of Bioscience and Biotechnology. It may be acremonium chrysogenum D (accession number KCTC 13922BP) deposited with the Center for Biological Resources on August 19, 2019.

In the present invention, the'transformation' is to introduce an expression vector into the strain, and can be performed by any means commonly used for this purpose. For example, the expression vector is not limited thereto, but calcium chloride (CaCl ₂ ) and heat shock method, particle gun bombardment, silicon carbide whiskers, sonication , Electroporation (electroporation), PEG (polyethylenglycol) can be introduced into the strain by a method such as precipitation.

The step (c) is Amicolatopsis Lactam Durance ( Amycolatopsis lactamdurans ), Gordonian Lubrifertincta ( Gordonia rubripertincta ), Mycobacterium abscess ( Mycobacterium abscessus ), Microbacterium Hydrocarbon Oxidance ( Microbacterium hydrocarbonoxydans ), Nannocistis Access ( Nannocystis exedens ), Sudo Monas Shillinge ( Pseudomonas synringae ), Streptomyces clabulizerus ( Streptomyces clavuligerus ) And Sphingomonas Dokdonensis ( Sphingomonas dokdonensis This is a step of transforming the strain of step (b) with an expression vector containing the CefE gene nucleotide sequence of the microorganism selected from the group consisting of ).

In the step (b), by introducing the CefE gene of various bacteria into the acremonium chrysogenum strain in which the CefEF and CefG genes are deleted, the expandase encoded by CefE can convert isopenicillin N and penicillin N to DAOC. This is the step of inducing them to be.

In the step (c) of the present invention, the CefE gene is Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydro Microbacterium hydrocarbonoxydans , Nannocystis exedens , Pseudomonas synringae , Streptomyces clavuligerus and Sphingomonas dokdonensis It may be a CefE gene of a strain selected from the group consisting of, and preferably, a strain selected from the group consisting of Amycoratosis lactamdurans, Mycobacterium absesus, Streptomyces clabulizerus and Sphingomonas dokdonensis It may be a CefE gene, and most preferably a CefE gene of Mycobacterium absesus.

According to an embodiment of the present invention, the Amycolatopsis lactamdurans is NCBI txid1913, Gordonia rubripertincta is NCBI txid1077975, Mycobacterium abscessus is NCBI txid1185650, Microbacterium hydrocarbonoxydans are NCBI txid1223527, Nannocystis exedens is NCBI txid1366054, Sudomonas Pseudomonas synringae is NCBI txid1189623, ( Streptomyces clavuligerus ) is NCBI txid443255 and Sphingomonas dokdonensis may be a strain of NCBI txid344880.

In the present invention, the CefE gene may include the nucleotide sequence of the CefE gene derived from the strain as it is, but as long as the original function of the gene encoding an expandase is maintained, mutations such as substitution, deletion, insertion, etc. May include the derived nucleotide sequence. Preferably, the CefE gene of the strain may be transduced into acremonium chrysogenum to include a codon-optimized nucleotide sequence so that the desired protein can be expressed.

In the present invention, the term "codon optimization" refers to a method of enhancing protein production by highlighting the preferred codon and modifying the rare codon among the amino acid codons of the protein-encoding site. Say. The codon optimization process only changes the base sequence of the gene, but does not change the amino acid sequence of the protein.

The codon optimization method is well known in the art, and a person skilled in the art performs codon optimization so that the protein encoded by the CefE gene of the strain can be well expressed in acremonium chrysogenum according to a method known in the art. Can be done.

According to one embodiment of the present invention, the CefE gene derived from Amycolatopsis lactamdurans is genbank NID Z13974.1, and the CefE gene derived from Gordonia rubripertincta is genbank NID CP022580. 1, The CefE gene derived from Mycobacterium abscessus is genbank NID FVPM01000026.1, the CefE gene derived from Microbacterium hydrocarbonoxydans is genbank NID JYJB01000010.1, Nannosystis Exedence The CefE gene from ( Nannocystis exedens ) is genbank NID FOMX01000018.1, the gene from Pseudomonas synringae is genbank NID AOJT01001469.1, and the gene from Streptomyces clavuligerus is genbank NID The CefE gene derived from DS570624.1 and Sphingomonas dokdonensis was codon-optimized for the nucleotide sequence of genbank NID NBBI01000005.1.

Preferably, in the present invention, the CefE gene of step (c) may be composed of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 8, more preferably SEQ ID NOs: 1, 3, 5, 7 and 8. It may be composed of a nucleotide sequence selected from the group consisting of, more preferably, it may be composed of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 7 and 8, and most preferably, the nucleotide sequence of SEQ ID NO: 3 It may have been done.

In one embodiment of the present invention, based on the CefE protein sequences of the eight kinds of bacteria searched for amino acid homology, 8 kinds of CefE genes were obtained through gene synthesis after codon optimization according to the codon of acremonium chrysogenum. , By operably linking this with a promoter to prepare an expression vector configured to be expressed, and transforming it into the defective strain of step (b), DAOC high-productivity acremonium was prepared.

In the present invention, the'expression vector' refers to a plasmid, virus or other mediator, preferably a plasmid expression vector, in which a polynucleotide encoding an expandase (expandase) has been cloned. The polynucleotide sequence cloned in the present invention may be operably linked to an appropriate expression control sequence, and the operably linked gene sequence and the expression control sequence are one containing a selection marker and a replication origin. It may be included in the expression vector of. “Operably linked” means that the polynucleotide sequence is linked to the expression control sequence in a manner that allows gene expression. The'expression control sequence' refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a specific host cell. Such regulatory sequences include one or more selected from the group consisting of a promoter for carrying out transcription, an arbitrary operator sequence for controlling transcription, a sequence encoding a suitable mRNA liposome binding site, and a sequence controlling termination of transcription and translation, and the like. Can include.

In the present invention, the term'promoter' refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a specific host cell, and'operably linked to the promoter' refers to the following by the function of the promoter. It means that the expression of the protein encoded by the expression cassette of the nucleic acid fragment is affected. As the promoter, a promoter (constitutive promoter) that induces the expression of the target gene at all times or a promoter (inducible promoter) that induces the expression of the target gene at a specific position or time may be used.

Preferably, in the present invention, the promoter is a promoter for high protein expression capable of improving the expression of a protein in acremonium chrysogenum, and may be a promoter consisting of nucleotide sequences of SEQ ID NOs: 9 to 13, and most preferably May be a promoter consisting of the nucleotide sequence of SEQ ID NO: 11.

In addition, the terminator sequence among the expression control sequences included in the expression vector of the present invention may be a trpC terminator, but is not limited thereto.

The vector used as the parent vector of the expression vector is not particularly limited, and all plasmids, viruses, or other mediators commonly used for expression in microorganisms used as host cells in the technical field to which this invention pertains can be used. . For example, the plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50), pBluescript series And the like, but is not limited thereto.

Preferably, the expression vector of the present invention may be a vector represented by the sequence of SEQ ID NOs: 14 to 21 cloned so that the CefE gene, which is a polynucleotide encoding an expandase, is operably linked between a promoter and a terminator, DAOC can be produced by transformation of the acremonium chrysogenum-deficient strain using these expression vectors.

In the present invention, the'expandase' encoded by the CefE gene includes both wild-type or variant types, but preferably may be a protein represented by the amino acid sequence of SEQ ID NOs: 22 to 29, which is the aforementioned SEQ ID NO: 1 It can be expressed by a polynucleotide sequence consisting of the nucleotide sequence of 8 to.

According to an embodiment of the present invention, the strain prepared according to the method of the present invention has a DAOC-producing ability of 30 g/L, and a strain having a DAOC-producing ability of this scale has not yet been disclosed, and the present inventors This is the first disclosure through the invention.

On the other hand, the method of the present invention may further include a process of selecting a DAOC high-producing strain by culturing the transformed cells after step (c) in a selective medium or in a test tube.

The present invention also comprises the steps of (a1) preparing a CPC (cephalosporin C) highly productive acremonium chrysogenum strain; (b1) deleting the CefEF and CefG genes of the strain; (c1) Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , Microbacterium hydrocarbonoxydans , I shi seutiseu ekse dense (Nannocystis exedens), also Pseudomonas sealing it (Pseudomonas synringae), Streptomyces Cloud disadvantage claim Russ (Streptomyces clavuligerus) and Sphingomonas islets norbornene sheath of the second microorganism or more member selected from the group consisting of (Sphingomonas dokdonensis) Transforming the strain of step (b) with each expression vector including the CefE gene sequence; And (d1) protoplast fusion of each of the transformed strains. It provides a method for producing a high-productivity acremonium chrysogenum strain, including DAOC (Deacetoxycephalosporin C).

Steps (a1) to (c1) in the method may refer to steps (a) to (c) of the above-described method.

The step (c1) of the present invention is Amycolatopsis lactamdurans , Gordonia rubripertincta , Mycobacterium abscessus , microbacterium hydrocarbon oxidans ( Microbacterium hydrocarbonoxydans ), Nannocystis exedens , Pseudomonas synringae , Streptomyces clavuligerus , and Sphingomonas dokdonensis from the group consisting of This is a step of transforming the strain of step (b1) with each expression vector including the CefE gene nucleotide sequence of two or more selected microorganisms.

For a description of the eight microorganisms from which the CefE gene is derived in step (c1), reference may be made to step (c).

In the step (c1), preferably, Amycolatopsis lactamdurans , Mycobacterium abscessus , Streptomyces clavuligerus ) and Sphingomonas dokdonensis ( Sphingomonas dokdonensis ) two or more microorganisms selected from the group consisting of, more preferably Mycobacterium abscessus , Streptomyces clavuligerus and Sphingomonas dokdonensis Two or more kinds of microorganisms selected from the group consisting of, most preferably Mycobacterium abscessus and Sphingomonas dokdonensis CefE gene base sequence of each of the expression vectors containing the ( Each strain of step b1) can be transformed.

The step (d1) is a step of protoplast fusion of each strain transformed with each expression vector including the CefE gene sequence in step (c1).

The protoplast fusion refers to an induced or spontaneous association, such as somatic cell hybridization, between two or more protoplasts (cells whose cell walls have been removed by enzymatic treatment) to produce cells with a single double- or multi-nucleus.

The protoplast fusion method in step (d1) of the present invention can be applied to the present invention without limitation, as long as it is a method known in the art.

On the other hand, the method of the present invention may further include the step of selecting a DAOC high-producing strain by culturing the protoplast-fused microorganism in a selection medium after the step (d1).

In one embodiment of the present invention, the inventors of the present invention are Amycolatopsis lactamdurans , Mycobacterium abscessus , Streptomyces clavuligerus , and Sphingomonas dokdonene . Cis ( Sphingomonas dokdonensis ) transformed microorganisms with each expression vector containing the CefE gene nucleotide sequence were first selected for DAOC high-producing strains, and two or more of the selected DAOC high-producing strains were fused to protoplasts. . As a result, it was confirmed that DAOC productivity was remarkably improved in the protoplasm-fused microorganism.

Therefore, the DAOC production ability of the DAOC high-productivity acremonium chrysogenum strain prepared according to the method comprising the steps (a1) to (d1) of the present invention is 25g/L or more, 26g/L or more, and 27g/L More than, 28g/L or more, 29g/L or more, 30g/L or more, 31g/L or more, 32g/L or more, 33g/L or more, 34g/L or more, 35g/L or more, preferably 30g/L It may be more than, most preferably more than 35g/L. The upper limit of the production capacity can be adjusted by the growth conditions of the recombinant acremonium chrysogenum, the amount of raw materials in the medium, etc., and can be easily set by those of ordinary skill in the art.

The present invention also provides a DAOC high productivity acremonium chrysogenum strain prepared according to the above method.

The present invention also includes the steps of: (i) culturing the DAOC high-productivity acremonium cresogenum strain under conditions capable of producing DAOC; And (ii) it provides a method of producing DAOC comprising the step of recovering the DAOC from the medium.

The step (i) of the DAOC production method is a step of culturing the strain prepared by the method of the present invention under conditions capable of producing DAOC, and the condition capable of producing DAOC refers to the DAOC highly productive acremonium chrysogenum strain. It means culturing under a specific medium composition, culture temperature, culture time, and pH environment. Such culture conditions are well known in the art, and those skilled in the art can appropriately set from known techniques.

The step (ii) of the DAOC production method is a step of recovering DAOC from the medium, and means separating, harvesting, purifying or collecting DAOC produced by the strain of the present invention from the strain or its culture medium. Specifically, the method of recovering DAOC is not particularly limited thereto, but centrifugation, filtration, extraction, spraying, drying, distillation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation), chromatography (for example, For example, ion exchange, affinity, hydrophobicity, and size exclusion) can be used, which can be easily constructed by those skilled in the art.

In particular, in the DAOC production method of the present invention, the culture in step (i) may be a fermentation culture.

The fermentation culture of the transformed strain can be carried out by a known host cell culture method or a modified method thereof. Fermentation culture may be divided into 1 to 5 seed cultures and main cultures, but is not limited thereto. The medium composition may be a natural medium or a synthetic medium containing a carbon source, a nitrogen source, an inorganic salt, etc. that can be efficiently used by the transformed recombinant host cells. Carbon sources that can be used include carbohydrates such as glucose, fructose, sucrose, soy flour, peanut flour, wheat flour, corn flour, dextrin, cornmeal, and fructose syrup; Starch, a hydrolyzate of starch; Organic acids such as acetic acid and propionic acid; Alcohols such as ethanol, propanol, and glycerol; Contains oils such as soybean oil, olive oil, canola oil, peanut oil, and fish oil. The nitrogen source is ammonia; Ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; Peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean extract, soybean hydrolyzate; And various fermented cells and their degradation products. As amino acids, sodium glutamate, methionine, lysine, leucine, cysteine, valine, and the like are included. Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, calcium carbonate, and the like.

의 범위에서 행하고, 배양시간은 일반적으로 1일 내지 20일, 바람직하게는 3일 내지 12일간 행한다. 배지의 pH는 배양 중에서 바람직하게는 3.0 내지 9.0의 범위를 유지한다. 배지의 pH는 무기 또는 유기산, 알칼리 용액, 우레아, 칼슘 카보네이트, 암모니아 등으로 조절할 수 있다. 배양 중에는 필요한 경우 형질전환체의 선별 및 오염의 방지를 위해 하이그로마이신 B, 제네티신, 플레오마이신, 베노밀, 암피실린, 스트렙토마이신, 클로람페니콜, 카나마이신 및 테트라사이클린과 같은 항생제를 첨가할 수 있다. Fermentation culture is usually carried out under aerobic conditions such as shaking culture or rotation by a rotating machine. The culture temperature is preferably 5 to 30

The cultivation time is generally 1 to 20 days, preferably 3 to 12 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 in culture. The pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like. During cultivation, if necessary, antibiotics such as hygromycin B, genetisin, pleomycin, benomyl, ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added to select transformants and prevent contamination.

In an embodiment of the present invention, the process was completed by evaluating the fermentation productivity of each DAOC-producing strain introduced with one expression vector selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 21, and the maximum DAOC fermentation productivity is SEQ ID NO: 16 It was evaluated as about 33.5 g/L in the strain into which the expression vector of was introduced. The produced DAOC was compared with the standard DAOC obtained by requesting a synthesis from MedKoo (Morrisville, North Carolina, USA) and confirmed to be the same material through HPLC and mass spectrometry.

The present invention also provides a polynucleotide for high DAOC expression in the acremonium cresogenum strain, consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 8, and preferably SEQ ID NOs: 1, 3, 5, 7 And a nucleotide sequence selected from the group consisting of 8, more preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 7 and 8, and most preferably an acremonium cresogenum strain consisting of the nucleotide sequence of SEQ ID NO: 3. It provides a polynucleotide for high expression of DAOC in.

The present invention also provides an expression vector for high DAOC expression in an acremonium cresogenum strain consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14 to 21, and preferably SEQ ID NOs: 14, 16, 18, 20 and In the acremonium cresogenum strain consisting of a nucleotide sequence selected from the group consisting of 21, more preferably a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18 and 20, and most preferably the nucleotide sequence of SEQ ID NO: 16 It provides an expression vector for high expression of DAOC.

The present invention also includes the steps of (I) culturing the strain under conditions capable of producing DAOC; (II) recovering DAOC from the medium; And (III) it provides a method for producing 7-ADCA (7-aminodeacetoxycephalosporanic acid) comprising the step of treating CPC (Cephalosporin C) acylase on the recovered DAOC.

In the above method, steps (I) and (II) are the same as described for (i) and (ii) of the above-described DAOC production method.

Step (III) is a step of preparing 7-ADCA by treating the DAOC culture medium obtained in step (II) with CPC acylase.

의 범위에서 행하고, 반응시간은 일반적으로 1분 내지 300분, 바람직하게는 1분 내지 120분 동안 행한다. 반응 pH는 바람직하게는 3.0 내지 9.0의 범위를 유지한다. 반응 pH는 무기 또는 유기산, 알칼리 용액, 우레아, 칼슘 카보네이트, 암모니아 등으로 조절할 수 있다. The conversion reaction using CPC acylase is usually carried out under aerobic conditions such as by shaking culture or rotation by a rotating machine. The conversion reaction temperature is preferably 5 to 30

And the reaction time is generally 1 minute to 300 minutes, preferably 1 minute to 120 minutes. The reaction pH is preferably maintained in the range of 3.0 to 9.0. The reaction pH can be adjusted with inorganic or organic acids, alkali solutions, urea, calcium carbonate, ammonia, and the like.

In one embodiment of the present invention, most of the conversion reactions were completed within 60 minutes after treatment with CPC acylase, and about 18 g/L for 20.08 g/L 7-ADCA produced when 33.5 g/L of DAOC was completely converted. Was produced to show a high yield of about 90%. The produced 7-ADCA was verified through mass spectrometry, and it was confirmed to be the same material by comparing it with the standard 7-ADCA (Sigma-Aldrich A8398, USA) through HPLC and mass spectrometry.

The 7-ADCA production method of the present invention may include the step of recovering 7-ADCA from the reaction solution after the step (III), which is a reaction solution of 7-ADCA produced by the enzyme reaction method of the present invention. Means to separate, harvest, purify or collect from. Specifically, the recovery method of 7-ADCA is not particularly limited thereto, but centrifugation, filtration, extraction, spraying, drying, distillation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation), chromatography ( For example, ion exchange, affinity, hydrophobicity and size exclusion) can be used, which can be easily constructed by those skilled in the art.

According to the method of the present invention, it is possible to produce 7-ADCA in an environmentally friendly and efficient manner without toxic by-products only by fermentation and culture of the strain provided by the present invention and a one-step enzymatic conversion method.

According to the present invention, an acremonium chrysogenum strain having high DAOC production ability can be prepared, and by treating DAOC produced by these strains with CPC acylase, 7-ADCA can be produced in an environmentally friendly and efficient manner without toxic by-products. I can.

1 is a structure of an expression vector introducing a CefE gene encoding various bacterial expansion enzymes. The vector contains the antibiotic resistance gene cassette, the Flp cassette, and the bacterial CefE cassette.
2 is a result of confirming DAOC productivity according to in vitro culture of acremonium recombined to produce DAOC by introducing the CefE gene of 8 kinds of bacteria.
3 is a result of evaluating DAOC productivity according to the 5-liter fermentation culture of the selected acremonium strains (1-38 strains, 3-7 strains, 7-23 strains, 7-53 strains, 8-60 strains).
4 is a result of mass spectrometry of DAOC produced from recombinant acremonium strain.
Figure 5 shows the production of 7-ADCA by treating CPC acylase in DAOC culture medium produced by 5 strains of 1-38 strains, 3-7 strains, 7-23 strains, 7-53 strains, and 8-60 strains. It is the result of an enzymatic reaction.
6 is a result of mass spectrometry of 7-ADCA contained in a reaction solution in which DAOC-produced fermentation broth was converted to CPC acylase.
7 is a result of evaluating DAOC productivity according to a 5-liter fermentation culture of strains selected from strains prepared according to the protoplast fusion method.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Test method>

Enzymes and kits

As a cloning method, gene amplification by PCR was performed according to the enclosed manual at the time of purchase of DNA polymerase, unless otherwise specified. In general, DNA polymerase was Pfu-X polymerase (Solgent, Korea). Restriction enzyme, T4 DNA ligase, and Klenow fragment were purchased from NEB (USA) and used according to the manual of the enzyme. QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, and QIAquick Gel Extraction Kit (Qiagen, Netherlands) were used for a series of cloning methods such as PCR, purification of plasmid DNA, and extraction from agarose gel.

Transformation

, 4000 rpm)로 균체를 회수하여, 얼음으로 냉각한 0.1 M 염화칼슘용액으로 4회 세척하여 competent cell을 제조하였다. 준비한 100 내지 500 ng의 plasmid DNA를 100 μL의 competent cell과 섞은 후 얼음에서 30분간 방치하고, 42

에서 30 초간 열충격을 주고 다시 얼음에서 2분간 방치한 후 1 mL의 LB를 넣어 37

1시간 배양 후 항생제를 갖는 LB 평판배지에 도말하였다.The transformation of E. coli DH5alpha for cloning was carried out by washing with calcium chloride and then subjecting to heat shock. Specifically, E. coli was inoculated into LB, cultured to OD0.6, and centrifuged (4

, 4000 rpm), and washed 4 times with 0.1 M calcium chloride solution cooled with ice to prepare a competent cell. After mixing the prepared 100 to 500 ng of plasmid DNA with 100 μL of competent cells, let stand on ice for 30 minutes, 42

After 30 seconds of thermal shock at the table, let stand on ice for 2 minutes, and then add 1 mL of LB to 37

After 1 hour incubation, it was spread on LB plate medium with antibiotics.

에서 6 내지 8일 배양하여 균락을 얻은 후 이를 100 mL TB배지 (12 g/L 트립톤, 24 g/L 효모추출물, 9.4 g/L K₂HPO₄, 2.2 g/L KH₂PO₄, 4 g/L 글리세롤)를 넣고, LB 평판배지에서 균락을 취하여 접종하였다. 균락의 접종은 LB 평판배지 상에 있는 균락을 메스를 이용하여 한 변이 5 내지 7 mm가 되게 정사각형으로 자른 후 균락의 크기에 따라 4 내지 6개를 취하여 접종하고 28

, 150 rpm 조건에서 3 내지 4일 배양하였다. 성장이 완료된 배양액을 4

, 4,000 rpm에서 원심분리한 후 상등액을 버리고 0.6 M MgSO₄ 용액으로 1회 세척하였다. Transformation into a fungal host was performed using a transformation method using PEG (polyethylene glycol), and in detail, the host strain was plated on an LB plate medium.

After culturing in 6 to 8 days to obtain colonies, this was obtained in 100 mL TB medium (12 g/L tryptone, 24 g/L yeast extract, 9.4 g/LK ₂ HPO ₄ , 2.2 g/L KH ₂ PO ₄ , 4 g /L glycerol) was added, and the bacteria were taken and inoculated on an LB plate medium. To inoculate the fungus, cut the fungus on the LB plate medium into a square with a side of 5 to 7 mm using a scalpel, and then take 4 to 6 according to the size of the fungus.

, Incubated for 3 to 4 days at 150 rpm. The growth is completed culture medium 4

, After centrifugation at 4,000 rpm, the supernatant was discarded and washed once with 0.6 M MgSO ₄ solution.

, 100 rpm에서 3시간 반응시켰다. 이 후 반응액에 동량의 separation buffer A(0.6 M sorbitol, 100 mM Tris-Cl, pH 7.0)를 overlay한 뒤, 1,800 g, 4

조건으로 10분간 원심분리하였다. 상등액 및 경계면을 새 원심분리관에 옮겨 동량의 separation buffer B(1.2 M sorbitol, 100 mM Tris-Cl, pH 7.5)를 넣고 1,800 g, 4

조건으로 10분간 원심분리하였다. 상등액을 버리고 침전물을 다시 MSC(1 M sorbitol, 10 mM MOPS, pH 6.5, 10 mM CaCl₂)로 세척하였다. 현미경으로 관찰하여 형성된 원형질체를 계수한 후 형질전환을 위해 1×10⁷개의 원형질체에 1 내지 5 μg DNA, 50 μL의 60% PEG용액(MSC에 polyethyleneglycol 6000을 60% 농도로 만듦)을 섞은 후, 얼음에서 20분 방치하였다. 다시 500 μL의 60% PEG 용액을 넣고 섞은 뒤 상온에서 20분 방치한 후, 적절한 항생제를 함유한 LB-sucrose 평판배지(0.8 M sucrose, 2% agar, 하이그로마이신(hygromycin)의 경우 100 mg/L, 제네티신의 경우 200 mg/L)에 도말하였다. 평판배지는 28

에서 10일 내지 30일 동안 균락이 형성될 때까지 배양하였다. To produce protoplasts, 30 lysing enzymes (Sigma-Aldrich, L1412, USA) of about 4 times the weight of the cells were treated.

And reacted at 100 rpm for 3 hours. After that, the same amount of separation buffer A (0.6 M sorbitol, 100 mM Tris-Cl, pH 7.0) was overlaid on the reaction solution, followed by 1,800 g, 4

Centrifuged for 10 minutes under conditions. Transfer the supernatant and the interface to a new centrifuge tube, add the same amount of separation buffer B (1.2 M sorbitol, 100 mM Tris-Cl, pH 7.5), and add 1,800 g, 4

Centrifuged for 10 minutes under conditions. The supernatant was discarded and the precipitate was washed again with MSC (1 M sorbitol, 10 mM MOPS, pH 6.5, 10 mM CaCl ₂ ). After counting the protoplasts formed by observation under a microscope, for transformation, 1 to 5 μg DNA and 50 μL of 60% PEG solution (polyethyleneglycol 6000 at 60% concentration in MSC) were mixed in 1×10 ⁷ protoplasts for transformation. It was left on ice for 20 minutes. Add 500 μL of 60% PEG solution again, mix and leave for 20 minutes at room temperature, and then LB-sucrose plate medium (0.8 M sucrose, 2% agar, hygromycin) containing an appropriate antibiotic 100 mg/ L, in the case of geneticin, 200 mg/L). 28 reputation badges

Incubated for 10 to 30 days until colonies were formed.

In vitro culture

정치배양기에서 14일동안 LB, 0.6M MgSO4, 2% agar 배지에서 키운 균락을 약 100개 정도 골라 화염멸균한 메스로 도려낸 뒤, 집게로 집어 1.5mL e-tube에 담았다. 0.2mL의 NaCl를 넣고 페슬로 균락을 부순 뒤, 포자 배지(녹말 24 g/L, 글라이신 1.2 g/L, 폴리펩톤 4 g/L, 효모 추출물 0.3 g/L, 카제인 8 g/L, 황산암모늄 6 g/L, 인산이칼륨 1.2 g/L, 황산마그네슘 0.6 g/L, 한천 20 g/L, pH 7.0,)에 도말하여 28

정치배양기에서 10일 내지 14일 배양하였다. 형성된 균락을 백금이로 긁어서 2 mL의 20% 글리세롤이 들어있는 15 mL 캡튜브에 담고, 잘 현탁한 뒤, 일정량씩 1.5mL e-tube에 담아 -80

냉동고에 보관하면서 사용하였다.28

About 100 fungi grown in LB, 0.6M MgSO4, 2% agar medium for 14 days in a stationary incubator were cut out with a flame sterilized scalpel, picked up with forceps, and placed in a 1.5mL e-tube. Add 0.2 mL of NaCl and crush the fungus with Pessloe, and then spore medium (starch 24 g/L, glycine 1.2 g/L, polypeptone 4 g/L, yeast extract 0.3 g/L, casein 8 g/L, ammonium sulfate). 6 g/L, dipotassium phosphate 1.2 g/L, magnesium sulfate 0.6 g/L, agar 20 g/L, pH 7.0,)

Cultured for 10 to 14 days in a stationary incubator. Scrape the formed fungus with platinum teeth and place it in a 15 mL cap tube containing 2 mL of 20% glycerol, suspend well, and put a certain amount in 1.5 mL e-tubes -80

It was used while being stored in a freezer.

냉동고에 보관한 stock 0.3 mL을 5 mL 배지가 들어있는 시험관에 접종 한 뒤, 28

, 200rpm 조건으로 진탕 배양기에서 4일 배양하였다. 본배양은 배지 5mL이 담긴 시험관에 종배양액 0.5 mL을 접종하고, 28

, 200rpm 조건으로 진탕 배양기에서 4일 배양한 뒤, 배양액 0.5 mL을 채취하여 상등액을 HPLC로 분석하였다. In vitro culture for strain selection was carried out in the stages of seed culture and main culture. A wrinkled test tube with a diameter of 2 cm and a length of 20 cm was used for the culture, and the medium composition and culture conditions of the seed culture and the main culture were the same. Medium ingredients are sugar 15 g/L, soytone 15 g/L, ammonium sulfate 5 g/L, methionine 10 g/L, calcium carbonate 10 g/L, yeast extract 10 g/L, glucose 5 g/L, magnesium sulfate It is 2 g/L, methyl oleate 50 g/L. Species culture is -80

Inoculate 0.3 mL of stock stored in the freezer into a test tube containing 5 mL of medium.

, And cultured for 4 days in a shaking incubator under conditions of 200 rpm. For the main culture, 0.5 mL of seed culture solution was inoculated into a test tube containing 5 mL of medium.

After incubation for 4 days in a shaking incubator under conditions of 200 rpm, 0.5 mL of the culture solution was collected and the supernatant was analyzed by HPLC.

Analysis of DAOC and 7-ADCA

, UV 검출기 220 nm를 이용하였다. Analysis of DAOC productivity or 7-ADCA after enzymatic conversion of the production strain was performed using HPLC.In detail, 25 μL of the supernatant was taken by centrifuging the culture or enzyme reaction solution, and then diluted 40 times by mixing with 975 μL of tertiary distilled water. I did. The diluted culture solution was filtered through a 0.2 μm filter and analyzed with Shimadzu LC10Avp. Analysis conditions were ZORBAX Eclipse Plus C18 (Analytical 4.6 mm × 250 mm, 5-Micron) column, mobile phase 20 mM ammonium acetate: acetonitrile (95:5), pH 7.0, flow rate 0.8 mL/min, column temperature 40

, UV detector 220 nm was used.

Other experimental methods used in the present invention will be described in detail in <Example>.

<Example 1>

Production of defective strains of CefEF and CefG

<1-1> Construction of plasmid vector for deletion

Among the CPC biosynthetic pathways, CefEF and CefG have bidirectional promoters that are expressed in opposite directions at adjacent locations on the genome, so about 2.5 kb each is homologous recombination outward from both terminal locations of CefEF and CefG. ) Was amplified by selecting it as the binding site. The left junction was amplified using primers 5'-GAGTAGTTTGGCCTTGATGGGACG-3' (SEQ ID NO: 30) and primer 5'-CCCTTTAGTGAGGGTTAATTGCTATGGAGGTGTCAGCCTGCC-3' (SEQ ID NO: 31), and 5'-GCCCTATAGTGAGTCGTATTACCGCCGATCGAGTAATAAATCT The right junction was amplified using 5'-GTGCTTCTCGGTTCAGTGAGTCGG-3' (SEQ ID NO: 33). A geneticin resistance gene was used as the selection marker gene, which was primer 5'-AATTAACCCTCACTAAAGGG-3' (SEQ ID NO: 34) and primer 5'-TAATACGACTCACTATAGGGC- from pB-GcastF specified in Korean Patent No. 10-1808192. It was amplified using 3'(SEQ ID NO: 35). Since the amplified left and right junction sites and the selection marker gene cassette were added with homologous portions, each gene was used as a template and ligated through sewing PCR. The gene fragment containing the junction site and the selection marker was inserted into the EcoRV restriction site of the pBluescript II SK+ (Stratagene, USA) plasmid vector by blunt-end ligation to construct a plasmid vector for CefEF and CefG deletion.

<1-2> Selection of defective strains

In the present invention, the acremonium chrysogenum strain (accession number KCTC13079BP) with improved CPC production ability prepared in the inventor's previous study (Korean Patent No. 10-1808192) was used for transformation. The plasmid vector for deletion was transformed into the strain by the PEG transformation method as introduced in the above <Experimental Method>, and the presence or absence of deletion of each colony lacks CPC productivity through the in vitro culture mentioned in the above <Experimental Method>. After finding the colony, the genomic DNA of the corresponding colony was extracted and confirmed through PCR. In detail, about 3 kb of primer 5'-CGATGTAGGAGGTTGACGGTGC-3' (SEQ ID NO: 36) located outside the deletion site and primer 5'-CGATAAGGGCCAGTTCCGCC-3' (SEQ ID NO: 37) inherent in PgpdA, the promoter of the marker gene, was used. The product was confirmed, and the defect was confirmed by selecting a colony that did not amplify in the primer outside the defect site and the primer 5′-GGTACCAGGCCCAGAAGTTTGC-3′ (SEQ ID NO: 38), which is inherent in CefG.

The CefEF and CefG gene-deficient CPC high-productivity acremonium chrysogenum strain was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (KCTC) on August 19, 2019 (accession number: KCTC 13922BP).

<Example 2>

Selection of DAOC-producing strains through the introduction of CefE of various bacteria

<2-1> Search and cloning of bacterial CefE

In this step, the search for CefE of bacteria is based on the amino acid sequence (SEQ ID NO: 22) encoded by the CefE gene of Streptomyces clavulizerus, which is a commonly known Sepa antibiotic-producing actinomycetes, BLAST (National Center for Biotechnology Information) (NCBI). The amino acid sequence of bacteria with high homology was secured through amino acid homology search by Basic Local Alignment Search Tool). In detail, 3 species of gamma-proteobacteria, delta-proteobacteria, and 1 species of alpha-proteobacteria in Gram-negative proteobacteria, and Pseudonocardiales of high GC gram-positive actinomycetes Five species of 1-2 species each were selected from Streptomycetales, Corynebacteriales, and Micrococcales to obtain the amino acid sequence of a total of 8 bacteria CefE. Thereafter, based on the sequence of each amino acid, the codon was optimized according to the codon of acremonium, and the gene synthesis was requested (Cosmo Genetech, Korea) to secure 8 kinds of CefE genes of bacteria. Each bacterial CefE gene was named E1 (SEQ ID NO: 1) as CefE of Amycoltopsis lactamdurans, and named E2 (SEQ ID NO: 2) as CefE of Gordonia rubripertincta. It was named E3 (SEQ ID NO: 3) as CefE of Mycobacterium abscessus, and named E4 (SEQ ID NO: 4) as CefE of Microbacterium hydrocarbonoxydans. It was named as E5 (SEQ ID NO: 5) as CefE of Nannocystis exedens, and named as E6 (SEQ ID NO: 6) as CefE of Pseudomonas synringae, and Streptomyces clabulizerus ( Streptomyces clavuligerus) was named E7 (SEQ ID NO: 7), and as CefE of Sphingomonas dokdonensis, it was named E8 (SEQ ID NO: 8).

Genes of E1 to E8 were amplified from each template obtained by requesting for synthesis,

E1 with primers 5'-CGCTTGAGCAGACATCACCATGACCGACGCCACCGTGCC-3' (SEQ ID NO: 39) and 5'-GCTAAGCTTTTATCAGCGGGCGGCGGC-3' (SEQ ID NO: 40), 5'-CGCTTGAGCAGACATCACTGCATGACCGACAGCCCGATTC-3' (SEQ ID NO: 41GGATCT-3' (SEQ ID NO: 41GG-AG) E2 using 3'(SEQ ID NO: 42), E2 using 5'-CGCTTGAGCAGACATCACCATGACGGACATCGGTGAAC-3' (SEQ ID NO: 43) and 5'-GCTAAGCTTTTATCAGCCGACGGTTATGGC-3' (SEQ ID NO: 44), E2 using 5'-CGCTTGAGCAGACATCGCGT E4 using'(SEQ ID NO: 45) and 5'-GCTAAGCTTTTATCACGCGCTCGCGCCT-3' (SEQ ID NO: 46), 5'-CGCTTGAGCAGACATCACCATGTCAAGCGCCATCATCGCC-3' (SEQ ID NO: 47) and 5'-GCTAAGCTTTTATCAGACGTTCAGGTT (SEQ ID NO: 48) E5, 5'-CGCTTGAGCAGACATCACCATGACCCTCCAGTACGTCCC-3' (SEQ ID NO: 49) and 5'-GCTAAGCTTTTATCAGGCGACCTTGGACC-3' (SEQ ID NO: 50), 5'-CGCTTGAGCAGACATCACCATGGACACCACCGTCCGAGACACCACCGT Amplify E7 using 5'-GCTAAGCTTTTACTACGCCTTCGAGGTCCGCCG-3' (SEQ ID NO: 52) and 5'-CGCTTGAGCAGACATCACCATGCATCGCGCGGGCGGC-3' (SEQ ID NO: 53) and 5'-GCTAAGCTTTTATCACTTCTTGATGAGAC-3' (SEQ ID NO: 54, respectively). I did. Promoter PEP3 is amplified from pB-HCXEP3 (SEQ ID NO: 15 of Korean Patent No. 10-1808192) using primers 5'-GCAACTAGTGCGGCCGCCCTTGTATCTCTACACACAGGC-3' (SEQ ID NO: 55) and 5'-GGTGATGTCTGCTCAAGCG-3' (SEQ ID NO: 56). I did.

Since the E1 to E8 genes amplified above and the promoter PEP3 have sequences that can overlap with each other, they were ligated by performing overlapping PCR (sewing PCR). PCR was performed using the PEP3 fragment and E1 fragment amplified above as a template, and primers SEQ ID NO: 55 and SEQ ID NO: 40 to obtain an E1 gene fragment to which a promoter is linked. In the same way, the PEP3 fragment and the E2 fragment were used as templates, and the promoter-linked E2 gene fragment was obtained using primers SEQ ID NO: 55 and SEQ ID NO: 42. PEP3 fragment and E3 fragment were used as templates, and primers SEQ ID NO: 55 and SEQ ID NO: 44. A promoter-linked E3 gene fragment was obtained using PEP3 fragment and E4 fragment as a template, and a promoter-linked E4 gene fragment was obtained using primers SEQ ID NO: 55 and SEQ ID NO: 46, and PEP3 fragment and E5 fragment were used as templates. And primers SEQ ID NO: 55 and SEQ ID NO: 48 to obtain a promoter-linked E5 gene fragment, PEP3 fragment and E6 fragment as templates, and primers SEQ ID NO: 55 and SEQ ID NO: 50 to obtain a promoter-linked E6 gene fragment. A PEP3 fragment and an E7 fragment were used as templates, and a promoter-linked E7 gene fragment was obtained using primers SEQ ID NO: 55 and SEQ ID NO: 52. PEP3 fragment and E8 fragment were used as templates, and primers SEQ ID NO: 55 and SEQ ID NO: 54. Was used to obtain a promoter-linked E8 gene fragment.

In order to have a terminator in the E1 to E8 genes having the promoter PEP3, a vector into which a terminator was introduced was first constructed. The terminator is a trpC terminator, amplified using primers 5'-CGACTCGAGACTTAACGTTACTGAAATCATCAAACAGC-3' (SEQ ID NO: 57) and 5'-GCAGGTACCATCGAGTGGAGATGTGGAGTGGG-3' (SEQ ID NO: 58) from pAN7-1 (GenBank Z32698) and cut with XhoI and KpnI Then, pB-TtrpC (SEQ ID NO: 59) was prepared by inserting it into the same site of pBluescript II SK+. The E1 to E8 gene fragments having the promoter PEP3 were introduced into the pB-TtrpC plasmid vector to complete a cassette of each gene having a promoter and a terminator.

Specifically, the E1 gene fragment linked to PEP3 was cut with NotI and HindIII and inserted into the same site of pB-TtrpC to complete pB-E1cast (SEQ ID NO: 60), and the E2 gene fragment linked to PEP3 was cut with SpeI and HindIII. pB-E2cast (SEQ ID NO: 61) was completed by inserting into the same site of pB-TtrpC, and the E3 gene fragment linked to PEP3 was cut with NotI and HindIII, inserted into the same site of pB-TtrpC, and pB-E3cast (SEQ ID NO: 62). ) Was completed, and the E4 gene fragment linked to PEP3 was cut with NotI and HindIII and inserted into the same site of pB-TtrpC to complete pB-E4cast (SEQ ID NO: 63), and the E5 gene fragment linked to PEP3 was converted to NotI and HindIII. After cutting and inserting into the same site of pB-TtrpC, pB-E5cast (SEQ ID NO: 64) was completed.The E6 gene fragment linked to PEP3 was cut with NotI and HindIII and inserted into the same site of pB-TtrpC, and pB-E6cast (sequence No. 65) was completed, and the E7 gene fragment linked to PEP3 was cut with NotI and HindIII and inserted into the same site of pB-TtrpC to complete pB-E7cast (SEQ ID NO: 66), and the E8 gene fragment linked to PEP3 with NotI and It was cut with HindIII and inserted into the same site of pB-TtrpC to complete pB-E8cast (SEQ ID NO: 67).

After amplifying each of the gene cassettes using primers T3 (SEQ ID NO: 34) and T7 (SEQ ID NO: 35) from the plasmid vector having the completed 8 bacterial CefE gene cassettes, the sequence specified in Korean Patent No. 10-1808192 It was inserted into the PmeI restriction site of pB-HF, number 5, by blunt-end ligation. Each completed plasmid is pB-HFE1 (SEQ ID NO: 14), pB-HFE2 (SEQ ID NO: 15), pB-HFE3 (SEQ ID NO: 16), pB-HFE4 (SEQ ID NO: 17), pB-HFE5 (SEQ ID NO: 18), It was finally named pB-HFE6 (SEQ ID NO: 19), pB-HFE7 (SEQ ID NO: 20), and pB-HFE8 (SEQ ID NO: 21). The configuration of the completed expression vector is shown in Figure 1. Each plasmid vector has a hygromycin antibiotic marker, and is configured to remove the marker by the flp-FRT system (Korean Patent No. 10-1808192).

<2-2> Selection of DAOC producing strains

In vitro culture for a number of colonies obtained after transforming and introducing the eight vectors constructed in <Example 2-1> into the D strain, the CefEF and CefG-deficient strains produced in <Example 1>. The high-productivity strains were selected for DAOC production and fermentation tests.

Transformation, in vitro culture, and HPLC analysis of the product followed the method shown in the above <Experimental Method>. In particular, the standard products used for the following HPLC analysis and mass spectrometry were synthesized by commissioning MedKoo Biosciences, Inc (Morrisville, North Carolina, USA). For each of the eight species into which the expression vectors of pB-HFE1 to pB-HFE8 were introduced, 60 colonies were cultured in vitro, and the results are shown in FIG. 2. In the strains into which the eight bacterial CefE genes were introduced, the activity was significantly different depending on the introduced gene, and even if the same gene was introduced, the difference in activity was found to be large depending on the fungus. In particular, in the case of transformants into which CefE2 and CefE4 were introduced, there were few strains with DAOC productivity, and the group with significant DAOC productivity was CefE1 of Amicolatopsis Lactam Durance, and Mycobacterium absesus. These were CefE3, CefE5 from Nannocystis exedence, CefE6 from Capital Monas Shillinge, CefE7 from Streptomyces clabulizerus, and CefE8 from Sphingomonas Dokdonensis. When CefE3 was introduced, the production of about 3.77 g/L was shown in 3-7 strains, the highest among all strains, and up to 3.35 g/L in 7-53 strains introduced with CefE7 of Streptomyces clabulizerus, In the case of 8-60 strains introduced with CefE8 from Pingomonas Dokdonensis, it was confirmed that the maximum production of about 3.5 g/L was relatively high.

<Example 3>

Evaluation of fermentation productivity of DAOC producing strains

In the test tube culture of <Example 2>, 5 strains were selected mainly from different strains among strains having high DAOC productivity, and fermentation productivity was evaluated. Fermentation productivity evaluation targets were 1-38 strains with CefE1 introduced, 3-7 strains with CefE3, 7-23 and 7-53 strains with CefE7, and 8-60 strains with CefE8.

, 200rpm에서 4 내지 5일 배양하였다. 2차 종배양은 1차 종배양액을 2차 종배양 배지(1차 종배양 배지와 동일하나 콩기름을 5 mL/L 추가함)가 담긴 발효조에 모두 접종하고, 30

, 35% DO, 400 rpm, 공기 1.0 vvm의 조건을 시작으로 균체 성장에 따라 6% 포도당을 feeding하고 교반속도를 단계적으로 올리면서 4 내지 5일 배양하였다. 본배양은 2차 종배양액 200 mL을 본 배양 배지(땅콩가루 23 g/L, 옥수수침지액 50 mL/L, 메티오닌 1.5 g/L, 덱스트린 70 g/L, 콘밀 35 g/L, 황산칼슘 13 g/L, 콩기름 60 mL/L, 황산암모늄 13 g/L, 과당 시럽 9 g/L, 탄산칼슘 10 g/L, 소포제 0.5 mL/L)가 담긴 발효조에 접종하고 28

, 35% DO, 400 rpm, 공기 1.0 vvm의 조건을 시작으로 배양하였고, pH는 암모니아수를 이용하여 5.4 내지 5.7로 조절하였으며, 균체 성장에 따라 교반속도를 단계적으로 올리면서 배양하였다. 배양 2일 후에 배양온도를 28

에서 25

로 낮춘 후, 균체성장에 따라 5% 내지 10%의 콩기름을 단계적으로 공급하면서 5일 내지 8일째 발효를 완료하였다. Fermentation of DAOC-producing strains was carried out in the stages of primary culture, secondary culture, and main culture. For the first seed culture, 4 to 6 colonies were used as the primary seed culture medium (soy flour 28.5 g/L, corn steep solution 25 mL/L, sucrose 35 g/L, glucose 5 g/L, calcium carbonate 5). g/L, antifoaming agent 0.8 mL/L) 30 after inoculation

, Incubated for 4 to 5 days at 200 rpm. For the second seed culture, inoculate all of the first seed culture solution into a fermenter containing the second seed culture medium (same as the first seed culture medium, but add 5 mL/L of soybean oil), and 30

Starting with the conditions of 35% DO, 400 rpm, and 1.0 vvm of air, 6% glucose was fed according to the growth of the cells, and the agitation speed was gradually increased and cultured for 4 to 5 days. This culture was carried out using 200 mL of the secondary seed culture medium (peanut powder 23 g/L, corn steep solution 50 mL/L, methionine 1.5 g/L, dextrin 70 g/L, corn mill 35 g/L, calcium sulfate 13 g/L, soybean oil 60 mL/L, ammonium sulfate 13 g/L, fructose syrup 9 g/L, calcium carbonate 10 g/L, antifoam 0.5 mL/L)

, 35% DO, 400 rpm, air was cultured starting with the conditions of 1.0 vvm, and the pH was adjusted to 5.4 to 5.7 using aqueous ammonia, and the agitation speed was gradually increased according to the growth of the cells. After 2 days of incubation, the incubation temperature was increased to 28

25 in

After lowering to, the fermentation was completed on the 5th to the 8th day while supplying 5% to 10% of soybean oil in stages depending on the cell growth.

For analysis of DAOC from the fermentation broth, 1 mL of the fermentation broth was diluted 100 times, centrifuged at 14,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 m syringe filter and analyzed by HPLC.

The fermentation results are shown in FIG. 3.

3-7 strains containing CefE (CefE3) derived from Mycobacterium abcesus produced 33.5 g/L DAOC in 140 hours, the highest, and CefE (CefE7) of Stratomyces clavulozerus was introduced. One 7-23 and 7-53 showed similar productivity at 140 hours, about 27.0 and 28.1 g/L, respectively. The 8-60 strains introduced with CefE (CefE8) of Sphingomonas Dokdonensis were fermented until about 183 hours due to delayed production, and after 160 hours, the increase rate was insignificant and finally produced 28.5 g/L.

The produced DAOC was confirmed through mass spectrometry, and mass spectrometry was conducted by requesting an external company, EZ Mass (Jinju City, Gyeongnam, Korea), and the mass spectrometry results are shown in Fig. 4, and it was confirmed that it was consistent with the standard DAOC.

<Example 4>

Conversion of DAOC culture to 7-ADCA

After DAOC production and fermentation, the fermentation broth was treated with CPC acylase to perform an enzymatic reaction. The CPC acylase was used as a mutant CPC acylase disclosed in Korean Patent Application Laid-Open No. 10-2014-0094150.

After the DAOC fermentation was completed, a temperature of 15° C., a stirring speed of 800 rpm, and a pH of 8.0 were adjusted using 14% aqueous ammonia, and then liquid CPC acylase was added to each fermenter at a final concentration of 30 U/mL. The reaction time was a total of 2 hours, and samples were taken and analyzed at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes.

The results are shown in FIG. 5.

There was a slight difference in each fermentation tank, but most of the reactions ended within 60 minutes and reached an almost maximum. The molar ratio of DAOC and 7-ADCA was about 59.94%, showing a conversion rate of 90%, and up to about 18 g/L of 7-ADCA could be obtained from the fermentation broth of 3-7 strains.

The produced 7-ADCA was confirmed through mass spectrometry, and mass spectrometry was conducted by requesting an external company, EZ Mass (Jinju City, Gyeongnam, Korea), as in DAOC analysis, and the mass spectrometry results are shown in Fig. 6, and the standard product 7- It was confirmed that it was consistent with ADCA (Sigma-Aldrich A8398, USA).

As a result, it was confirmed that DAOC produced by fermentation was converted to 7-ADCA with high efficiency by CPC acylase.

<Example 5>

Increase DAOC productivity through protoplast fusion

To increase DAOC productivity, genome shuffling was performed by protoplast fusion between strains with excellent DAOC productivity. Protoplasts of the strains 1-38, 3-7, 7-23, 7-53, and 8-60, which are the fermentation test strains selected in Example 3, were prepared and fused at random, and then a plurality of colonies were selected to increase DAOC productivity. Tested.

에서 10일 내지 30일 동안 균락이 형성될 때까지 배양하였다. Protoplast fusion was performed by modifying the PEG (polyethylene glycol) transformation method described above. Preparation of protoplasts for each of the five strains 1-38, 3-7, 7-23, 7-53, and 8-60 was the same as in the above transformation method. After counting the prepared protoplasts, 250 μL of 60% PEG solution was mixed with 1×10 ⁷ protoplasts for each of the five strains, and then left on ice for 40 minutes. Thereafter, 2.5 mL of 60% PEG solution was added, mixed, and allowed to stand at room temperature for 40 minutes, and then spread on LB-sucrose plate medium containing antibiotics. 28 reputation badges

Incubated for 10 to 30 days until colonies were formed.

After taking about 100 colonies formed and in vitro culture by the above method, the top 5 strains with high DAOC production were selected to extract genomic DNA, and then the presence or absence of CefE1 using primers SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: The presence or absence of CefE3 using 43 and SEQ ID NO: 44, the presence or absence of CefE7 using SEQ ID NO: 51 and SEQ ID NO: 52, and the presence or absence of CefE8 using SEQ ID NO: 53 and SEQ ID NO: 54 respectively It was confirmed how the strain was fused. In addition, a fermentation test was conducted at the same time to define productivity.

7 is a result of fermentation of strains selected by protoplast fusion. Among the highly productive selection strains, one strain did not fusion, and three strains were fused. CefE1 and CefE7 (E1-E7), CefE1 and CefE8 (E1-E8), CefE3 and CefE8 ( E3-E8) was fused, and three strains of CefE3, CefE7, and CefE8 were fused (E3-E7-E8) in one strain. Fermentation productivity was the highest at 38.7 g/L at 149 hours when the CefE3-introducing strain and the CefE8-introducing strain were fused. When the CefE3, CefE7, and CefE8 introduced strains were fused, the productivity was significantly higher at 35.4 g/L at 140 hours.

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

According to the present invention, an acremonium chrysogenum strain having high DAOC production ability can be prepared, and by treating DAOC produced by these strains with CPC acylase, 7-ADCA can be produced in an environmentally friendly and efficient manner without toxic by-products. Can be used in the industry is very high.

Korea Research Institute of Bioscience and Biotechnology KCTC13922BP 20190819

<110> AMICOGEN, INC. <120> Methods for preparing recombinant Acremonium chrysogenum producing deacetoxycephalosporin C with high concentration and Acremonium chrysogenum prepared thereby as bioprocess for 7-ADCA preparation <130> NP19-0090 <160> 67 <170> KoPatentIn 3.0 <210> 1 <211> 948 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Amycolatopsis lactamdurans (NCBI:txid1913) <400> 1 atgaccgacg ccaccgtgcc caccttcgac ctggccgagc tccgggaggg tctgcaccag 60 gaggagttca ggcattgcct ccgcgaaaag ggtgtcttct acctgaaggg caccggcctg 120 cccgccgagg ccgaccacgc atccgggcgg gagatcgcgg tggacttctt cgaccacggc 180 accgaggccg agaagaaggc ggtgatgacg cccatcccta ccatccgccg gggctacgcg 240 ggcctggaga gcgagtccac cgcgcagatc acgaacaccg gcaagtacac cgactactcc 300 atgtcctatt cgatgggtac cgcggacaac ctcttcccca gcgccgagtt cgagaaggcc 360 tgggaggact atttcgcccg tatgtaccgc gcctcccagg acgtggcgcg gcaggtgctg 420 acgtcggtcg gggcggagcc ggaagtgggc atggacgcct tcctggactg tgagccgctt 480 ctgcgccttc ggtacttccc tgaggtcccc gaggaccgcg tggccgagga acagccgctc 540 cgcatggcgc cgcactacga cctgagcatc gtcacgctga tccaccagac cccgtgcgcc 600 aacggcttcg tgtccctgca ggtggaggtg gacggcagct acgtcgacat ccccgcccag 660 ccgggcgccg tgctggtctt ctgcggggcc gtcgcgacgc tggtggcgga cggggcgatc 720 aaggccccga agcaccacgt ggccgcgccc ggcgccgaca agcgcgtagg ctccagccgc 780 acgtccagcg tgttcttcct ccgtccgaac ggcgacttcc ggttcagcgt cccccgtgcg 840 cgcgaatgcg gcttcgacgt gtccatcccc gccgagaccg cgaccttcga cgactggatc 900 gggggcaact acatcaacat ccgcaagacg gccgccgccc gctgataa 948 <210> 2 <211> 975 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Gordonia rubripertincta CWB2 NBRC 101908 (NCBI:txid1077975) <400> 2 atgaccgaca gcccgatctt gaccgtcgac ctgtcccgct ggcgggccgg tggcgcggac 60 gcggctgccg tgtgcgccgc cgtcgacgag tcgctccaga aggccggctt cctgctggtg 120 accggccacg ggatcgaccc ggtgctcccc gcggagctgc gggcggcagc ccgcgccttc 180 ttcgcgctgc cggccgaggt gaagcagaag tacgcggtcg gggtcggggg ccggggctgg 240 atcgggcccg gcatggaggc caacgcctac gccgagggta ccgagacccc cccggacctg 300 aaggagacct ataactcggg ggcgcaaacg ccggtcggcg taccggaggt ggacgactac 360 tggttcgcac ccgacgtctg gcccattgaa gcgccgcggc tcaaggagct cttcacggca 420 tggaccgcac agtgcaagac actcagcgac gacctcctgg cgctgatggc cgcctccctc 480 ggcttcgccg ccgcggacaa ccccttccgc gacctggctg gcaacgccac ctggacctcc 540 aacatcaacc actacccggc gatggccgtc gtgggcgagc ccgagccggg gcagtaccgg 600 atcggcccgc acaccgactt cggcacggtg accgtcctcg accgcgaacc cggttccggc 660 ggcctgcagg tctacagcga agacggggga tgggctgacg cgccgtacga ccccgcggcc 720 ctcacggtca acatcgggga cctcctcgag tactggtccg gtggtcggtg gccggccggg 780 cgccaccggg tgctgccgcc ccagccggac gcccccgagg aggacctgat gtccctgatc 840 ttcttctatg aactggacca tgacgccgtc gtcacgccga tcggtaagcc cctggggcgc 900 cgcgccggcc tggagcctgt ggtctccggc gagttcatcc gggagcgcct ggacgcgatc 960 accgtcggct gataa 975 <210> 3 <211> 414 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Mycobacteroides abscessus subsp. abscessus (NCBI:txid1185650) <400> 3 atgacggaca tcggtgaacc ggagcctggt cagtaccgga tcggcccgca cacggacttc 60 ggcaccgtca cgatcctgga ccgggagccg ggagccggag ggctgcaggt gtactccgag 120 cagggcggct gggccgacgc cccgtacgac ccgtcggccc tgacggtcaa catcggagac 180 ctgctcgagt actggtcggg gggcaggtgg ccggcgggcc gccaccgggt gctcccgcca 240 caggcggacg ctccggagga agacctggtg tctctcatct tcttctacga actcgaccac 300 gacgccgtcg tgaccccgct cgaccccccg gtcggaaaga tcgccggccg gccgccggtc 360 atcggcggcg atttcatccg ggagaggctg gacgccataa ccgtcggctg ataa 414 <210> 4 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Microbacterium hydrocarbonoxydans NBRC 103074 (NCBI:txid1223527) <400> 4 atggccctgc cgcccgtcat gctgaggaac acgcgggtct ccccccggat gcgcatctac 60 tcccgggtga tgtcggagct gtcgctgcct atcctggacc tctcgcagct ggacgccggc 120 ccggaagccg ccgcccggtt ccgggacgac ctgagggcgg ccacccacga cgtcgggttc 180 ttctacctca ccggcacggg cgtcagtccc gagctcgaag cgcgcctgca ccgcgccgct 240 ctcgacttct tcgcgctgcc ggaggcggag aagctggcga tcgagaacat caactcgccg 300 cacttccggg gctacactcg ggtgggtggg gagcgcaccc agggcgaggt cgactggcgg 360 gagcagatcg acatcggccc cgagcgcgac gccgtagatg gtggcccggc ctacaatcgg 420 ctgatcgggc cgaacctgtg gcccgcggcc cagccggagc tgaaggaagt ggtggcggag 480 tggcattcca cgctcagcga ggtcagccgg cgcctcctcc gcgcgtgggc ggagaccctg 540 ggggcggagc ctagctactt cgacgagcac ttcggagagc cctccaccct catcaagatc 600 gtccggtacc ccggcaccca cgagcccgag ccgcagcagg gtgtcggggc gcataaagac 660 tcaggtgtgc tcacgctcct gtgggtggaa cccggcaagg gtggcctgca ggtggagcgg 720 gacggaacgt gggttgacgc cccctccgtg cccggtgcgt tcgtcgtcaa catcggcgaa 780 ctcctggagt acgccaccgg gggctacctg aaggcgacca accaccgggt ggtctccccg 840 aaggcccccg acgagaggat ctccatcccc ttcttcttca atccggccct cgataagcgg 900 ctgccgctga tcgagctgcc cgcgcccctc gcggccgagg cgaccggggt gaccgacgac 960 ccgcgtaacc cgatccacgc cctgtacggc gagaacgccc tgaagtcacg gctgcgtgcc 1020 caccccgacg tggccgccat ccaccacgcg gacctggtag gcgcgagcgc gtgataa 1077 <210> 5 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Nannocystis exedens subsp. cinnabaria (NCBI:txid1366054) <400> 5 atgtcaagcg ccatcatcgc cgaaatcccg gtgatcgacc tgggcccatt cggagacggg 60 gacgcgccgg gggcggcgga agtggccgcg gcattcgacc gggcctgcag ggacctgggg 120 ttcgtggtcg tcgtggggca cggcgtgccc caggccgtca tcgacgccgc gcaccggagt 180 gcccgcgcgt tctttgacct ggacctcgct acccgcgagc ggtacgcggc cccgcccggc 240 gggttcgtcg ggtaccgcgg gctcggagcc gaggggctgt cttacagtct ggaccaggag 300 gctgcgccgg acttcaagga gacatatacc gtcggacgca tcgatcgcgg agacgagcct 360 tacttcacgt cccagctggg gcggatgtac atgccggacc ctacgtggcc ggcggaggtc 420 cccgagttcg cagcgtcgtg ggccgagttc tatcgccaga tggaccgggt cgcctggcgc 480 ctgatgcgcg cgttcgccac cgcgctggcc ctcccccgcg agttcttcga cgacaagatt 540 gatcggaaca tctcgtgcct gcgcgcgctg aactaccccc acccgtccac acccccccag 600 cccggccagc tgcgcgcggg cgcccacacg gactacggct ccctcacgct gctcagcatg 660 gccgacgcgc ccgggggcct cgaggtgcag cgggacgacg ggtcgtgggc gccggtccgg 720 gtcccggcag acgccttcgt gatgaacgtg ggcgacctta tggcgcagtg gaccaacgac 780 cgttggcgct cgtcgatgca ccgcgtgacg aatccccccc cggacgcgat cggcgacacg 840 cggcggcaga gcctggcgtt cttccaccag cccaactatg acgccgaggt catcccgctc 900 gcgtcctgct gcggcccggc caaccccccg aagtacgccc gcaccacgag cggcgagcac 960 ctgttcatga agatgaccaa ggcgaagaac ctgaacgtct gataa 1005 <210> 6 <211> 1017 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Pseudomonas syringae pv. actinidiae ICMP 18886 (NCBI:txid1189623) <400> 6 atgaccctcc agtacgtccc catcatcaac ctggcgccct atttctcggg cgagccggac 60 ggcaaggcgg cggtcgccca ggcggtgaac caggcgtgca aggacatcgg cttcctcgtc 120 atcaccgagc accagatccc aaccgagctg atcgaccgcg tctcgcgcct cacgcggcaa 180 ttcttcgatc tgccgctcgc cgaaaagagg aaggtcgacc gcccttcgcc ggagatggtc 240 cgggggtatt ccgccgtggc ggaggagtcg ctgtcctact cgctcgagga gtcggcgccg 300 ggggacctga aggagtcctt ctccatcggc ccgagcgacg tcccgaacga ggactactac 360 cacaacgccg aagcggggcc ccacttcgcg ccgaacgtgt ggccggccaa cgaacgggtc 420 cctggcttcc aggaggccta tcaggcctac ttcggggcaa tgtcccagct cgcgcagtcc 480 ctcatgcgct tgttcgccct ggccctcgag ctcgacgaat gcttcttcga cgaccgcatt 540 gaccgccaca tctccatgtt ccggtcgctc tcctacccgg acatcaagac cgaggtcgag 600 gccggccagc tgcgcgcgtc cgcccacacc gactacggct ccctgacgat cgtacgcccc 660 gacaacgccc tgggaggcct ccaggttcgg aaccagcagg gcgagtgggt ggacgttccg 720 tacgtggaga acggcttcgt cgtgaacatc ggtgatctga tgatgcagtg gaccaacgac 780 cagtggatca gcaccctgca cagggtcgtg aatccgccca tgacgagcga gcaggacaat 840 cggcggcagt ccctggtctt cttccaccag ccgaactatg acaccctgat cgagtgcctc 900 cccggctgcc tgctgccggg taccacgccc cgccacgccc ccgtcaccag cggcgaccac 960 ctcctcgcca agtttgtcaa gcagacgacg ttcggagggt ccaaggtcgc ctgataa 1017 <210> 7 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Streptomyces clavuligerus ATCC 27064 (NCBI:txid443255) <400> 7 atggacacca ccgtgccgac cttctccctc gccgagctcc agcagggtct gcaccaggac 60 gagttccggc ggtgtctgcg cgacaagggg ctgttctacc tgaccgactg cggcctaaca 120 gacacggagc tcaagtccgc caaggatatc gtcatcgact ttttcgagca cgggagcgag 180 gccgagaagc gggcggtcac cagccccgtc ccgacgatgc gccgcggctt caccggtctg 240 gagtccgagt cgaccgccca aatcaccaac accggatcct actccgacta cagcatgtgt 300 tactccatgg ggacggccga caacctcttc ccctccgggg atttcgagcg catctggacc 360 cagtacttcg accgccagta caccgccagc agggccgtgg ccagggaggt cctgcgcgcc 420 accgggacgg aaccggacgg cggggtggag gccttcctgg actgcgaacc gcttctgcgg 480 ttccggtact tcccccaggt ccccgagcat cggtccgccg aggagcagcc gctccggatg 540 gccccccact acgacctgtc catggtgacc ctcatccagc agaccccgtg cgcaaacggg 600 ttcgtgtcgc tccaggccga ggtgggcggc gcgttcacag acctccctta ccgacccgac 660 gcggtgttgg tcttctgcgg cgccatcgcc accctcgtga cgggcggcca ggtcaaggcc 720 ccccggcacc acgtggccgc cccgcggcgt gaccagatcg ccggaagcag ccgcacgtcg 780 tccgtgttct tcctgcgccc gaacgcggac ttcacattct ccgtccccct cgcgcgggag 840 tgcggcttcg acgtctccct ggacggggag accgccacgt tccaggactg gatcggcggc 900 aactacgtca acatccggcg gacctcgaag gcgtgataa 939 <210> 8 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> Codon optimized CefE of Sphingomonas dokdonensis (NCBI:txid344880) <400> 8 atgcatcgcg cgggcggcag cgggctcgtc cccagtggcc gcatcgccgg ctattggcga 60 tgcgagcggc cctggcgcat gcagcgccga tcccgctgct cgaccaaggc cctggtgatg 120 ctggacaccc ccctggcgaa ggtgccgacc ctcagtctcg cagaccaggc agtcgatccc 180 gaccgcttcg ccgccgacct gggcggttcg ttcaggcggt tcggcttcgc catcgtcgcg 240 gaccatggca tcccggacga cctcatcgcc cgggcctggg cgcagaccga ggccctgttc 300 gcccagccgg acgccgacaa gcggggctac tacgtggaag gcgcgggcgg ggcccgcggc 360 tacaccccgt tcaagaccga gatcgcgaag gacgccaagc acgtcgacct gaaggagttc 420 tggcacgtgg ggcgagagct gcccacgggc caccggtacg cgggtgagat ggccccgaac 480 atctggcccg cccagcccga tggcttcaag gagaccttcg tcgagctgtt ccgcgctttc 540 gacacggcgg gcgacaagct cctgagcgcc atcgcccggg acctcgggct ggcgccggac 600 tggttcgacc tggcggtcaa ggacggcaac tccgtcctcc gcctgctgca ctaccccccg 660 atcccgatgg acgcggaggg cgtccgcgcg ggagcccatg aggacatcaa cctgatcacc 720 cttctcctgg gtgccgagga ggctggactg gagctgctcg accgcgacag cgggaattgg 780 ctggccatca agcccccgga gggcgcgatg gtagtgaacg tcggggacat gctgcagcgg 840 ttgaccaacc acgtcctgcc gtccaccacc caccgcgtcg tcaatccgcc cccggagcgc 900 cgggcccact cccgctacag catgcccttc tttctgcacc cggcccccga cttcctgatc 960 aagaccctgc cccagaccgt cacctcctcc aacccgaacc ggtaccccac cccgatcacg 1020 gcgcacgact acctccatga gcggcttgtg gagatcggtc tcatcaagaa gtgataa 1077 <210> 9 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> Promoter EP1 <400> 9 tccaaatatc gtgcctctcc tgctttgcca ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctctcctca ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 10 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> Promoter EP2 <400> 10 tccaaatatc gtgcctctcc tgctttgctc ggtgtatgaa accggatagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt taccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctctcccca ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 11 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> Promoter EP3 <400> 11 tccaaatatc gtgcctctcc tgctttgcca ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctttcccca ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 12 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> Promoter EP4 <400> 12 tccaaatatc gttcctctcc tgctttgccc ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tctgtccaac atttgttgcc atattttcct 240 cctctcccta ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 13 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> Promoter EP5 <400> 13 tccaaatatc gtgcctctcc tgctttgccc ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cattccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctctcccca ccagctgctc ttttcgtttc tctttctttt ccca 284 <210> 14 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE1 vector <400> 14 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaattaa 2220 ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgcc cttgtatctc 2280 tacacacagg ctcaaatcaa taagaagaac ggttcgtctt tttcgtttat atcttgcatc 2340 gtcccaaagc tattggcggg atattctgtt tgcagttggc tgacttgaag taatctctgc 2400 agatctttcg acactgaaat acgtcgagcc tgctccgctt ggaagcggcg aggagcctcg 2460 tcctgtcaca actaccaaca tggagtacga taagggccag ttccgccagc tcattaagag 2520 ccagttcatg ggcgttggca tgatggccgt catgcatctg tacttcaagt acaccaacgc 2580 tcttctgatc cagtcgatca tccgctgaag gcgctttcga atctggttaa gatccacgtc 2640 ttcgggaagc cagcgactgg tgacctccag cgtcccttta aggctgccaa cagctttctc 2700 agccagggcc agcccaagac cgacaaggcc tccctccaga acgccgagaa gaactggagg 2760 ggtggtgtca aggaggagta agctccttat tgaagtcgga ggacggagcg gtgtcaagag 2820 gatattcttc gactctgtat tatagataag atgatgagga attggaggta gcatagcttc 2880 atttggattt gctttccagg ctgagactct agcttggagc atagagggtc ctttggcttt 2940 caatattctc aagtatctcg agtttgaact tattccctgt gaacctttta ttcaccaatg 3000 agcattggaa tgaacatgaa tctgaggact gcaatcgcca tgaggttttc gaaatacatc 3060 cggatgtcga aggcttgggg cacctgcgtt ggttgaattt agaacgtggc actattgatc 3120 atccgatagc tctgcaaagg gcgttgcaca atgcaagtca aacgttgcta gcagttccag 3180 gtggaatgtt atgatgagca ttgtattaaa tcaggagata tagcatgatc tctagttagc 3240 tcaccacaaa agtcagacgg cgtaaccaaa agtcacacaa cacaagctgt aaggatttcg 3300 gcacggctac ggaagacgga gaagccacct tcagtggact cgagtaccat ttaattctat 3360 ttgtgtttga tcgagaccta atacagcccc tacaacgacc atcaaagtcg tatagctacc 3420 agtgaggaag tggactcaaa tcgacttcag caacatctcc tggataaact ttaagcctaa 3480 actatacaga ataagatagg tggagagctt ataccgagct cccaaatctg tccagatcat 3540 ggttgaccgg tgcctggatc ttcctataga atcatcctta ttcgttgacc tagctgattc 3600 tggagtgacc cagagggtca tgacttgagc ctaaaatccg ccgcctccac catttgtaga 3660 aaaatgtgac gaactcgtga gctctgtaca gtgaccggtg actctttctg gcatgcggag 3720 agacggacgg acgcagagag aagggctgag taataagcca ctggccagac agctctggcg 3780 gctctgaggt gcagtggatg attattaatc cgggaccggc cgcccctccg ccccgaagtg 3840 gaaaggctgg tgtgcccctc gttgaccaag aatctattgc atcatcggag aatatggagc 3900 ttcatcgaat caccggcagt aagcgaagga gaatgtgaag ccaggggtgt atagccgtcg 3960 gcgaaatagc atgccattaa cctaggtaca gaagtccaat tgcttccgat ctggtaaaag 4020 attcacgaga tagtaccttc tccgaagtag gtagagcgag tacccggcgc gtaagctccc 4080 taattggcga attcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt 4140 tgccaggtgt atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa 4200 cacaagctgg cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc 4260 cctggtaggc agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg 4320 ttgcgtcagt ccaacatttg ttgccatatt ttcctgcttt ccccaccagc tgctcttttc 4380 ttttctcttt cttttcccat cttcagtata ttcatcttcc catatgcatc caagaacctt 4440 tatttcccct aagtaagtac tttgctacat ccatactcca tccttcccat cccttattcc 4500 tttgaacctt tcagttcgag ctttcccact tcatcgcagc ttgactaaca gctaccccgc 4560 ttgagcagac atcaccatga ccgacgccac cgtgcccacc ttcgacctgg ccgagctccg 4620 ggagggtctg caccaggagg agttcaggca ttgcctccgc gaaaagggtg tcttctacct 4680 gaagggcacc ggcctgcccg ccgaggccga ccacgcatcc gggcgggaga tcgcggtgga 4740 cttcttcgac cacggcaccg aggccgagaa gaaggcggtg atgacgccca tccctaccat 4800 ccgccggggc tacgcgggcc tggagagcga gtccaccgcg cagatcacga acaccggcaa 4860 gtacaccgac tactccatgt cctattcgat gggtaccgcg gacaacctct tccccagcgc 4920 cgagttcgag aaggcctggg aggactattt cgcccgtatg taccgcgcct cccaggacgt 4980 ggcgcggcag gtgctgacgt cggtcggggc ggagccggaa gtgggcatgg acgccttcct 5040 ggactgtgag ccgcttctgc gccttcggta cttccctgag gtccccgagg accgcgtggc 5100 cgaggaacag ccgctccgca tggcgccgca ctacgacctg agcatcgtca cgctgatcca 5160 ccagaccccg tgcgccaacg gcttcgtgtc cctgcaggtg gaggtggacg gcagctacgt 5220 cgacatcccc gcccagccgg gcgccgtgct ggtcttctgc ggggccgtcg cgacgctggt 5280 ggcggacggg gcgatcaagg ccccgaagca ccacgtggcc gcgcccggcg ccgacaagcg 5340 cgtaggctcc agccgcacgt ccagcgtgtt cttcctccgt ccgaacggcg acttccggtt 5400 cagcgtcccc cgtgcgcgcg aatgcggctt cgacgtgtcc atccccgccg agaccgcgac 5460 cttcgacgac tggatcgggg gcaactacat caacatccgc aagacggccg ccgcccgctg 5520 ataaaagctt atcgataccg tcgacctcga gacttaacgt tactgaaatc atcaaacagc 5580 ttgacgaatc tggatataag atcgttggtg tcgatgtcag ctccggagtt gagacaaatg 5640 gtgttcagga tctcgataag atacgttcat ttgtccaagc agcaaagagt gccttctagt 5700 gatttaatag ctccatgtca acaagaataa aacgcgtttt cgggtttacc tcttccagat 5760 acagctcatc tgcaatgcat taatgcattg actgcaacct agtaacgcct ttcaggctcc 5820 ggcgaagaga agaatagctt agcagagcta ttttcatttt cgggagacga gatcaagcag 5880 atcaacggtc gtcaagagac ctacgagact gaggaatccg ctcttggctc cacgcgacta 5940 tatatttgtc tctaattgta ctttgacatg ctcctcttct ttactctgat agcttgacta 6000 tgaaaattcc gtcaccagcc cctgggttcg caaagataat tgcatgtttc ttccttgaac 6060 tctcaagcct acaggacaca cattcatcgt aggtataaac ctcgaaatca gttcctacta 6120 agatggtata caatagtaac catgcatggt tgcctagtga atgctccgta acacccaata 6180 cgccggccga aactttttta caactctcct atgagtcgtt tacccagaat gcacaggtac 6240 acttgtttag aggtaatcct tctttctaga agtcctcgtg tactgtgtaa gcgcccactc 6300 cacatctcca ctcgatggta cccaattcgc cctatagtga gtcgtattaa aaccccgggt 6360 acgtaaatta accctcacta aagggaacaa aagctgggca aagcttgagg ccggacaaat 6420 tcagcctgca ggctaaagtt tctattacat gatatttgtc gtggacaagg gcgtctgggg 6480 taaaaaggaa tgtgggcaga gagctctcgg cttaaagacc ggcttcggag ttccagaccc 6540 actcgaactg aaaacgatga ttagagataa gtagatctat ttggacagta taagaatcct 6600 agtcggctag tatttctcca cttttacttt aaaattcccc agagtaaggg ggcaggttcg 6660 tcgtcacggg tagcctcgtc tcgaggcatc gcggatagca ttatcttggg gtatcacgag 6720 taacttcatc gtggcattat cgataggtac tgtaagtact gtaggaactt atccgggcat 6780 ggatatgtat aggtacatac gatgcactaa aagtggagaa atactagccg agtagagatc 6840 ttataccatc cgaatagatt tactccttat aatcaagggg caacaatcag ttgtgacgat 6900 gtcggctgtg ggagtgcagt ttaggggtca tcaaagcaca ggaaaacgtg ttgtatggat 6960 gcctatcgtc ttgactatta gaaaggaaga ggggaaaaac cctcatcagg agcgattgta 7020 agttcggcca tagtaatgat attggccatt aatggacgtg tgtatgtata catacacggt 7080 tcatttgccg cattcggacc aaaaccgctg ctgccgcaga cacagtgctt ccatggcctc 7140 gaacaaccgg ttattcttct cttccatgtc ctcgggacga aggaaatttc ggagccctaa 7200 cagccgtacc ggacttgcgt taacatggtt gcgaagaact aaagactgat ggggtcatga 7260 ttggcagtgt tgacacttca tgtcttgaac ctagctagca gaaaaatgaa tgctctatgt 7320 gacactggta gatctcaacg catcatgttc cacctttagt gtaaacacgg caagctgtcc 7380 gcaacgtcgg taccaacgtt tgaacacaca accttctgcc actccaggag tttagcccac 7440 ggcttactgg atgaaacgta atctcccgct accccctcaa ttccattggg gtctagagta 7500 gtcggaagaa cggctgtcgc cttgcgctgc ggttaaagca cccaccgtgg tggctactgc 7560 ttctcgggag gagaaatcct gggtactcga gcacttgcgc taggggatga aacatccatg 7620 gcatattggc gaaagcaatg cgccatgttc ggcgcggcac atgacactct tgagaagccg 7680 tattcttcgc aaactttcct atttacattc agcctctaac tataacacga tcggaaatgc 7740 ctgaacgagc ccgccctcat gtgcctagcg tcaaacaacg ccaagcacac aacgcctcct 7800 gctattcttc cctcttctca ctgccatcac tgggcgcaag ctgcaatgtc cttctcgaat 7860 tcaagcgggc aaatcaagat tgggaaaatg cgccatcttg gtttggatct tgatgtctat 7920 ccgcatcaag gtcttggttt ggtcgtttat ttagtagcaa aggcaaatca aaacgtgcgc 7980 gccaagtgcc agggtccaga ctccagactg gagcatttag cctgaatatc gtctctagaa 8040 tctggtgcta ttattcagga tatagtaact atgatctact cgggccatgt tcggctcctc 8100 cgcacggcca agttgtctgc cggcgtctgg accggactca tggcttggct tctccaggga 8160 tagcatggca ttggcaaaac tctccgccac ga 8192 <210> 15 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE2 vector <400> 15 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaattaa 2220 ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagaactag 2280 tgcggccgcc cttgtatctc tacacacagg ctcaaatcaa taagaagaac ggttcgtctt 2340 tttcgtttat atcttgcatc gtcccaaagc tattggcggg atattctgtt tgcagttggc 2400 tgacttgaag taatctctgc agatctttcg acactgaaat acgtcgagcc tgctccgctt 2460 ggaagcggcg aggagcctcg tcctgtcaca actaccaaca tggagtacga taagggccag 2520 ttccgccagc tcattaagag ccagttcatg ggcgttggca tgatggccgt catgcatctg 2580 tacttcaagt acaccaacgc tcttctgatc cagtcgatca tccgctgaag gcgctttcga 2640 atctggttaa gatccacgtc ttcgggaagc cagcgactgg tgacctccag cgtcccttta 2700 aggctgccaa cagctttctc agccagggcc agcccaagac cgacaaggcc tccctccaga 2760 acgccgagaa gaactggagg ggtggtgtca aggaggagta agctccttat tgaagtcgga 2820 ggacggagcg gtgtcaagag gatattcttc gactctgtat tatagataag atgatgagga 2880 attggaggta gcatagcttc atttggattt gctttccagg ctgagactct agcttggagc 2940 atagagggtc ctttggcttt caatattctc aagtatctcg agtttgaact tattccctgt 3000 gaacctttta ttcaccaatg agcattggaa tgaacatgaa tctgaggact gcaatcgcca 3060 tgaggttttc gaaatacatc cggatgtcga aggcttgggg cacctgcgtt ggttgaattt 3120 agaacgtggc actattgatc atccgatagc tctgcaaagg gcgttgcaca atgcaagtca 3180 aacgttgcta gcagttccag gtggaatgtt atgatgagca ttgtattaaa tcaggagata 3240 tagcatgatc tctagttagc tcaccacaaa agtcagacgg cgtaaccaaa agtcacacaa 3300 cacaagctgt aaggatttcg gcacggctac ggaagacgga gaagccacct tcagtggact 3360 cgagtaccat ttaattctat ttgtgtttga tcgagaccta atacagcccc tacaacgacc 3420 atcaaagtcg tatagctacc agtgaggaag tggactcaaa tcgacttcag caacatctcc 3480 tggataaact ttaagcctaa actatacaga ataagatagg tggagagctt ataccgagct 3540 cccaaatctg tccagatcat ggttgaccgg tgcctggatc ttcctataga atcatcctta 3600 ttcgttgacc tagctgattc tggagtgacc cagagggtca tgacttgagc ctaaaatccg 3660 ccgcctccac catttgtaga aaaatgtgac gaactcgtga gctctgtaca gtgaccggtg 3720 actctttctg gcatgcggag agacggacgg acgcagagag aagggctgag taataagcca 3780 ctggccagac agctctggcg gctctgaggt gcagtggatg attattaatc cgggaccggc 3840 cgcccctccg ccccgaagtg gaaaggctgg tgtgcccctc gttgaccaag aatctattgc 3900 atcatcggag aatatggagc ttcatcgaat caccggcagt aagcgaagga gaatgtgaag 3960 ccaggggtgt atagccgtcg gcgaaatagc atgccattaa cctaggtaca gaagtccaat 4020 tgcttccgat ctggtaaaag attcacgaga tagtaccttc tccgaagtag gtagagcgag 4080 tacccggcgc gtaagctccc taattggcga attcccatcc ggcatctgta gggcgtccaa 4140 atatcgtgcc tctcctgctt tgccaggtgt atgaaaccgg aaaggccgct caggagctgg 4200 ccagcggcgc agaccgggaa cacaagctgg cagtcgaccc atccggtgct ctgcactcga 4260 cctgctgagg tccctcagtc cctggtaggc agctttgccc cgtctgtccg cccggtgtgt 4320 cggcggggtt gacaaggtcg ttgcgtcagt ccaacatttg ttgccatatt ttcctgcttt 4380 ccccaccagc tgctcttttc ttttctcttt cttttcccat cttcagtata ttcatcttcc 4440 catatgcatc caagaacctt tatttcccct aagtaagtac tttgctacat ccatactcca 4500 tccttcccat cccttattcc tttgaacctt tcagttcgag ctttcccact tcatcgcagc 4560 ttgactaaca gctaccccgc ttgagcagac atcaccatga ccgacagccc gatcttgacc 4620 gtcgacctgt cccgctggcg ggccggtggc gcggacgcgg ctgccgtgtg cgccgccgtc 4680 gacgagtcgc tccagaaggc cggcttcctg ctggtgaccg gccacgggat cgacccggtg 4740 ctccccgcgg agctgcgggc ggcagcccgc gccttcttcg cgctgccggc cgaggtgaag 4800 cagaagtacg cggtcggggt cgggggccgg ggctggatcg ggcccggcat ggaggccaac 4860 gcctacgccg agggtaccga gacccccccg gacctgaagg agacctataa ctcgggggcg 4920 caaacgccgg tcggcgtacc ggaggtggac gactactggt tcgcacccga cgtctggccc 4980 attgaagcgc cgcggctcaa ggagctcttc acggcatgga ccgcacagtg caagacactc 5040 agcgacgacc tcctggcgct gatggccgcc tccctcggct tcgccgccgc ggacaacccc 5100 ttccgcgacc tggctggcaa cgccacctgg acctccaaca tcaaccacta cccggcgatg 5160 gccgtcgtgg gcgagcccga gccggggcag taccggatcg gcccgcacac cgacttcggc 5220 acggtgaccg tcctcgaccg cgaacccggt tccggcggcc tgcaggtcta cagcgaagac 5280 gggggatggg ctgacgcgcc gtacgacccc gcggccctca cggtcaacat cggggacctc 5340 ctcgagtact ggtccggtgg tcggtggccg gccgggcgcc accgggtgct gccgccccag 5400 ccggacgccc ccgaggagga cctgatgtcc ctgatcttct tctatgaact ggaccatgac 5460 gccgtcgtca cgccgatcgg taagcccctg gggcgccgcg ccggcctgga gcctgtggtc 5520 tccggcgagt tcatccggga gcgcctggac gcgatcaccg tcggctgata aaagcttatc 5580 gataccgtcg acctcgagac ttaacgttac tgaaatcatc aaacagcttg acgaatctgg 5640 atataagatc gttggtgtcg atgtcagctc cggagttgag acaaatggtg ttcaggatct 5700 cgataagata cgttcatttg tccaagcagc aaagagtgcc ttctagtgat ttaatagctc 5760 catgtcaaca agaataaaac gcgttttcgg gtttacctct tccagataca gctcatctgc 5820 aatgcattaa tgcattgact gcaacctagt aacgcctttc aggctccggc gaagagaaga 5880 atagcttagc agagctattt tcattttcgg gagacgagat caagcagatc aacggtcgtc 5940 aagagaccta cgagactgag gaatccgctc ttggctccac gcgactatat atttgtctct 6000 aattgtactt tgacatgctc ctcttcttta ctctgatagc ttgactatga aaattccgtc 6060 accagcccct gggttcgcaa agataattgc atgtttcttc cttgaactct caagcctaca 6120 ggacacacat tcatcgtagg tataaacctc gaaatcagtt cctactaaga tggtatacaa 6180 tagtaaccat gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc cggccgaaac 6240 ttttttacaa ctctcctatg agtcgtttac ccagaatgca caggtacact tgtttagagg 6300 taatccttct ttctagaagt cctcgtgtac tgtgtaagcg cccactccac atctccactc 6360 gatggtaccc aattcgccct atagtgagtc gtattaaaac cccgggtacg taaattaacc 6420 ctcactaaag ggaacaaaag ctgggcaaag cttgaggccg gacaaattca gcctgcaggc 6480 taaagtttct attacatgat atttgtcgtg gacaagggcg tctggggtaa aaaggaatgt 6540 gggcagagag ctctcggctt aaagaccggc ttcggagttc cagacccact cgaactgaaa 6600 acgatgatta gagataagta gatctatttg gacagtataa gaatcctagt cggctagtat 6660 ttctccactt ttactttaaa attccccaga gtaagggggc aggttcgtcg tcacgggtag 6720 cctcgtctcg aggcatcgcg gatagcatta tcttggggta tcacgagtaa cttcatcgtg 6780 gcattatcga taggtactgt aagtactgta ggaacttatc cgggcatgga tatgtatagg 6840 tacatacgat gcactaaaag tggagaaata ctagccgagt agagatctta taccatccga 6900 atagatttac tccttataat caaggggcaa caatcagttg tgacgatgtc ggctgtggga 6960 gtgcagttta ggggtcatca aagcacagga aaacgtgttg tatggatgcc tatcgtcttg 7020 actattagaa aggaagaggg gaaaaaccct catcaggagc gattgtaagt tcggccatag 7080 taatgatatt ggccattaat ggacgtgtgt atgtatacat acacggttca tttgccgcat 7140 tcggaccaaa accgctgctg ccgcagacac agtgcttcca tggcctcgaa caaccggtta 7200 ttcttctctt ccatgtcctc gggacgaagg aaatttcgga gccctaacag ccgtaccgga 7260 cttgcgttaa catggttgcg aagaactaaa gactgatggg gtcatgattg gcagtgttga 7320 cacttcatgt cttgaaccta gctagcagaa aaatgaatgc tctatgtgac actggtagat 7380 ctcaacgcat catgttccac ctttagtgta aacacggcaa gctgtccgca acgtcggtac 7440 caacgtttga acacacaacc ttctgccact ccaggagttt agcccacggc ttactggatg 7500 aaacgtaatc tcccgctacc ccctcaattc cattggggtc tagagtagtc ggaagaacgg 7560 ctgtcgcctt gcgctgcggt taaagcaccc accgtggtgg ctactgcttc tcgggaggag 7620 aaatcctggg tactcgagca cttgcgctag gggatgaaac atccatggca tattggcgaa 7680 agcaatgcgc catgttcggc gcggcacatg acactcttga gaagccgtat tcttcgcaaa 7740 ctttcctatt tacattcagc ctctaactat aacacgatcg gaaatgcctg aacgagcccg 7800 ccctcatgtg cctagcgtca aacaacgcca agcacacaac gcctcctgct attcttccct 7860 cttctcactg ccatcactgg gcgcaagctg caatgtcctt ctcgaattca agcgggcaaa 7920 tcaagattgg gaaaatgcgc catcttggtt tggatcttga tgtctatccg catcaaggtc 7980 ttggtttggt cgtttattta gtagcaaagg caaatcaaaa cgtgcgcgcc aagtgccagg 8040 gtccagactc cagactggag catttagcct gaatatcgtc tctagaatct ggtgctatta 8100 ttcaggatat agtaactatg atctactcgg gccatgttcg gctcctccgc acggccaagt 8160 tgtctgccgg cgtctggacc ggactcatgg ct 8192 <210> 16 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE3 vector <400> 16 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaattaa 2220 ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgcg tttaaaccct 2280 tgtatctcta cacacaggct caaatcaata agaagaacgg ttcgtctttt tcgtttatat 2340 cttgcatcgt cccaaagcta ttggcgggat attctgtttg cagttggctg acttgaagta 2400 atctctgcag atctttcgac actgaaatac gtcgagcctg ctccgcttgg aagcggcgag 2460 gagcctcgtc ctgtcacaac taccaacatg gagtacgata agggccagtt ccgccagctc 2520 attaagagcc agttcatggg cgttggcatg atggccgtca tgcatctgta cttcaagtac 2580 accaacgctc ttctgatcca gtcgatcatc cgctgaaggc gctttcgaat ctggttaaga 2640 tccacgtctt cgggaagcca gcgactggtg acctccagcg tccctttaag gctgccaaca 2700 gctttctcag ccagggccag cccaagaccg acaaggcctc cctccagaac gccgagaaga 2760 actggagggg tggtgtcaag gaggagtaag ctccttattg aagtcggagg acggagcggt 2820 gtcaagagga tattcttcga ctctgtatta tagataagat gatgaggaat tggaggtagc 2880 atagcttcat ttggatttgc tttccaggct gagactctag cttggagcat agagggtcct 2940 ttggctttca atattctcaa gtatctcgag tttgaactta ttccctgtga accttttatt 3000 caccaatgag cattggaatg aacatgaatc tgaggactgc aatcgccatg aggttttcga 3060 aatacatccg gatgtcgaag gcttggggca cctgcgttgg ttgaatttag aacgtggcac 3120 tattgatcat ccgatagctc tgcaaagggc gttgcacaat gcaagtcaaa cgttgctagc 3180 agttccaggt ggaatgttat gatgagcatt gtattaaatc aggagatata gcatgatctc 3240 tagttagctc accacaaaag tcagacggcg taaccaaaag tcacacaaca caagctgtaa 3300 ggatttcggc acggctacgg aagacggaga agccaccttc agtggactcg agtaccattt 3360 aattctattt gtgtttgatc gagacctaat acagccccta caacgaccat caaagtcgta 3420 tagctaccag tgaggaagtg gactcaaatc gacttcagca acatctcctg gataaacttt 3480 aagcctaaac tatacagaat aagataggtg gagagcttat accgagctcc caaatctgtc 3540 cagatcatgg ttgaccggtg cctggatctt cctatagaat catccttatt cgttgaccta 3600 gctgattctg gagtgaccca gagggtcatg acttgagcct aaaatccgcc gcctccacca 3660 tttgtagaaa aatgtgacga actcgtgagc tctgtacagt gaccggtgac tctttctggc 3720 atgcggagag acggacggac gcagagagaa gggctgagta ataagccact ggccagacag 3780 ctctggcggc tctgaggtgc agtggatgat tattaatccg ggaccggccg cccctccgcc 3840 ccgaagtgga aaggctggtg tgcccctcgt tgaccaagaa tctattgcat catcggagaa 3900 tatggagctt catcgaatca ccggcagtaa gcgaaggaga atgtgaagcc aggggtgtat 3960 agccgtcggc gaaatagcat gccattaacc taggtacaga agtccaattg cttccgatct 4020 ggtaaaagat tcacgagata gtaccttctc cgaagtaggt agagcgagta cccggcgcgt 4080 aagctcccta attggcgaat tcccatccgg catctgtagg gcgtccaaat atcgtgcctc 4140 tcctgctttg ccaggtgtat gaaaccggaa aggccgctca ggagctggcc agcggcgcag 4200 accgggaaca caagctggca gtcgacccat ccggtgctct gcactcgacc tgctgaggtc 4260 cctcagtccc tggtaggcag ctttgccccg tctgtccgcc cggtgtgtcg gcggggttga 4320 caaggtcgtt gcgtcagtcc aacatttgtt gccatatttt cctgctttcc ccaccagctg 4380 ctcttttctt ttctctttct tttcccatct tcagtatatt catcttccca tatgcatcca 4440 agaaccttta tttcccctaa gtaagtactt tgctacatcc atactccatc cttcccatcc 4500 cttattcctt tgaacctttc agttcgagct ttcccacttc atcgcagctt gactaacagc 4560 taccccgctt gagcagacat caccatgacg gacatcggtg aaccggagcc tggtcagtac 4620 cggatcggcc cgcacacgga cttcggcacc gtcacgatcc tggaccggga gccgggagcc 4680 ggagggctgc aggtgtactc cgagcagggc ggctgggccg acgccccgta cgacccgtcg 4740 gccctgacgg tcaacatcgg agacctgctc gagtactggt cggggggcag gtggccggcg 4800 ggccgccacc gggtgctccc gccacaggcg gacgctccgg aggaagacct ggtgtctctc 4860 atcttcttct acgaactcga ccacgacgcc gtcgtgaccc cgctcgaccc cccggtcgga 4920 aagatcgccg gccggccgcc ggtcatcggc ggcgatttca tccgggagag gctggacgcc 4980 ataaccgtcg gctgataaaa gcttatcgat accgtcgacc tcgagactta acgttactga 5040 aatcatcaaa cagcttgacg aatctggata taagatcgtt ggtgtcgatg tcagctccgg 5100 agttgagaca aatggtgttc aggatctcga taagatacgt tcatttgtcc aagcagcaaa 5160 gagtgccttc tagtgattta atagctccat gtcaacaaga ataaaacgcg ttttcgggtt 5220 tacctcttcc agatacagct catctgcaat gcattaatgc attgactgca acctagtaac 5280 gcctttcagg ctccggcgaa gagaagaata gcttagcaga gctattttca ttttcgggag 5340 acgagatcaa gcagatcaac ggtcgtcaag agacctacga gactgaggaa tccgctcttg 5400 gctccacgcg actatatatt tgtctctaat tgtactttga catgctcctc ttctttactc 5460 tgatagcttg actatgaaaa ttccgtcacc agcccctggg ttcgcaaaga taattgcatg 5520 tttcttcctt gaactctcaa gcctacagga cacacattca tcgtaggtat aaacctcgaa 5580 atcagttcct actaagatgg tatacaatag taaccatgca tggttgccta gtgaatgctc 5640 cgtaacaccc aatacgccgg ccgaaacttt tttacaactc tcctatgagt cgtttaccca 5700 gaatgcacag gtacacttgt ttagaggtaa tccttctttc tagaagtcct cgtgtactgt 5760 gtaagcgccc actccacatc tccactcgat ggtacccaat tcgccctata gtgagtcgta 5820 ttaaaacccc gggtacgtaa attaaccctc actaaaggga acaaaagctg ggcaaagctt 5880 gaggccggac aaattcagcc tgcaggctaa agtttctatt acatgatatt tgtcgtggac 5940 aagggcgtct ggggtaaaaa ggaatgtggg cagagagctc tcggcttaaa gaccggcttc 6000 ggagttccag acccactcga actgaaaacg atgattagag ataagtagat ctatttggac 6060 agtataagaa tcctagtcgg ctagtatttc tccactttta ctttaaaatt ccccagagta 6120 agggggcagg ttcgtcgtca cgggtagcct cgtctcgagg catcgcggat agcattatct 6180 tggggtatca cgagtaactt catcgtggca ttatcgatag gtactgtaag tactgtagga 6240 acttatccgg gcatggatat gtataggtac atacgatgca ctaaaagtgg agaaatacta 6300 gccgagtaga gatcttatac catccgaata gatttactcc ttataatcaa ggggcaacaa 6360 tcagttgtga cgatgtcggc tgtgggagtg cagtttaggg gtcatcaaag cacaggaaaa 6420 cgtgttgtat ggatgcctat cgtcttgact attagaaagg aagaggggaa aaaccctcat 6480 caggagcgat tgtaagttcg gccatagtaa tgatattggc cattaatgga cgtgtgtatg 6540 tatacataca cggttcattt gccgcattcg gaccaaaacc gctgctgccg cagacacagt 6600 gcttccatgg cctcgaacaa ccggttattc ttctcttcca tgtcctcggg acgaaggaaa 6660 tttcggagcc ctaacagccg taccggactt gcgttaacat ggttgcgaag aactaaagac 6720 tgatggggtc atgattggca gtgttgacac ttcatgtctt gaacctagct agcagaaaaa 6780 tgaatgctct atgtgacact ggtagatctc aacgcatcat gttccacctt tagtgtaaac 6840 acggcaagct gtccgcaacg tcggtaccaa cgtttgaaca cacaaccttc tgccactcca 6900 ggagtttagc ccacggctta ctggatgaaa cgtaatctcc cgctaccccc tcaattccat 6960 tggggtctag agtagtcgga agaacggctg tcgccttgcg ctgcggttaa agcacccacc 7020 gtggtggcta ctgcttctcg ggaggagaaa tcctgggtac tcgagcactt gcgctagggg 7080 atgaaacatc catggcatat tggcgaaagc aatgcgccat gttcggcgcg gcacatgaca 7140 ctcttgagaa gccgtattct tcgcaaactt tcctatttac attcagcctc taactataac 7200 acgatcggaa atgcctgaac gagcccgccc tcatgtgcct agcgtcaaac aacgccaagc 7260 acacaacgcc tcctgctatt cttccctctt ctcactgcca tcactgggcg caagctgcaa 7320 tgtccttctc gaattcaagc gggcaaatca agattgggaa aatgcgccat cttggtttgg 7380 atcttgatgt ctatccgcat caaggtcttg gtttggtcgt ttatttagta gcaaaggcaa 7440 atcaaaacgt gcgcgccaag tgccagggtc cagactccag actggagcat ttagcctgaa 7500 tatcgtctct agaatctggt gctattattc aggatatagt aactatgatc tactcgggcc 7560 atgttcggct cctccgcacg gccaagttgt ctgccggcgt ctggaccgga ctcatggctt 7620 ggcttctcca gggatagcat ggcattggca aaactctccg ccacgattcg agcatagcta 7680 ccgcagccac gcggcattgt ttccagattg agatatgtca ctattagccg gaatgcaaac 7740 atcgagctga cctgcgcaac atctccatga gttccgaagt cgacctccga agcaatattg 7800 gtctagtatt acggatgata tagtggtccc cggacccaat gagccgtgtg ttttttaccg 7860 cctttgctct gaggattaac aagattcgtg caattgggga ctgtgcggag taactgggct 7920 ggtttactgc tttgccgacg aaagaaacgt ataaacagcc gactcgtcca ctgccactcg 7980 ggacgatctc ccaacccaga cagcttccca gaacccatct tcaacaccct gacaccatgc 8040 cccagttcga catcctatgc aagactcctc cgaaggtgct cgtccgccag ttcgtggagc 8100 gattcgagcg cccctcgggc gaaaagatcg ccttgtgtgc cgctgagctt acctacctct 8160 gctggatgat cacccacaac ggcaccgcca tc 8192 <210> 17 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE4 vector <400> 17 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaattaa 2220 ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgcc cttgtatctc 2280 tacacacagg ctcaaatcaa taagaagaac ggttcgtctt tttcgtttat atcttgcatc 2340 gtcccaaagc tattggcggg atattctgtt tgcagttggc tgacttgaag taatctctgc 2400 agatctttcg acactgaaat acgtcgagcc tgctccgctt ggaagcggcg aggagcctcg 2460 tcctgtcaca actaccaaca tggagtacga taagggccag ttccgccagc tcattaagag 2520 ccagttcatg ggcgttggca tgatggccgt catgcatctg tacttcaagt acaccaacgc 2580 tcttctgatc cagtcgatca tccgctgaag gcgctttcga atctggttaa gatccacgtc 2640 ttcgggaagc cagcgactgg tgacctccag cgtcccttta aggctgccaa cagctttctc 2700 agccagggcc agcccaagac cgacaaggcc tccctccaga acgccgagaa gaactggagg 2760 ggtggtgtca aggaggagta agctccttat tgaagtcgga ggacggagcg gtgtcaagag 2820 gatattcttc gactctgtat tatagataag atgatgagga attggaggta gcatagcttc 2880 atttggattt gctttccagg ctgagactct agcttggagc atagagggtc ctttggcttt 2940 caatattctc aagtatctcg agtttgaact tattccctgt gaacctttta ttcaccaatg 3000 agcattggaa tgaacatgaa tctgaggact gcaatcgcca tgaggttttc gaaatacatc 3060 cggatgtcga aggcttgggg cacctgcgtt ggttgaattt agaacgtggc actattgatc 3120 atccgatagc tctgcaaagg gcgttgcaca atgcaagtca aacgttgcta gcagttccag 3180 gtggaatgtt atgatgagca ttgtattaaa tcaggagata tagcatgatc tctagttagc 3240 tcaccacaaa agtcagacgg cgtaaccaaa agtcacacaa cacaagctgt aaggatttcg 3300 gcacggctac ggaagacgga gaagccacct tcagtggact cgagtaccat ttaattctat 3360 ttgtgtttga tcgagaccta atacagcccc tacaacgacc atcaaagtcg tatagctacc 3420 agtgaggaag tggactcaaa tcgacttcag caacatctcc tggataaact ttaagcctaa 3480 actatacaga ataagatagg tggagagctt ataccgagct cccaaatctg tccagatcat 3540 ggttgaccgg tgcctggatc ttcctataga atcatcctta ttcgttgacc tagctgattc 3600 tggagtgacc cagagggtca tgacttgagc ctaaaatccg ccgcctccac catttgtaga 3660 aaaatgtgac gaactcgtga gctctgtaca gtgaccggtg actctttctg gcatgcggag 3720 agacggacgg acgcagagag aagggctgag taataagcca ctggccagac agctctggcg 3780 gctctgaggt gcagtggatg attattaatc cgggaccggc cgcccctccg ccccgaagtg 3840 gaaaggctgg tgtgcccctc gttgaccaag aatctattgc atcatcggag aatatggagc 3900 ttcatcgaat caccggcagt aagcgaagga gaatgtgaag ccaggggtgt atagccgtcg 3960 gcgaaatagc atgccattaa cctaggtaca gaagtccaat tgcttccgat ctggtaaaag 4020 attcacgaga tagtaccttc tccgaagtag gtagagcgag tacccggcgc gtaagctccc 4080 taattggcga attcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt 4140 tgccaggtgt atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa 4200 cacaagctgg cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc 4260 cctggtaggc agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg 4320 ttgcgtcagt ccaacatttg ttgccatatt ttcctgcttt ccccaccagc tgctcttttc 4380 ttttctcttt cttttcccat cttcagtata ttcatcttcc catatgcatc caagaacctt 4440 tatttcccct aagtaagtac tttgctacat ccatactcca tccttcccat cccttattcc 4500 tttgaacctt tcagttcgag ctttcccact tcatcgcagc ttgactaaca gctaccccgc 4560 ttgagcagac atcaccatgg ccctgccgcc cgtcatgctg aggaacacgc gggtctcccc 4620 ccggatgcgc atctactccc gggtgatgtc ggagctgtcg ctgcctatcc tggacctctc 4680 gcagctggac gccggcccgg aagccgccgc ccggttccgg gacgacctga gggcggccac 4740 ccacgacgtc gggttcttct acctcaccgg cacgggcgtc agtcccgagc tcgaagcgcg 4800 cctgcaccgc gccgctctcg acttcttcgc gctgccggag gcggagaagc tggcgatcga 4860 gaacatcaac tcgccgcact tccggggcta cactcgggtg ggtggggagc gcacccaggg 4920 cgaggtcgac tggcgggagc agatcgacat cggccccgag cgcgacgccg tagatggtgg 4980 cccggcctac aatcggctga tcgggccgaa cctgtggccc gcggcccagc cggagctgaa 5040 ggaagtggtg gcggagtggc attccacgct cagcgaggtc agccggcgcc tcctccgcgc 5100 gtgggcggag accctggggg cggagcctag ctacttcgac gagcacttcg gagagccctc 5160 caccctcatc aagatcgtcc ggtaccccgg cacccacgag cccgagccgc agcagggtgt 5220 cggggcgcat aaagactcag gtgtgctcac gctcctgtgg gtggaacccg gcaagggtgg 5280 cctgcaggtg gagcgggacg gaacgtgggt tgacgccccc tccgtgcccg gtgcgttcgt 5340 cgtcaacatc ggcgaactcc tggagtacgc caccgggggc tacctgaagg cgaccaacca 5400 ccgggtggtc tccccgaagg cccccgacga gaggatctcc atccccttct tcttcaatcc 5460 ggccctcgat aagcggctgc cgctgatcga gctgcccgcg cccctcgcgg ccgaggcgac 5520 cggggtgacc gacgacccgc gtaacccgat ccacgccctg tacggcgaga acgccctgaa 5580 gtcacggctg cgtgcccacc ccgacgtggc cgccatccac cacgcggacc tggtaggcgc 5640 gagcgcgtga taaaagctta tcgataccgt cgacctcgag acttaacgtt actgaaatca 5700 tcaaacagct tgacgaatct ggatataaga tcgttggtgt cgatgtcagc tccggagttg 5760 agacaaatgg tgttcaggat ctcgataaga tacgttcatt tgtccaagca gcaaagagtg 5820 ccttctagtg atttaatagc tccatgtcaa caagaataaa acgcgttttc gggtttacct 5880 cttccagata cagctcatct gcaatgcatt aatgcattga ctgcaaccta gtaacgcctt 5940 tcaggctccg gcgaagagaa gaatagctta gcagagctat tttcattttc gggagacgag 6000 atcaagcaga tcaacggtcg tcaagagacc tacgagactg aggaatccgc tcttggctcc 6060 acgcgactat atatttgtct ctaattgtac tttgacatgc tcctcttctt tactctgata 6120 gcttgactat gaaaattccg tcaccagccc ctgggttcgc aaagataatt gcatgtttct 6180 tccttgaact ctcaagccta caggacacac attcatcgta ggtataaacc tcgaaatcag 6240 ttcctactaa gatggtatac aatagtaacc atgcatggtt gcctagtgaa tgctccgtaa 6300 cacccaatac gccggccgaa acttttttac aactctccta tgagtcgttt acccagaatg 6360 cacaggtaca cttgtttaga ggtaatcctt ctttctagaa gtcctcgtgt actgtgtaag 6420 cgcccactcc acatctccac tcgatggtac ccaattcgcc ctatagtgag tcgtattaaa 6480 accccgggta cgtaaattaa ccctcactaa agggaacaaa agctgggcaa agcttgaggc 6540 cggacaaatt cagcctgcag gctaaagttt ctattacatg atatttgtcg tggacaaggg 6600 cgtctggggt aaaaaggaat gtgggcagag agctctcggc ttaaagaccg gcttcggagt 6660 tccagaccca ctcgaactga aaacgatgat tagagataag tagatctatt tggacagtat 6720 aagaatccta gtcggctagt atttctccac ttttacttta aaattcccca gagtaagggg 6780 gcaggttcgt cgtcacgggt agcctcgtct cgaggcatcg cggatagcat tatcttgggg 6840 tatcacgagt aacttcatcg tggcattatc gataggtact gtaagtactg taggaactta 6900 tccgggcatg gatatgtata ggtacatacg atgcactaaa agtggagaaa tactagccga 6960 gtagagatct tataccatcc gaatagattt actccttata atcaaggggc aacaatcagt 7020 tgtgacgatg tcggctgtgg gagtgcagtt taggggtcat caaagcacag gaaaacgtgt 7080 tgtatggatg cctatcgtct tgactattag aaaggaagag gggaaaaacc ctcatcagga 7140 gcgattgtaa gttcggccat agtaatgata ttggccatta atggacgtgt gtatgtatac 7200 atacacggtt catttgccgc attcggacca aaaccgctgc tgccgcagac acagtgcttc 7260 catggcctcg aacaaccggt tattcttctc ttccatgtcc tcgggacgaa ggaaatttcg 7320 gagccctaac agccgtaccg gacttgcgtt aacatggttg cgaagaacta aagactgatg 7380 gggtcatgat tggcagtgtt gacacttcat gtcttgaacc tagctagcag aaaaatgaat 7440 gctctatgtg acactggtag atctcaacgc atcatgttcc acctttagtg taaacacggc 7500 aagctgtccg caacgtcggt accaacgttt gaacacacaa ccttctgcca ctccaggagt 7560 ttagcccacg gcttactgga tgaaacgtaa tctcccgcta ccccctcaat tccattgggg 7620 tctagagtag tcggaagaac ggctgtcgcc ttgcgctgcg gttaaagcac ccaccgtggt 7680 ggctactgct tctcgggagg agaaatcctg ggtactcgag cacttgcgct aggggatgaa 7740 acatccatgg catattggcg aaagcaatgc gccatgttcg gcgcggcaca tgacactctt 7800 gagaagccgt attcttcgca aactttccta tttacattca gcctctaact ataacacgat 7860 cggaaatgcc tgaacgagcc cgccctcatg tgcctagcgt caaacaacgc caagcacaca 7920 acgcctcctg ctattcttcc ctcttctcac tgccatcact gggcgcaagc tgcaatgtcc 7980 ttctcgaatt caagcgggca aatcaagatt gggaaaatgc gccatcttgg tttggatctt 8040 gatgtctatc cgcatcaagg tcttggtttg gtcgtttatt tagtagcaaa ggcaaatcaa 8100 aacgtgcgcg ccaagtgcca gggtccagac tccagactgg agcatttagc ctgaatatcg 8160 tctctagaat ctggtgctat tattcaggat at 8192 <210> 18 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE5 vector <400> 18 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 60 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 120 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 180 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa 240 caaaagctgg gcaaagctgt ttaattaacc ctcactaaag ggaacaaaag ctggagctcc 300 accgcggtgg cggccgccct tgtatctcta cacacaggct caaatcaata agaagaacgg 360 ttcgtctttt tcgtttatat cttgcatcgt cccaaagcta ttggcgggat attctgtttg 420 cagttggctg acttgaagta atctctgcag atctttcgac actgaaatac gtcgagcctg 480 ctccgcttgg aagcggcgag gagcctcgtc ctgtcacaac taccaacatg gagtacgata 540 agggccagtt ccgccagctc attaagagcc agttcatggg cgttggcatg atggccgtca 600 tgcatctgta cttcaagtac accaacgctc ttctgatcca gtcgatcatc cgctgaaggc 660 gctttcgaat ctggttaaga tccacgtctt cgggaagcca gcgactggtg acctccagcg 720 tccctttaag gctgccaaca gctttctcag ccagggccag cccaagaccg acaaggcctc 780 cctccagaac gccgagaaga actggagggg tggtgtcaag gaggagtaag ctccttattg 840 aagtcggagg acggagcggt gtcaagagga tattcttcga ctctgtatta tagataagat 900 gatgaggaat tggaggtagc atagcttcat ttggatttgc tttccaggct gagactctag 960 cttggagcat agagggtcct ttggctttca atattctcaa gtatctcgag tttgaactta 1020 ttccctgtga accttttatt caccaatgag cattggaatg aacatgaatc tgaggactgc 1080 aatcgccatg aggttttcga aatacatccg gatgtcgaag gcttggggca cctgcgttgg 1140 ttgaatttag aacgtggcac tattgatcat ccgatagctc tgcaaagggc gttgcacaat 1200 gcaagtcaaa cgttgctagc agttccaggt ggaatgttat gatgagcatt gtattaaatc 1260 aggagatata gcatgatctc tagttagctc accacaaaag tcagacggcg taaccaaaag 1320 tcacacaaca caagctgtaa ggatttcggc acggctacgg aagacggaga agccaccttc 1380 agtggactcg agtaccattt aattctattt gtgtttgatc gagacctaat acagccccta 1440 caacgaccat caaagtcgta tagctaccag tgaggaagtg gactcaaatc gacttcagca 1500 acatctcctg gataaacttt aagcctaaac tatacagaat aagataggtg gagagcttat 1560 accgagctcc caaatctgtc cagatcatgg ttgaccggtg cctggatctt cctatagaat 1620 catccttatt cgttgaccta gctgattctg gagtgaccca gagggtcatg acttgagcct 1680 aaaatccgcc gcctccacca tttgtagaaa aatgtgacga actcgtgagc tctgtacagt 1740 gaccggtgac tctttctggc atgcggagag acggacggac gcagagagaa gggctgagta 1800 ataagccact ggccagacag ctctggcggc tctgaggtgc agtggatgat tattaatccg 1860 ggaccggccg cccctccgcc ccgaagtgga aaggctggtg tgcccctcgt tgaccaagaa 1920 tctattgcat catcggagaa tatggagctt catcgaatca ccggcagtaa gcgaaggaga 1980 atgtgaagcc aggggtgtat agccgtcggc gaaatagcat gccattaacc taggtacaga 2040 agtccaattg cttccgatct ggtaaaagat tcacgagata gtaccttctc cgaagtaggt 2100 agagcgagta cccggcgcgt aagctcccta attggcgaat tcccatccgg catctgtagg 2160 gcgtccaaat atcgtgcctc tcctgctttg ccaggtgtat gaaaccggaa aggccgctca 2220 ggagctggcc agcggcgcag accgggaaca caagctggca gtcgacccat ccggtgctct 2280 gcactcgacc tgctgaggtc cctcagtccc tggtaggcag ctttgccccg tctgtccgcc 2340 cggtgtgtcg gcggggttga caaggtcgtt gcgtcagtcc aacatttgtt gccatatttt 2400 cctgctttcc ccaccagctg ctcttttctt ttctctttct tttcccatct tcagtatatt 2460 catcttccca tatgcatcca agaaccttta tttcccctaa gtaagtactt tgctacatcc 2520 atactccatc cttcccatcc cttattcctt tgaacctttc agttcgagct ttcccacttc 2580 atcgcagctt gactaacagc taccccgctt gagcagacat caccatgtca agcgccatca 2640 tcgccgaaat cccggtgatc gacctgggcc cattcggaga cggggacgcg ccgggggcgg 2700 cggaagtggc cgcggcattc gaccgggcct gcagggacct ggggttcgtg gtcgtcgtgg 2760 ggcacggcgt gccccaggcc gtcatcgacg ccgcgcaccg gagtgcccgc gcgttctttg 2820 acctggacct cgctacccgc gagcggtacg cggccccgcc cggcgggttc gtcgggtacc 2880 gcgggctcgg agccgagggg ctgtcttaca gtctggacca ggaggctgcg ccggacttca 2940 aggagacata taccgtcgga cgcatcgatc gcggagacga gccttacttc acgtcccagc 3000 tggggcggat gtacatgccg gaccctacgt ggccggcgga ggtccccgag ttcgcagcgt 3060 cgtgggccga gttctatcgc cagatggacc gggtcgcctg gcgcctgatg cgcgcgttcg 3120 ccaccgcgct ggccctcccc cgcgagttct tcgacgacaa gattgatcgg aacatctcgt 3180 gcctgcgcgc gctgaactac ccccacccgt ccacaccccc ccagcccggc cagctgcgcg 3240 cgggcgccca cacggactac ggctccctca cgctgctcag catggccgac gcgcccgggg 3300 gcctcgaggt gcagcgggac gacgggtcgt gggcgccggt ccgggtcccg gcagacgcct 3360 tcgtgatgaa cgtgggcgac cttatggcgc agtggaccaa cgaccgttgg cgctcgtcga 3420 tgcaccgcgt gacgaatccc cccccggacg cgatcggcga cacgcggcgg cagagcctgg 3480 cgttcttcca ccagcccaac tatgacgccg aggtcatccc gctcgcgtcc tgctgcggcc 3540 cggccaaccc cccgaagtac gcccgcacca cgagcggcga gcacctgttc atgaagatga 3600 ccaaggcgaa gaacctgaac gtctgataaa agcttatcga taccgtcgac ctcgagactt 3660 aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 3720 gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 3780 caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 3840 gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 3900 aacctagtaa cgcctttcag gctccggcga agagaagaat agcttagcag agctattttc 3960 attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 4020 atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 4080 cttctttact ctgatagctt gactatgaaa attccgtcac cagcccctgg gttcgcaaag 4140 ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 4200 taaacctcga aatcagttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 4260 agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 4320 tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 4380 tcgtgtactg tgtaagcgcc cactccacat ctccactcga tggtacccaa ttcgccctat 4440 agtgagtcgt attaaaaccc cgggtacgta aattaaccct cactaaaggg aacaaaagct 4500 gggcaaagct tgaggccgga caaattcagc ctgcaggcta aagtttctat tacatgatat 4560 ttgtcgtgga caagggcgtc tggggtaaaa aggaatgtgg gcagagagct ctcggcttaa 4620 agaccggctt cggagttcca gacccactcg aactgaaaac gatgattaga gataagtaga 4680 tctatttgga cagtataaga atcctagtcg gctagtattt ctccactttt actttaaaat 4740 tccccagagt aagggggcag gttcgtcgtc acgggtagcc tcgtctcgag gcatcgcgga 4800 tagcattatc ttggggtatc acgagtaact tcatcgtggc attatcgata ggtactgtaa 4860 gtactgtagg aacttatccg ggcatggata tgtataggta catacgatgc actaaaagtg 4920 gagaaatact agccgagtag agatcttata ccatccgaat agatttactc cttataatca 4980 aggggcaaca atcagttgtg acgatgtcgg ctgtgggagt gcagtttagg ggtcatcaaa 5040 gcacaggaaa acgtgttgta tggatgccta tcgtcttgac tattagaaag gaagagggga 5100 aaaaccctca tcaggagcga ttgtaagttc ggccatagta atgatattgg ccattaatgg 5160 acgtgtgtat gtatacatac acggttcatt tgccgcattc ggaccaaaac cgctgctgcc 5220 gcagacacag tgcttccatg gcctcgaaca accggttatt cttctcttcc atgtcctcgg 5280 gacgaaggaa atttcggagc cctaacagcc gtaccggact tgcgttaaca tggttgcgaa 5340 gaactaaaga ctgatggggt catgattggc agtgttgaca cttcatgtct tgaacctagc 5400 tagcagaaaa atgaatgctc tatgtgacac tggtagatct caacgcatca tgttccacct 5460 ttagtgtaaa cacggcaagc tgtccgcaac gtcggtacca acgtttgaac acacaacctt 5520 ctgccactcc aggagtttag cccacggctt actggatgaa acgtaatctc ccgctacccc 5580 ctcaattcca ttggggtcta gagtagtcgg aagaacggct gtcgccttgc gctgcggtta 5640 aagcacccac cgtggtggct actgcttctc gggaggagaa atcctgggta ctcgagcact 5700 tgcgctaggg gatgaaacat ccatggcata ttggcgaaag caatgcgcca tgttcggcgc 5760 ggcacatgac actcttgaga agccgtattc ttcgcaaact ttcctattta cattcagcct 5820 ctaactataa cacgatcgga aatgcctgaa cgagcccgcc ctcatgtgcc tagcgtcaaa 5880 caacgccaag cacacaacgc ctcctgctat tcttccctct tctcactgcc atcactgggc 5940 gcaagctgca atgtccttct cgaattcaag cgggcaaatc aagattggga aaatgcgcca 6000 tcttggtttg gatcttgatg tctatccgca tcaaggtctt ggtttggtcg tttatttagt 6060 agcaaaggca aatcaaaacg tgcgcgccaa gtgccagggt ccagactcca gactggagca 6120 tttagcctga atatcgtctc tagaatctgg tgctattatt caggatatag taactatgat 6180 ctactcgggc catgttcggc tcctccgcac ggccaagttg tctgccggcg tctggaccgg 6240 actcatggct tggcttctcc agggatagca tggcattggc aaaactctcc gccacgattc 6300 gagcatagct accgcagcca cgcggcattg tttccagatt gagatatgtc actattagcc 6360 ggaatgcaaa catcgagctg acctgcgcaa catctccatg agttccgaag tcgacctccg 6420 aagcaatatt ggtctagtat tacggatgat atagtggtcc ccggacccaa tgagccgtgt 6480 gttttttacc gcctttgctc tgaggattaa caagattcgt gcaattgggg actgtgcgga 6540 gtaactgggc tggtttactg ctttgccgac gaaagaaacg tataaacagc cgactcgtcc 6600 actgccactc gggacgatct cccaacccag acagcttccc agaacccatc ttcaacaccc 6660 tgacaccatg ccccagttcg acatcctatg caagactcct ccgaaggtgc tcgtccgcca 6720 gttcgtggag cgattcgagc gcccctcggg cgaaaagatc gccttgtgtg ccgctgagct 6780 tacctacctc tgctggatga tcacccacaa cggcaccgcc atcaagcggg ctacctttat 6840 gagttacaat accatcatta gcaactccct ctcgtttgac atcgtcaaca agtccctgca 6900 gttcaagtac aagacccaaa aggccaccat cctcgaggcc tcgctgaaga agctgatccc 6960 cgcatgggaa ttcactatca tcccctacta tggtcagaag catcagtctg acatcaccga 7020 cattgtaagc agtctccagc tccagtttga gtcctccgag gaggccgata aaggcaactc 7080 ccactccaag aagatgctca aggctttgct gagtgaaggc gagtccatct gggaaataac 7140 cgaaaagatc ttgaacagtt tcgagtacac cagccgcttc acgaagacca agacgctgta 7200 ccagttcctg ttcctcgcca cattcatcaa ttgcggacgc ttcagtgata tcaagaacgt 7260 ggacccgaag tcattcaagc tggttcagaa caagtacctc ggtgtcatca ttcagtgctt 7320 ggtcaccgag accaagacat ccgtcagccg tcacatctac ttcttttccg caagagggcg 7380 tatcgacccc ctagtgtacc tagacgaatt cctgcgcaac tcggagcccg tcctaaagcg 7440 cgtcaaccgt accggcaact ctagcagcaa caagcaggag taccagctct tgaaggataa 7500 cctcgtccga tcatacaaca aggcgctcaa gaagaacgct ccatacagca tctttgctat 7560 caagaatggg ccaaagtcac acatcggccg ccacctcatg acctcgtttc tgtcgatgaa 7620 gggcctcact gagcttacca acgttgttgg caactggtcc gataagcgcg ccagcgccgt 7680 ggcgcggacg acttacactc atcagatcac agctatccct gaccactact tcgccttggt 7740 cagtcgctat tacgcctacg acccaattag taaagagatg atcgctctca aggacgaaac 7800 caaccccatc gaggagtggc aacacatcga gcagcttaaa ggctccgccg agggctcgat 7860 ccggtacccc gcctggaacg gtatcatatc gcaggaggtc ttggattacc tgagcagtta 7920 tatcaatcgc cgcatatgat aaggatccac ttaacgttac tgaaatcatc aaacagcttg 7980 acgaatctgg atataagatc gttggtgtcg atgtcagctc cggagttgag acaaatggtg 8040 ttcaggatct cgataagata cgttcatttg tccaagcagc aaagagtgcc ttctagtgat 8100 ttaatagctc catgtcaaca agaataaaac gcgttttcgg gtttacctct tccagataca 8160 gctcatctgc aatgcattaa tgcattgact gc 8192 <210> 19 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE6 vector <400> 19 taagccactg gccagacagc tctggcggct ctgaggtgca gtggatgatt attaatccgg 60 gaccggccgc ccctccgccc cgaagtggaa aggctggtgt gcccctcgtt gaccaagaat 120 ctattgcatc atcggagaat atggagcttc atcgaatcac cggcagtaag cgaaggagaa 180 tgtgaagcca ggggtgtata gccgtcggcg aaatagcatg ccattaacct aggtacagaa 240 gtccaattgc ttccgatctg gtaaaagatt cacgagatag taccttctcc gaagtaggta 300 gagcgagtac ccggcgcgta agctccctaa ttggcccatc cggcatctgt agggcgtcca 360 aatatcgtgc ctctcctgct ttgcccggtg tatgaaaccg gaaaggccgc tcaggagctg 420 gccagcggcg cagaccggga acacaagctg gcagtcgacc catccggtgc tctgcactcg 480 acctgctgag gtccctcagt ccctggtagg cagctttgcc ccgtctgtcc gcccggtgtg 540 tcggcggggt tgacaaggtc gttgcgtcag tccaacattt gttgccatat tttcctgctc 600 tccccaccag ctgctctttt cttttctctt tcttttccca tcttcagtat attcatcttc 660 ccatccaaga acctttattt cccctaagta agtactttgc tacatccata ctccatcctt 720 cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc gcagcttgac 780 taacagctac cccgcttgag cagacatcac catgcctgaa ctcaccgcga cgtctgtcga 840 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga 900 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag 960 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct 1020 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc 1080 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct 1140 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg 1200 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg 1260 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc 1320 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg 1380 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac 1440 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat 1500 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag 1560 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga 1620 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg 1680 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag 1740 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg 1800 ccccagcact cgtccgaggg caaaggaata gagtagatgc cgaccgcggg atccacttaa 1860 cgttactgaa atcatcaaac agcttgacga atctggatat aagatcgttg gtgtcgatgt 1920 cagctccgga gttgagacaa atggtgttca ggatctcgat aagatacgtt catttgtcca 1980 agcagcaaag agtgccttct agtgatttaa tagctccatg tcaacaagaa taaaacgcgt 2040 tttcgggttt acctcttcca gatacagctc atctgcaatg cattaatgca ttgactgcaa 2100 cctagtaacg ccttncaggc tccggcgaag agaagaatag cttagcagag ctattttcat 2160 tttcgggaga cgagatcaag cagatcaacg gtcgtcaaga gacctacgag actgaggaat 2220 ccgctcttgg ctccacgcga ctatatattt gtctctaatt gtactttgac atgctcctct 2280 tctttactct gatagcttga ctatgaaaat tccgtcacca gcncctgggt tcgcaaagat 2340 aattgcatgt ttcttccttg aactctcaag cctacaggac acacattcat cgtaggtata 2400 aacctcgaaa tcanttccta ctaagatggt atacaatagt aaccatgcat ggttgcctag 2460 tgaatgctcc gtaacaccca atacgccggc cgaaactttt ttacaactct cctatgagtc 2520 gtttacccag aatgcacagg tacacttgtt tagaggtaat ccttctttct agaagtcctc 2580 gtgtactgtg taagcgccca ctccacatct ccactcgacc tgcagttcga agttcctatt 2640 ctctagaaag tataggaact tcagagcgct ttccaattcg ccctatagtg agtcgtatta 2700 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 2760 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 2820 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag 2880 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 2940 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 3000 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 3060 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 3120 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 3180 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 3240 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 3300 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct 3360 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 3420 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3480 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3540 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3600 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3660 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3720 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3780 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3840 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3900 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 3960 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 4020 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 4080 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 4140 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 4200 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 4260 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 4320 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 4380 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4440 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4500 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4560 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4620 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4680 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4740 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4800 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4860 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4920 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4980 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5040 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5100 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5160 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5220 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5280 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5340 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5400 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5460 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa 5520 aagctgggca aagctgttta attaaccctc actaaaggga acaaaagctg gagctccacc 5580 gcggtggcgg ccgcccttgt atctctacac acaggctcaa atcaataaga agaacggttc 5640 gtctttttcg tttatatctt gcatcgtccc aaagctattg gcgggatatt ctgtttgcag 5700 ttggctgact tgaagtaatc tctgcagatc tttcgacact gaaatacgtc gagcctgctc 5760 cgcttggaag cggcgaggag cctcgtcctg tcacaactac caacatggag tacgataagg 5820 gccagttccg ccagctcatt aagagccagt tcatgggcgt tggcatgatg gccgtcatgc 5880 atctgtactt caagtacacc aacgctcttc tgatccagtc gatcatccgc tgaaggcgct 5940 ttcgaatctg gttaagatcc acgtcttcgg gaagccagcg actggtgacc tccagcgtcc 6000 ctttaaggct gccaacagct ttctcagcca gggccagccc aagaccgaca aggcctccct 6060 ccagaacgcc gagaagaact ggaggggtgg tgtcaaggag gagtaagctc cttattgaag 6120 tcggaggacg gagcggtgtc aagaggatat tcttcgactc tgtattatag ataagatgat 6180 gaggaattgg aggtagcata gcttcatttg gatttgcttt ccaggctgag actctagctt 6240 ggagcataga gggtcctttg gctttcaata ttctcaagta tctcgagttt gaacttattc 6300 cctgtgaacc ttttattcac caatgagcat tggaatgaac atgaatctga ggactgcaat 6360 cgccatgagg ttttcgaaat acatccggat gtcgaaggct tggggcacct gcgttggttg 6420 aatttagaac gtggcactat tgatcatccg atagctctgc aaagggcgtt gcacaatgca 6480 agtcaaacgt tgctagcagt tccaggtgga atgttatgat gagcattgta ttaaatcagg 6540 agatatagca tgatctctag ttagctcacc acaaaagtca gacggcgtaa ccaaaagtca 6600 cacaacacaa gctgtaagga tttcggcacg gctacggaag acggagaagc caccttcagt 6660 ggactcgagt accatttaat tctatttgtg tttgatcgag acctaataca gcccctacaa 6720 cgaccatcaa agtcgtatag ctaccagtga ggaagtggac tcaaatcgac ttcagcaaca 6780 tctcctggat aaactttaag cctaaactat acagaataag ataggtggag agcttatacc 6840 gagctcccaa atctgtccag atcatggttg accggtgcct ggatcttcct atagaatcat 6900 ccttattcgt tgacctagct gattctggag tgacccagag ggtcatgact tgagcctaaa 6960 atccgccgcc tccaccattt gtagaaaaat gtgacgaact cgtgagctct gtacagtgac 7020 cggtgactct ttctggcatg cggagagacg gacggacgca gagagaaggg ctgagtaata 7080 agccactggc cagacagctc tggcggctct gaggtgcagt ggatgattat taatccggga 7140 ccggccgccc ctccgccccg aagtggaaag gctggtgtgc ccctcgttga ccaagaatct 7200 attgcatcat cggagaatat ggagcttcat cgaatcaccg gcagtaagcg aaggagaatg 7260 tgaagccagg ggtgtatagc cgtcggcgaa atagcatgcc attaacctag gtacagaagt 7320 ccaattgctt ccgatctggt aaaagattca cgagatagta ccttctccga agtaggtaga 7380 gcgagtaccc ggcgcgtaag ctccctaatt ggcgaattcc catccggcat ctgtagggcg 7440 tccaaatatc gtgcctctcc tgctttgcca ggtgtatgaa accggaaagg ccgctcagga 7500 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 7560 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 7620 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 7680 gctttcccca ccagctgctc ttttcttttc tctttctttt cccatcttca gtatattcat 7740 cttcccatat gcatccaaga acctttattt cccctaagta agtactttgc tacatccata 7800 ctccatcctt cccatccctt attcctttga acctttcagt tcgagctttc ccacttcatc 7860 gcagcttgac taacagctac cccgcttgag cagacatcac catgaccctc cagtacgtcc 7920 ccatcatcaa cctggcgccc tatttctcgg gcgagccgga cggcaaggcg gcggtcgccc 7980 aggcggtgaa ccaggcgtgc aaggacatcg gcttcctcgt catcaccgag caccagatcc 8040 caaccgagct gatcgaccgc gtctcgcgcc tcacgcggca attcttcgat ctgccgctcg 8100 ccgaaaagag gaaggtcgac cgcccttcgc cggagatggt ccgggggtat tccgccgtgg 8160 cggaggagtc gctgtcctac tcgctcgagg ag 8192 <210> 20 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE7 vector <400> 20 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaattaa 2220 ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgcc cttgtatctc 2280 tacacacagg ctcaaatcaa taagaagaac ggttcgtctt tttcgtttat atcttgcatc 2340 gtcccaaagc tattggcggg atattctgtt tgcagttggc tgacttgaag taatctctgc 2400 agatctttcg acactgaaat acgtcgagcc tgctccgctt ggaagcggcg aggagcctcg 2460 tcctgtcaca actaccaaca tggagtacga taagggccag ttccgccagc tcattaagag 2520 ccagttcatg ggcgttggca tgatggccgt catgcatctg tacttcaagt acaccaacgc 2580 tcttctgatc cagtcgatca tccgctgaag gcgctttcga atctggttaa gatccacgtc 2640 ttcgggaagc cagcgactgg tgacctccag cgtcccttta aggctgccaa cagctttctc 2700 agccagggcc agcccaagac cgacaaggcc tccctccaga acgccgagaa gaactggagg 2760 ggtggtgtca aggaggagta agctccttat tgaagtcgga ggacggagcg gtgtcaagag 2820 gatattcttc gactctgtat tatagataag atgatgagga attggaggta gcatagcttc 2880 atttggattt gctttccagg ctgagactct agcttggagc atagagggtc ctttggcttt 2940 caatattctc aagtatctcg agtttgaact tattccctgt gaacctttta ttcaccaatg 3000 agcattggaa tgaacatgaa tctgaggact gcaatcgcca tgaggttttc gaaatacatc 3060 cggatgtcga aggcttgggg cacctgcgtt ggttgaattt agaacgtggc actattgatc 3120 atccgatagc tctgcaaagg gcgttgcaca atgcaagtca aacgttgcta gcagttccag 3180 gtggaatgtt atgatgagca ttgtattaaa tcaggagata tagcatgatc tctagttagc 3240 tcaccacaaa agtcagacgg cgtaaccaaa agtcacacaa cacaagctgt aaggatttcg 3300 gcacggctac ggaagacgga gaagccacct tcagtggact cgagtaccat ttaattctat 3360 ttgtgtttga tcgagaccta atacagcccc tacaacgacc atcaaagtcg tatagctacc 3420 agtgaggaag tggactcaaa tcgacttcag caacatctcc tggataaact ttaagcctaa 3480 actatacaga ataagatagg tggagagctt ataccgagct cccaaatctg tccagatcat 3540 ggttgaccgg tgcctggatc ttcctataga atcatcctta ttcgttgacc tagctgattc 3600 tggagtgacc cagagggtca tgacttgagc ctaaaatccg ccgcctccac catttgtaga 3660 aaaatgtgac gaactcgtga gctctgtaca gtgaccggtg actctttctg gcatgcggag 3720 agacggacgg acgcagagag aagggctgag taataagcca ctggccagac agctctggcg 3780 gctctgaggt gcagtggatg attattaatc cgggaccggc cgcccctccg ccccgaagtg 3840 gaaaggctgg tgtgcccctc gttgaccaag aatctattgc atcatcggag aatatggagc 3900 ttcatcgaat caccggcagt aagcgaagga gaatgtgaag ccaggggtgt atagccgtcg 3960 gcgaaatagc atgccattaa cctaggtaca gaagtccaat tgcttccgat ctggtaaaag 4020 attcacgaga tagtaccttc tccgaagtag gtagagcgag tacccggcgc gtaagctccc 4080 taattggcga attcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt 4140 tgccaggtgt atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa 4200 cacaagctgg cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc 4260 cctggtaggc agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg 4320 ttgcgtcagt ccaacatttg ttgccatatt ttcctgcttt ccccaccagc tgctcttttc 4380 ttttctcttt cttttcccat cttcagtata ttcatcttcc catatgcatc caagaacctt 4440 tatttcccct aagtaagtac tttgctacat ccatactcca tccttcccat cccttattcc 4500 tttgaacctt tcagttcgag ctttcccact tcatcgcagc ttgactaaca gctaccccgc 4560 ttgagcagac atcaccatgg acaccaccgt gccgaccttc tccctcgccg agctccagca 4620 gggtctgcac caggacgagt tccggcggtg tctgcgcgac aaggggctgt tctacctgac 4680 cgactgcggc ctaacagaca cggagctcaa gtccgccaag gatatcgtca tcgacttttt 4740 cgagcacggg agcgaggccg agaagcgggc ggtcaccagc cccgtcccga cgatgcgccg 4800 cggcttcacc ggtctggagt ccgagtcgac cgcccaaatc accaacaccg gatcctactc 4860 cgactacagc atgtgttact ccatggggac ggccgacaac ctcttcccct ccggggattt 4920 cgagcgcatc tggacccagt acttcgaccg ccagtacacc gccagcaggg ccgtggccag 4980 ggaggtcctg cgcgccaccg ggacggaacc ggacggcggg gtggaggcct tcctggactg 5040 cgaaccgctt ctgcggttcc ggtacttccc ccaggtcccc gagcatcggt ccgccgagga 5100 gcagccgctc cggatggccc cccactacga cctgtccatg gtgaccctca tccagcagac 5160 cccgtgcgca aacgggttcg tgtcgctcca ggccgaggtg ggcggcgcgt tcacagacct 5220 cccttaccga cccgacgcgg tgttggtctt ctgcggcgcc atcgccaccc tcgtgacggg 5280 cggccaggtc aaggcccccc ggcaccacgt ggccgccccg cggcgtgacc agatcgccgg 5340 aagcagccgc acgtcgtccg tgttcttcct gcgcccgaac gcggacttca cattctccgt 5400 ccccctcgcg cgggagtgcg gcttcgacgt ctccctggac ggggagaccg ccacgttcca 5460 ggactggatc ggcggcaact acgtcaacat ccggcggacc tcgaaggcgt agtaaaagct 5520 tatcgatacc gtcgacctcg agacttaacg ttactgaaat catcaaacag cttgacgaat 5580 ctggatataa gatcgttggt gtcgatgtca gctccggagt tgagacaaat ggtgttcagg 5640 atctcgataa gatacgttca tttgtccaag cagcaaagag tgccttctag tgatttaata 5700 gctccatgtc aacaagaata aaacgcgttt tcgggtttac ctcttccaga tacagctcat 5760 ctgcaatgca ttaatgcatt gactgcaacc tagtaacgcc tttcaggctc cggcgaagag 5820 aagaatagct tagcagagct attttcattt tcgggagacg agatcaagca gatcaacggt 5880 cgtcaagaga cctacgagac tgaggaatcc gctcttggct ccacgcgact atatatttgt 5940 ctctaattgt actttgacat gctcctcttc tttactctga tagcttgact atgaaaattc 6000 cgtcaccagc ccctgggttc gcaaagataa ttgcatgttt cttccttgaa ctctcaagcc 6060 tacaggacac acattcatcg taggtataaa cctcgaaatc agttcctact aagatggtat 6120 acaatagtaa ccatgcatgg ttgcctagtg aatgctccgt aacacccaat acgccggccg 6180 aaactttttt acaactctcc tatgagtcgt ttacccagaa tgcacaggta cacttgttta 6240 gaggtaatcc ttctttctag aagtcctcgt gtactgtgta agcgcccact ccacatctcc 6300 actcgatggt acccaattcg ccctatagtg agtcgtatta aaaccccggg tacgtaaatt 6360 aaccctcact aaagggaaca aaagctgggc aaagcttgag gccggacaaa ttcagcctgc 6420 aggctaaagt ttctattaca tgatatttgt cgtggacaag ggcgtctggg gtaaaaagga 6480 atgtgggcag agagctctcg gcttaaagac cggcttcgga gttccagacc cactcgaact 6540 gaaaacgatg attagagata agtagatcta tttggacagt ataagaatcc tagtcggcta 6600 gtatttctcc acttttactt taaaattccc cagagtaagg gggcaggttc gtcgtcacgg 6660 gtagcctcgt ctcgaggcat cgcggatagc attatcttgg ggtatcacga gtaacttcat 6720 cgtggcatta tcgataggta ctgtaagtac tgtaggaact tatccgggca tggatatgta 6780 taggtacata cgatgcacta aaagtggaga aatactagcc gagtagagat cttataccat 6840 ccgaatagat ttactcctta taatcaaggg gcaacaatca gttgtgacga tgtcggctgt 6900 gggagtgcag tttaggggtc atcaaagcac aggaaaacgt gttgtatgga tgcctatcgt 6960 cttgactatt agaaaggaag aggggaaaaa ccctcatcag gagcgattgt aagttcggcc 7020 atagtaatga tattggccat taatggacgt gtgtatgtat acatacacgg ttcatttgcc 7080 gcattcggac caaaaccgct gctgccgcag acacagtgct tccatggcct cgaacaaccg 7140 gttattcttc tcttccatgt cctcgggacg aaggaaattt cggagcccta acagccgtac 7200 cggacttgcg ttaacatggt tgcgaagaac taaagactga tggggtcatg attggcagtg 7260 ttgacacttc atgtcttgaa cctagctagc agaaaaatga atgctctatg tgacactggt 7320 agatctcaac gcatcatgtt ccacctttag tgtaaacacg gcaagctgtc cgcaacgtcg 7380 gtaccaacgt ttgaacacac aaccttctgc cactccagga gtttagccca cggcttactg 7440 gatgaaacgt aatctcccgc taccccctca attccattgg ggtctagagt agtcggaaga 7500 acggctgtcg ccttgcgctg cggttaaagc acccaccgtg gtggctactg cttctcggga 7560 ggagaaatcc tgggtactcg agcacttgcg ctaggggatg aaacatccat ggcatattgg 7620 cgaaagcaat gcgccatgtt cggcgcggca catgacactc ttgagaagcc gtattcttcg 7680 caaactttcc tatttacatt cagcctctaa ctataacacg atcggaaatg cctgaacgag 7740 cccgccctca tgtgcctagc gtcaaacaac gccaagcaca caacgcctcc tgctattctt 7800 ccctcttctc actgccatca ctgggcgcaa gctgcaatgt ccttctcgaa ttcaagcggg 7860 caaatcaaga ttgggaaaat gcgccatctt ggtttggatc ttgatgtcta tccgcatcaa 7920 ggtcttggtt tggtcgttta tttagtagca aaggcaaatc aaaacgtgcg cgccaagtgc 7980 cagggtccag actccagact ggagcattta gcctgaatat cgtctctaga atctggtgct 8040 attattcagg atatagtaac tatgatctac tcgggccatg ttcggctcct ccgcacggcc 8100 aagttgtctg ccggcgtctg gaccggactc atggcttggc ttctccaggg atagcatggc 8160 attggcaaaa ctctccgcca cgattcgagc at 8192 <210> 21 <211> 8192 <212> DNA <213> Artificial Sequence <220> <223> pB-HFE8 vector <400> 21 cgcgactata tatttgtctc taattgtact ttgacatgct cctcttcttt actctgatag 60 cttgactatg aaaattccgt caccagcncc tgggttcgca aagataattg catgtttctt 120 ccttgaactc tcaagcctac aggacacaca ttcatcgtag gtataaacct cgaaatcant 180 tcctactaag atggtataca atagtaacca tgcatggttg cctagtgaat gctccgtaac 240 acccaatacg ccggccgaaa cttttttaca actctcctat gagtcgttta cccagaatgc 300 acaggtacac ttgtttagag gtaatccttc tttctagaag tcctcgtgta ctgtgtaagc 360 gcccactcca catctccact cgacctgcag ttcgaagttc ctattctcta gaaagtatag 420 gaacttcaga gcgctttcca attcgcccta tagtgagtcg tattacgcgc gctcactggc 480 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 540 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 600 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 660 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 720 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 780 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 840 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 900 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 960 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 1020 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 1080 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 1140 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 1200 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 1260 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 1320 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 1380 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 1440 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 1500 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 1560 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 1620 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 1680 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 1740 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 1800 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 1860 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 1920 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 1980 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 2040 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 2100 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 2160 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 2220 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2280 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2340 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 2400 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 2460 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2520 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2580 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2640 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 2700 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 2760 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 2820 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 2880 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 2940 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 3000 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 3060 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 3120 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 3180 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 3240 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct 3300 gtttaattaa ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgcc 3360 cttgtatctc tacacacagg ctcaaatcaa taagaagaac ggttcgtctt tttcgtttat 3420 atcttgcatc gtcccaaagc tattggcggg atattctgtt tgcagttggc tgacttgaag 3480 taatctctgc agatctttcg acactgaaat acgtcgagcc tgctccgctt ggaagcggcg 3540 aggagcctcg tcctgtcaca actaccaaca tggagtacga taagggccag ttccgccagc 3600 tcattaagag ccagttcatg ggcgttggca tgatggccgt catgcatctg tacttcaagt 3660 acaccaacgc tcttctgatc cagtcgatca tccgctgaag gcgctttcga atctggttaa 3720 gatccacgtc ttcgggaagc cagcgactgg tgacctccag cgtcccttta aggctgccaa 3780 cagctttctc agccagggcc agcccaagac cgacaaggcc tccctccaga acgccgagaa 3840 gaactggagg ggtggtgtca aggaggagta agctccttat tgaagtcgga ggacggagcg 3900 gtgtcaagag gatattcttc gactctgtat tatagataag atgatgagga attggaggta 3960 gcatagcttc atttggattt gctttccagg ctgagactct agcttggagc atagagggtc 4020 ctttggcttt caatattctc aagtatctcg agtttgaact tattccctgt gaacctttta 4080 ttcaccaatg agcattggaa tgaacatgaa tctgaggact gcaatcgcca tgaggttttc 4140 gaaatacatc cggatgtcga aggcttgggg cacctgcgtt ggttgaattt agaacgtggc 4200 actattgatc atccgatagc tctgcaaagg gcgttgcaca atgcaagtca aacgttgcta 4260 gcagttccag gtggaatgtt atgatgagca ttgtattaaa tcaggagata tagcatgatc 4320 tctagttagc tcaccacaaa agtcagacgg cgtaaccaaa agtcacacaa cacaagctgt 4380 aaggatttcg gcacggctac ggaagacgga gaagccacct tcagtggact cgagtaccat 4440 ttaattctat ttgtgtttga tcgagaccta atacagcccc tacaacgacc atcaaagtcg 4500 tatagctacc agtgaggaag tggactcaaa tcgacttcag caacatctcc tggataaact 4560 ttaagcctaa actatacaga ataagatagg tggagagctt ataccgagct cccaaatctg 4620 tccagatcat ggttgaccgg tgcctggatc ttcctataga atcatcctta ttcgttgacc 4680 tagctgattc tggagtgacc cagagggtca tgacttgagc ctaaaatccg ccgcctccac 4740 catttgtaga aaaatgtgac gaactcgtga gctctgtaca gtgaccggtg actctttctg 4800 gcatgcggag agacggacgg acgcagagag aagggctgag taataagcca ctggccagac 4860 agctctggcg gctctgaggt gcagtggatg attattaatc cgggaccggc cgcccctccg 4920 ccccgaagtg gaaaggctgg tgtgcccctc gttgaccaag aatctattgc atcatcggag 4980 aatatggagc ttcatcgaat caccggcagt aagcgaagga gaatgtgaag ccaggggtgt 5040 atagccgtcg gcgaaatagc atgccattaa cctaggtaca gaagtccaat tgcttccgat 5100 ctggtaaaag attcacgaga tagtaccttc tccgaagtag gtagagcgag tacccggcgc 5160 gtaagctccc taattggcga attcccatcc ggcatctgta gggcgtccaa atatcgtgcc 5220 tctcctgctt tgccaggtgt atgaaaccgg aaaggccgct caggagctgg ccagcggcgc 5280 agaccgggaa cacaagctgg cagtcgaccc atccggtgct ctgcactcga cctgctgagg 5340 tccctcagtc cctggtaggc agctttgccc cgtctgtccg cccggtgtgt cggcggggtt 5400 gacaaggtcg ttgcgtcagt ccaacatttg ttgccatatt ttcctgcttt ccccaccagc 5460 tgctcttttc ttttctcttt cttttcccat cttcagtata ttcatcttcc catatgcatc 5520 caagaacctt tatttcccct aagtaagtac tttgctacat ccatactcca tccttcccat 5580 cccttattcc tttgaacctt tcagttcgag ctttcccact tcatcgcagc ttgactaaca 5640 gctaccccgc ttgagcagac atcaccatgc atcgcgcggg cggcagcggg ctcgtcccca 5700 gtggccgcat cgccggctat tggcgatgcg agcggccctg gcgcatgcag cgccgatccc 5760 gctgctcgac caaggccctg gtgatgctgg acacccccct ggcgaaggtg ccgaccctca 5820 gtctcgcaga ccaggcagtc gatcccgacc gcttcgccgc cgacctgggc ggttcgttca 5880 ggcggttcgg cttcgccatc gtcgcggacc atggcatccc ggacgacctc atcgcccggg 5940 cctgggcgca gaccgaggcc ctgttcgccc agccggacgc cgacaagcgg ggctactacg 6000 tggaaggcgc gggcggggcc cgcggctaca ccccgttcaa gaccgagatc gcgaaggacg 6060 ccaagcacgt cgacctgaag gagttctggc acgtggggcg agagctgccc acgggccacc 6120 ggtacgcggg tgagatggcc ccgaacatct ggcccgccca gcccgatggc ttcaaggaga 6180 ccttcgtcga gctgttccgc gctttcgaca cggcgggcga caagctcctg agcgccatcg 6240 cccgggacct cgggctggcg ccggactggt tcgacctggc ggtcaaggac ggcaactccg 6300 tcctccgcct gctgcactac cccccgatcc cgatggacgc ggagggcgtc cgcgcgggag 6360 cccatgagga catcaacctg atcacccttc tcctgggtgc cgaggaggct ggactggagc 6420 tgctcgaccg cgacagcggg aattggctgg ccatcaagcc cccggagggc gcgatggtag 6480 tgaacgtcgg ggacatgctg cagcggttga ccaaccacgt cctgccgtcc accacccacc 6540 gcgtcgtcaa tccgcccccg gagcgccggg cccactcccg ctacagcatg cccttctttc 6600 tgcacccggc ccccgacttc ctgatcaaga ccctgcccca gaccgtcacc tcctccaacc 6660 cgaaccggta ccccaccccg atcacggcgc acgactacct ccatgagcgg cttgtggaga 6720 tcggtctcat caagaagtga taaaagctta tcgataccgt cgacctcgag acttaacgtt 6780 actgaaatca tcaaacagct tgacgaatct ggatataaga tcgttggtgt cgatgtcagc 6840 tccggagttg agacaaatgg tgttcaggat ctcgataaga tacgttcatt tgtccaagca 6900 gcaaagagtg ccttctagtg atttaatagc tccatgtcaa caagaataaa acgcgttttc 6960 gggtttacct cttccagata cagctcatct gcaatgcatt aatgcattga ctgcaaccta 7020 gtaacgcctt tcaggctccg gcgaagagaa gaatagctta gcagagctat tttcattttc 7080 gggagacgag atcaagcaga tcaacggtcg tcaagagacc tacgagactg aggaatccgc 7140 tcttggctcc acgcgactat atatttgtct ctaattgtac tttgacatgc tcctcttctt 7200 tactctgata gcttgactat gaaaattccg tcaccagccc ctgggttcgc aaagataatt 7260 gcatgtttct tccttgaact ctcaagccta caggacacac attcatcgta ggtataaacc 7320 tcgaaatcag ttcctactaa gatggtatac aatagtaacc atgcatggtt gcctagtgaa 7380 tgctccgtaa cacccaatac gccggccgaa acttttttac aactctccta tgagtcgttt 7440 acccagaatg cacaggtaca cttgtttaga ggtaatcctt ctttctagaa gtcctcgtgt 7500 actgtgtaag cgcccactcc acatctccac tcgatggtac ccaattcgcc ctatagtgag 7560 tcgtattaaa accccgggta cgtaaattaa ccctcactaa agggaacaaa agctgggcaa 7620 agcttgaggc cggacaaatt cagcctgcag gctaaagttt ctattacatg atatttgtcg 7680 tggacaaggg cgtctggggt aaaaaggaat gtgggcagag agctctcggc ttaaagaccg 7740 gcttcggagt tccagaccca ctcgaactga aaacgatgat tagagataag tagatctatt 7800 tggacagtat aagaatccta gtcggctagt atttctccac ttttacttta aaattcccca 7860 gagtaagggg gcaggttcgt cgtcacgggt agcctcgtct cgaggcatcg cggatagcat 7920 tatcttgggg tatcacgagt aacttcatcg tggcattatc gataggtact gtaagtactg 7980 taggaactta tccgggcatg gatatgtata ggtacatacg atgcactaaa agtggagaaa 8040 tactagccga gtagagatct tataccatcc gaatagattt actccttata atcaaggggc 8100 aacaatcagt tgtgacgatg tcggctgtgg gagtgcagtt taggggtcat caaagcacag 8160 gaaaacgtgt tgtatggatg cctatcgtct tg 8192 <210> 22 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> CefE of Amycolatopsis lactamdurans (genbank PID CAA78376.1) <400> 22 Met Thr Asp Ala Thr Val Pro Thr Phe Asp Leu Ala Glu Leu Arg Glu 1 5 10 15 Gly Leu His Gln Glu Glu Phe Arg His Cys Leu Arg Glu Lys Gly Val 20 25 30 Phe Tyr Leu Lys Gly Thr Gly Leu Pro Ala Glu Ala Asp His Ala Ser 35 40 45 Gly Arg Glu Ile Ala Val Asp Phe Phe Asp His Gly Thr Glu Ala Glu 50 55 60 Lys Lys Ala Val Met Thr Pro Ile Pro Thr Ile Arg Arg Gly Tyr Ala 65 70 75 80 Gly Leu Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Lys Tyr 85 90 95 Thr Asp Tyr Ser Met Ser Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe 100 105 110 Pro Ser Ala Glu Phe Glu Lys Ala Trp Glu Asp Tyr Phe Ala Arg Met 115 120 125 Tyr Arg Ala Ser Gln Asp Val Ala Arg Gln Val Leu Thr Ser Val Gly 130 135 140 Ala Glu Pro Glu Val Gly Met Asp Ala Phe Leu Asp Cys Glu Pro Leu 145 150 155 160 Leu Arg Leu Arg Tyr Phe Pro Glu Val Pro Glu Asp Arg Val Ala Glu 165 170 175 Glu Gln Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Ile Val Thr 180 185 190 Leu Ile His Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Val 195 200 205 Glu Val Asp Gly Ser Tyr Val Asp Ile Pro Ala Gln Pro Gly Ala Val 210 215 220 Leu Val Phe Cys Gly Ala Val Ala Thr Leu Val Ala Asp Gly Ala Ile 225 230 235 240 Lys Ala Pro Lys His His Val Ala Ala Pro Gly Ala Asp Lys Arg Val 245 250 255 Gly Ser Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Gly Asp 260 265 270 Phe Arg Phe Ser Val Pro Arg Ala Arg Glu Cys Gly Phe Asp Val Ser 275 280 285 Ile Pro Ala Glu Thr Ala Thr Phe Asp Asp Trp Ile Gly Gly Asn Tyr 290 295 300 Ile Asn Ile Arg Lys Thr Ala Ala Ala Arg 305 310 <210> 23 <211> 323 <212> PRT <213> Artificial Sequence <220> <223> CefE of Gordonia rubripertincta (genbank PID ASR04298.1) <400> 23 Met Thr Asp Ser Pro Ile Leu Thr Val Asp Leu Ser Arg Trp Arg Ala 1 5 10 15 Gly Gly Ala Asp Ala Ala Ala Val Cys Ala Ala Val Asp Glu Ser Leu 20 25 30 Gln Lys Ala Gly Phe Leu Leu Val Thr Gly His Gly Ile Asp Pro Val 35 40 45 Leu Pro Ala Glu Leu Arg Ala Ala Ala Arg Ala Phe Phe Ala Leu Pro 50 55 60 Ala Glu Val Lys Gln Lys Tyr Ala Val Gly Val Gly Gly Arg Gly Trp 65 70 75 80 Ile Gly Pro Gly Met Glu Ala Asn Ala Tyr Ala Glu Gly Thr Glu Thr 85 90 95 Pro Pro Asp Leu Lys Glu Thr Tyr Asn Ser Gly Ala Gln Thr Pro Val 100 105 110 Gly Val Pro Glu Val Asp Asp Tyr Trp Phe Ala Pro Asp Val Trp Pro 115 120 125 Ile Glu Ala Pro Arg Leu Lys Glu Leu Phe Thr Ala Trp Thr Ala Gln 130 135 140 Cys Lys Thr Leu Ser Asp Asp Leu Leu Ala Leu Met Ala Ala Ser Leu 145 150 155 160 Gly Phe Ala Ala Ala Asp Asn Pro Phe Arg Asp Leu Ala Gly Asn Ala 165 170 175 Thr Trp Thr Ser Asn Ile Asn His Tyr Pro Ala Met Ala Val Val Gly 180 185 190 Glu Pro Glu Pro Gly Gln Tyr Arg Ile Gly Pro His Thr Asp Phe Gly 195 200 205 Thr Val Thr Val Leu Asp Arg Glu Pro Gly Ser Gly Gly Leu Gln Val 210 215 220 Tyr Ser Glu Asp Gly Gly Trp Ala Asp Ala Pro Tyr Asp Pro Ala Ala 225 230 235 240 Leu Thr Val Asn Ile Gly Asp Leu Leu Glu Tyr Trp Ser Gly Gly Arg 245 250 255 Trp Pro Ala Gly Arg His Arg Val Leu Pro Pro Gln Pro Asp Ala Pro 260 265 270 Glu Glu Asp Leu Met Ser Leu Ile Phe Phe Tyr Glu Leu Asp His Asp 275 280 285 Ala Val Val Thr Pro Ile Gly Lys Pro Leu Gly Arg Arg Ala Gly Leu 290 295 300 Glu Pro Val Val Ser Gly Glu Phe Ile Arg Glu Arg Leu Asp Ala Ile 305 310 315 320 Thr Val Gly <210> 24 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> CefE of Mycobacterium abscessus (genbank PID SKX81615.1) <400> 24 Met Thr Asp Ile Gly Glu Pro Glu Pro Gly Gln Tyr Arg Ile Gly Pro 1 5 10 15 His Thr Asp Phe Gly Thr Val Thr Ile Leu Asp Arg Glu Pro Gly Ala 20 25 30 Gly Gly Leu Gln Val Tyr Ser Glu Gln Gly Gly Trp Ala Asp Ala Pro 35 40 45 Tyr Asp Pro Ser Ala Leu Thr Val Asn Ile Gly Asp Leu Leu Glu Tyr 50 55 60 Trp Ser Gly Gly Arg Trp Pro Ala Gly Arg His Arg Val Leu Pro Pro 65 70 75 80 Gln Ala Asp Ala Pro Glu Glu Asp Leu Val Ser Leu Ile Phe Phe Tyr 85 90 95 Glu Leu Asp His Asp Ala Val Val Thr Pro Leu Asp Pro Pro Val Gly 100 105 110 Lys Ile Ala Gly Arg Pro Pro Val Ile Gly Gly Asp Phe Ile Arg Glu 115 120 125 Arg Leu Asp Ala Ile Thr Val Gly 130 135 <210> 25 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> CefE of Microbacterium hydrocarbonoxydans (genbank PID KJL46674.1) <400> 25 Met Ala Leu Pro Pro Val Met Leu Arg Asn Thr Arg Val Ser Pro Arg 1 5 10 15 Met Arg Ile Tyr Ser Arg Val Met Ser Glu Leu Ser Leu Pro Ile Leu 20 25 30 Asp Leu Ser Gln Leu Asp Ala Gly Pro Glu Ala Ala Ala Arg Phe Arg 35 40 45 Asp Asp Leu Arg Ala Ala Thr His Asp Val Gly Phe Phe Tyr Leu Thr 50 55 60 Gly Thr Gly Val Ser Pro Glu Leu Glu Ala Arg Leu His Arg Ala Ala 65 70 75 80 Leu Asp Phe Phe Ala Leu Pro Glu Ala Glu Lys Leu Ala Ile Glu Asn 85 90 95 Ile Asn Ser Pro His Phe Arg Gly Tyr Thr Arg Val Gly Gly Glu Arg 100 105 110 Thr Gln Gly Glu Val Asp Trp Arg Glu Gln Ile Asp Ile Gly Pro Glu 115 120 125 Arg Asp Ala Val Asp Gly Gly Pro Ala Tyr Asn Arg Leu Ile Gly Pro 130 135 140 Asn Leu Trp Pro Ala Ala Gln Pro Glu Leu Lys Glu Val Val Ala Glu 145 150 155 160 Trp His Ser Thr Leu Ser Glu Val Ser Arg Arg Leu Leu Arg Ala Trp 165 170 175 Ala Glu Thr Leu Gly Ala Glu Pro Ser Tyr Phe Asp Glu His Phe Gly 180 185 190 Glu Pro Ser Thr Leu Ile Lys Ile Val Arg Tyr Pro Gly Thr His Glu 195 200 205 Pro Glu Pro Gln Gln Gly Val Gly Ala His Lys Asp Ser Gly Val Leu 210 215 220 Thr Leu Leu Trp Val Glu Pro Gly Lys Gly Gly Leu Gln Val Glu Arg 225 230 235 240 Asp Gly Thr Trp Val Asp Ala Pro Ser Val Pro Gly Ala Phe Val Val 245 250 255 Asn Ile Gly Glu Leu Leu Glu Tyr Ala Thr Gly Gly Tyr Leu Lys Ala 260 265 270 Thr Asn His Arg Val Val Ser Pro Lys Ala Pro Asp Glu Arg Ile Ser 275 280 285 Ile Pro Phe Phe Phe Asn Pro Ala Leu Asp Lys Arg Leu Pro Leu Ile 290 295 300 Glu Leu Pro Ala Pro Leu Ala Ala Glu Ala Thr Gly Val Thr Asp Asp 305 310 315 320 Pro Arg Asn Pro Ile His Ala Leu Tyr Gly Glu Asn Ala Leu Lys Ser 325 330 335 Arg Leu Arg Ala His Pro Asp Val Ala Ala Ile His His Ala Asp Leu 340 345 350 Val Gly Ala Ser Ala 355 <210> 26 <211> 333 <212> PRT <213> Artificial Sequence <220> <223> CefE of Nannocystis exedens (genbank PID SFE71965.1) <400> 26 Met Ser Ser Ala Ile Ile Ala Glu Ile Pro Val Ile Asp Leu Gly Pro 1 5 10 15 Phe Gly Asp Gly Asp Ala Pro Gly Ala Ala Glu Val Ala Ala Ala Phe 20 25 30 Asp Arg Ala Cys Arg Asp Leu Gly Phe Val Val Val Val Gly His Gly 35 40 45 Val Pro Gln Ala Val Ile Asp Ala Ala His Arg Ser Ala Arg Ala Phe 50 55 60 Phe Asp Leu Asp Leu Ala Thr Arg Glu Arg Tyr Ala Ala Pro Pro Gly 65 70 75 80 Gly Phe Val Gly Tyr Arg Gly Leu Gly Ala Glu Gly Leu Ser Tyr Ser 85 90 95 Leu Asp Gln Glu Ala Ala Pro Asp Phe Lys Glu Thr Tyr Thr Val Gly 100 105 110 Arg Ile Asp Arg Gly Asp Glu Pro Tyr Phe Thr Ser Gln Leu Gly Arg 115 120 125 Met Tyr Met Pro Asp Pro Thr Trp Pro Ala Glu Val Pro Glu Phe Ala 130 135 140 Ala Ser Trp Ala Glu Phe Tyr Arg Gln Met Asp Arg Val Ala Trp Arg 145 150 155 160 Leu Met Arg Ala Phe Ala Thr Ala Leu Ala Leu Pro Arg Glu Phe Phe 165 170 175 Asp Asp Lys Ile Asp Arg Asn Ile Ser Cys Leu Arg Ala Leu Asn Tyr 180 185 190 Pro His Pro Ser Thr Pro Pro Gln Pro Gly Gln Leu Arg Ala Gly Ala 195 200 205 His Thr Asp Tyr Gly Ser Leu Thr Leu Leu Ser Met Ala Asp Ala Pro 210 215 220 Gly Gly Leu Glu Val Gln Arg Asp Asp Gly Ser Trp Ala Pro Val Arg 225 230 235 240 Val Pro Ala Asp Ala Phe Val Met Asn Val Gly Asp Leu Met Ala Gln 245 250 255 Trp Thr Asn Asp Arg Trp Arg Ser Ser Met His Arg Val Thr Asn Pro 260 265 270 Pro Pro Asp Ala Ile Gly Asp Thr Arg Arg Gln Ser Leu Ala Phe Phe 275 280 285 His Gln Pro Asn Tyr Asp Ala Glu Val Ile Pro Leu Ala Ser Cys Cys 290 295 300 Gly Pro Ala Asn Pro Pro Lys Tyr Ala Arg Thr Thr Ser Gly Glu His 305 310 315 320 Leu Phe Met Lys Met Thr Lys Ala Lys Asn Leu Asn Val 325 330 <210> 27 <211> 337 <212> PRT <213> Artificial Sequence <220> <223> CefE of Pseudomonas syringae (genbank PID EPM68633.1) <400> 27 Met Thr Leu Gln Tyr Val Pro Ile Ile Asn Leu Ala Pro Tyr Phe Ser 1 5 10 15 Gly Glu Pro Asp Gly Lys Ala Ala Val Ala Gln Ala Val Asn Gln Ala 20 25 30 Cys Lys Asp Ile Gly Phe Leu Val Ile Thr Glu His Gln Ile Pro Thr 35 40 45 Glu Leu Ile Asp Arg Val Ser Arg Leu Thr Arg Gln Phe Phe Asp Leu 50 55 60 Pro Leu Ala Glu Lys Arg Lys Val Asp Arg Pro Ser Pro Glu Met Val 65 70 75 80 Arg Gly Tyr Ser Ala Val Ala Glu Glu Ser Leu Ser Tyr Ser Leu Glu 85 90 95 Glu Ser Ala Pro Gly Asp Leu Lys Glu Ser Phe Ser Ile Gly Pro Ser 100 105 110 Asp Val Pro Asn Glu Asp Tyr Tyr His Asn Ala Glu Ala Gly Pro His 115 120 125 Phe Ala Pro Asn Val Trp Pro Ala Asn Glu Arg Val Pro Gly Phe Gln 130 135 140 Glu Ala Tyr Gln Ala Tyr Phe Gly Ala Met Ser Gln Leu Ala Gln Ser 145 150 155 160 Leu Met Arg Leu Phe Ala Leu Ala Leu Glu Leu Asp Glu Cys Phe Phe 165 170 175 Asp Asp Arg Ile Asp Arg His Ile Ser Met Phe Arg Ser Leu Ser Tyr 180 185 190 Pro Asp Ile Lys Thr Glu Val Glu Ala Gly Gln Leu Arg Ala Ser Ala 195 200 205 His Thr Asp Tyr Gly Ser Leu Thr Ile Val Arg Pro Asp Asn Ala Leu 210 215 220 Gly Gly Leu Gln Val Arg Asn Gln Gln Gly Glu Trp Val Asp Val Pro 225 230 235 240 Tyr Val Glu Asn Gly Phe Val Val Asn Ile Gly Asp Leu Met Met Gln 245 250 255 Trp Thr Asn Asp Gln Trp Ile Ser Thr Leu His Arg Val Val Asn Pro 260 265 270 Pro Met Thr Ser Glu Gln Asp Asn Arg Arg Gln Ser Leu Val Phe Phe 275 280 285 His Gln Pro Asn Tyr Asp Thr Leu Ile Glu Cys Leu Pro Gly Cys Leu 290 295 300 Leu Pro Gly Thr Thr Pro Arg His Ala Pro Val Thr Ser Gly Asp His 305 310 315 320 Leu Leu Ala Lys Phe Val Lys Gln Thr Thr Phe Gly Gly Ser Lys Val 325 330 335 Ala <210> 28 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> CefE of S. clavuligerus (genbank PID EDY47106.1) <400> 28 Met Asp Thr Thr Val Pro Thr Phe Ser Leu Ala Glu Leu Gln Gln Gly 1 5 10 15 Leu His Gln Asp Glu Phe Arg Arg Cys Leu Arg Asp Lys Gly Leu Phe 20 25 30 Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys 35 40 45 Asp Ile Val Ile Asp Phe Phe Glu His Gly Ser Glu Ala Glu Lys Arg 50 55 60 Ala Val Thr Ser Pro Val Pro Thr Met Arg Arg Gly Phe Thr Gly Leu 65 70 75 80 Glu Ser Glu Ser Thr Ala Gln Ile Thr Asn Thr Gly Ser Tyr Ser Asp 85 90 95 Tyr Ser Met Cys Tyr Ser Met Gly Thr Ala Asp Asn Leu Phe Pro Ser 100 105 110 Gly Asp Phe Glu Arg Ile Trp Thr Gln Tyr Phe Asp Arg Gln Tyr Thr 115 120 125 Ala Ser Arg Ala Val Ala Arg Glu Val Leu Arg Ala Thr Gly Thr Glu 130 135 140 Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Leu Arg 145 150 155 160 Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Ser Ala Glu Glu Gln 165 170 175 Pro Leu Arg Met Ala Pro His Tyr Asp Leu Ser Met Val Thr Leu Ile 180 185 190 Gln Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 205 Gly Gly Ala Phe Thr Asp Leu Pro Tyr Arg Pro Asp Ala Val Leu Val 210 215 220 Phe Cys Gly Ala Ile Ala Thr Leu Val Thr Gly Gly Gln Val Lys Ala 225 230 235 240 Pro Arg His His Val Ala Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser 245 250 255 Ser Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Asn Ala Asp Phe Thr 260 265 270 Phe Ser Val Pro Leu Ala Arg Glu Cys Gly Phe Asp Val Ser Leu Asp 275 280 285 Gly Glu Thr Ala Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn 290 295 300 Ile Arg Arg Thr Ser Lys Ala 305 310 <210> 29 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> CefE of Sphingomonas dokdonensis (genbank PID OWK28829.1) <400> 29 Met His Arg Ala Gly Gly Ser Gly Leu Val Pro Ser Gly Arg Ile Ala 1 5 10 15 Gly Tyr Trp Arg Cys Glu Arg Pro Trp Arg Met Gln Arg Arg Ser Arg 20 25 30 Cys Ser Thr Lys Ala Leu Val Met Leu Asp Thr Pro Leu Ala Lys Val 35 40 45 Pro Thr Leu Ser Leu Ala Asp Gln Ala Val Asp Pro Asp Arg Phe Ala 50 55 60 Ala Asp Leu Gly Gly Ser Phe Arg Arg Phe Gly Phe Ala Ile Val Ala 65 70 75 80 Asp His Gly Ile Pro Asp Asp Leu Ile Ala Arg Ala Trp Ala Gln Thr 85 90 95 Glu Ala Leu Phe Ala Gln Pro Asp Ala Asp Lys Arg Gly Tyr Tyr Val 100 105 110 Glu Gly Ala Gly Gly Ala Arg Gly Tyr Thr Pro Phe Lys Thr Glu Ile 115 120 125 Ala Lys Asp Ala Lys His Val Asp Leu Lys Glu Phe Trp His Val Gly 130 135 140 Arg Glu Leu Pro Thr Gly His Arg Tyr Ala Gly Glu Met Ala Pro Asn 145 150 155 160 Ile Trp Pro Ala Gln Pro Asp Gly Phe Lys Glu Thr Phe Val Glu Leu 165 170 175 Phe Arg Ala Phe Asp Thr Ala Gly Asp Lys Leu Leu Ser Ala Ile Ala 180 185 190 Arg Asp Leu Gly Leu Ala Pro Asp Trp Phe Asp Leu Ala Val Lys Asp 195 200 205 Gly Asn Ser Val Leu Arg Leu Leu His Tyr Pro Pro Ile Pro Met Asp 210 215 220 Ala Glu Gly Val Arg Ala Gly Ala His Glu Asp Ile Asn Leu Ile Thr 225 230 235 240 Leu Leu Leu Gly Ala Glu Glu Ala Gly Leu Glu Leu Leu Asp Arg Asp 245 250 255 Ser Gly Asn Trp Leu Ala Ile Lys Pro Pro Glu Gly Ala Met Val Val 260 265 270 Asn Val Gly Asp Met Leu Gln Arg Leu Thr Asn His Val Leu Pro Ser 275 280 285 Thr Thr His Arg Val Val Asn Pro Pro Pro Glu Arg Arg Ala His Ser 290 295 300 Arg Tyr Ser Met Pro Phe Phe Leu His Pro Ala Pro Asp Phe Leu Ile 305 310 315 320 Lys Thr Leu Pro Gln Thr Val Thr Ser Ser Asn Pro Asn Arg Tyr Pro 325 330 335 Thr Pro Ile Thr Ala His Asp Tyr Leu His Glu Arg Leu Val Glu Ile 340 345 350 Gly Leu Ile Lys Lys 355 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EFGLB-F <400> 30 gagtagtttg gccttgatgg gacg 24 <210> 31 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> EFGLB-R <400> 31 ccctttagtg agggttaatt gctatggagg tgtcagcctg cc 42 <210> 32 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> EFGRB-F <400> 32 gccctatagt gagtcgtatt accgccgatc gagtaataaa tctacg 46 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EFGRB-R <400> 33 gtgcttctcg gttcagtgag tcgg 24 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T3 <400> 34 aattaaccct cactaaaggg 20 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> T7 <400> 35 taatacgact cactataggg c 21 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Gout (patent) <400> 36 cgatgtagga ggttgacggt gc 22 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgpdup (patent) <400> 37 cgataagggc cagttccgcc 20 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Gdown (patent) <400> 38 ggtaccaggc ccagaagttt gc 22 <210> 39 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> E1-F <400> 39 cgcttgagca gacatcacca tgaccgacgc caccgtgcc 39 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> E1-R <400> 40 gctaagcttt tatcagcggg cggcggc 27 <210> 41 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> E2-F <400> 41 cgcttgagca gacatcacca tgaccgacag cccgatct 38 <210> 42 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E2-R <400> 42 gctaagcttt tatcagccga cggtgatcg 29 <210> 43 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> E3-F <400> 43 cgcttgagca gacatcacca tgacggacat cggtgaac 38 <210> 44 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> E3-R <400> 44 gctaagcttt tatcagccga cggttatggc 30 <210> 45 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> E4-F <400> 45 cgcttgagca gacatcacca tggccctgcc gcccgtcatg c 41 <210> 46 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> E4-R <400> 46 gctaagcttt tatcacgcgc tcgcgcct 28 <210> 47 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> E5-F <400> 47 cgcttgagca gacatcacca tgtcaagcgc catcatcgcc 40 <210> 48 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> E5-R <400> 48 gctaagcttt tatcagacgt tcaggttctt cgccttg 37 <210> 49 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> E6-F <400> 49 cgcttgagca gacatcacca tgaccctcca gtacgtccc 39 <210> 50 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E6-R <400> 50 gctaagcttt tatcaggcga ccttggacc 29 <210> 51 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> E7-F <400> 51 cgcttgagca gacatcacca tggacaccac cgtgccgac 39 <210> 52 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> E7-R <400> 52 gctaagcttt tactacgcct tcgaggtccg ccg 33 <210> 53 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> E8-F <400> 53 cgcttgagca gacatcacca tgcatcgcgc gggcggc 37 <210> 54 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> E8-R <400> 54 gctaagcttt tatcacttct tgatgagac 29 <210> 55 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> PEP3-F <400> 55 gcaactagtg cggccgccct tgtatctcta cacacaggc 39 <210> 56 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PEP3-R <400> 56 ggtgatgtct gctcaagcg 19 <210> 57 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> TtC-F <400> 57 cgactcgaga cttaacgtta ctgaaatcat caaacagc 38 <210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TtC-R <400> 58 gcaggtacca tcgagtggag atgtggagtg gg 32 <210> 59 <211> 3717 <212> DNA <213> Artificial Sequence <220> <223> pB-TtrpC <400> 59 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgctctag aactagtgga tcccccgggc tgcaggaatt cgatatcaag cttatcgata 2280 ccgtcgacct cgagacttaa cgttactgaa atcatcaaac agcttgacga atctggatat 2340 aagatcgttg gtgtcgatgt cagctccgga gttgagacaa atggtgttca ggatctcgat 2400 aagatacgtt catttgtcca agcagcaaag agtgccttct agtgatttaa tagctccatg 2460 tcaacaagaa taaaacgcgt tttcgggttt acctcttcca gatacagctc atctgcaatg 2520 cattaatgca ttgactgcaa cctagtaacg cctttcaggc tccggcgaag agaagaatag 2580 cttagcagag ctattttcat tttcgggaga cgagatcaag cagatcaacg gtcgtcaaga 2640 gacctacgag actgaggaat ccgctcttgg ctccacgcga ctatatattt gtctctaatt 2700 gtactttgac atgctcctct tctttactct gatagcttga ctatgaaaat tccgtcacca 2760 gcccctgggt tcgcaaagat aattgcatgt ttcttccttg aactctcaag cctacaggac 2820 acacattcat cgtaggtata aacctcgaaa tcagttccta ctaagatggt atacaatagt 2880 aaccatgcat ggttgcctag tgaatgctcc gtaacaccca atacgccggc cgaaactttt 2940 ttacaactct cctatgagtc gtttacccag aatgcacagg tacacttgtt tagaggtaat 3000 ccttctttct agaagtcctc gtgtactgtg taagcgccca ctccacatct ccactcgatg 3060 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa 3120 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 3180 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 3240 agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 3300 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 3360 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 3420 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 3480 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 3540 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 3600 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 3660 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttag 3717 <210> 60 <211> 6930 <212> DNA <213> Artificial Sequence <220> <223> pB-E1cast <400> 60 attcttcgac tctgtattat agataagatg atgaggaatt ggaggtagca tagcttcatt 60 tggatttgct ttccaggctg agactctagc ttggagcata gagggtcctt tggctttcaa 120 tattctcaag tatctcgagt ttgaacttat tccctgtgaa ccttttattc accaatgagc 180 attggaatga acatgaatct gaggactgca atcgccatga ggttttcgaa atacatccgg 240 atgtcgaagg cttggggcac ctgcgttggt tgaatttaga acgtggcact attgatcatc 300 cgatagctct gcaaagggcg ttgcacaatg caagtcaaac gttgctagca gttccaggtg 360 gaatgttatg atgagcattg tattaaatca ggagatatag catgatctct agttagctca 420 ccacaaaagt cagacggcgt aaccaaaagt cacacaacac aagctgtaag gatttcggca 480 cggctacgga agacggagaa gccaccttca gtggactcga gtaccattta attctatttg 540 tgtttgatcg agacctaata cagcccctac aacgaccatc aaagtcgtat agctaccagt 600 gaggaagtgg actcaaatcg acttcagcaa catctcctgg ataaacttta agcctaaact 660 atacagaata agataggtgg agagcttata ccgagctccc aaatctgtcc agatcatggt 720 tgaccggtgc ctggatcttc ctatagaatc atccttattc gttgacctag ctgattctgg 780 agtgacccag agggtcatga cttgagccta aaatccgccg cctccaccat ttgtagaaaa 840 atgtgacgaa ctcgtgagct ctgtacagtg accggtgact ctttctggca tgcggagaga 900 cggacggacg cagagagaag ggctgagtaa taagccactg gccagacagc tctggcggct 960 ctgaggtgca gtggatgatt attaatccgg gaccggccgc ccctccgccc cgaagtggaa 1020 aggctggtgt gcccctcgtt gaccaagaat ctattgcatc atcggagaat atggagcttc 1080 atcgaatcac cggcagtaag cgaaggagaa tgtgaagcca ggggtgtata gccgtcggcg 1140 aaatagcatg ccattaacct aggtacagaa gtccaattgc ttccgatctg gtaaaagatt 1200 cacgagatag taccttctcc gaagtaggta gagcgagtac ccggcgcgta agctccctaa 1260 ttggcgaatt cccatccggc atctgtaggg cgtccaaata tcgtgcctct cctgctttgc 1320 caggtgtatg aaaccggaaa ggccgctcag gagctggcca gcggcgcaga ccgggaacac 1380 aagctggcag tcgacccatc cggtgctctg cactcgacct gctgaggtcc ctcagtccct 1440 ggtaggcagc tttgccccgt ctgtccgccc ggtgtgtcgg cggggttgac aaggtcgttg 1500 cgtcagtcca acatttgttg ccatattttc ctgctttccc caccagctgc tcttttcttt 1560 tctctttctt ttcccatctt cagtatattc atcttcccat atgcatccaa gaacctttat 1620 ttcccctaag taagtacttt gctacatcca tactccatcc ttcccatccc ttattccttt 1680 gaacctttca gttcgagctt tcccacttca tcgcagcttg actaacagct accccgcttg 1740 agcagacatc accatgaccg acgccaccgt gcccaccttc gacctggccg agctccggga 1800 gggtctgcac caggaggagt tcaggcattg cctccgcgaa aagggtgtct tctacctgaa 1860 gggcaccggc ctgcccgccg aggccgacca cgcatccggg cgggagatcg cggtggactt 1920 cttcgaccac ggcaccgagg ccgagaagaa ggcggtgatg acgcccatcc ctaccatccg 1980 ccggggctac gcgggcctgg agagcgagtc caccgcgcag atcacgaaca ccggcaagta 2040 caccgactac tccatgtcct attcgatggg taccgcggac aacctcttcc ccagcgccga 2100 gttcgagaag gcctgggagg actatttcgc ccgtatgtac cgcgcctccc aggacgtggc 2160 gcggcaggtg ctgacgtcgg tcggggcgga gccggaagtg ggcatggacg ccttcctgga 2220 ctgtgagccg cttctgcgcc ttcggtactt ccctgaggtc cccgaggacc gcgtggccga 2280 ggaacagccg ctccgcatgg cgccgcacta cgacctgagc atcgtcacgc tgatccacca 2340 gaccccgtgc gccaacggct tcgtgtccct gcaggtggag gtggacggca gctacgtcga 2400 catccccgcc cagccgggcg ccgtgctggt cttctgcggg gccgtcgcga cgctggtggc 2460 ggacggggcg atcaaggccc cgaagcacca cgtggccgcg cccggcgccg acaagcgcgt 2520 aggctccagc cgcacgtcca gcgtgttctt cctccgtccg aacggcgact tccggttcag 2580 cgtcccccgt gcgcgcgaat gcggcttcga cgtgtccatc cccgccgaga ccgcgacctt 2640 cgacgactgg atcgggggca actacatcaa catccgcaag acggccgccg cccgctgata 2700 aaagcttatc gataccgtcg acctcgagac ttaacgttac tgaaatcatc aaacagcttg 2760 acgaatctgg atataagatc gttggtgtcg atgtcagctc cggagttgag acaaatggtg 2820 ttcaggatct cgataagata cgttcatttg tccaagcagc aaagagtgcc ttctagtgat 2880 ttaatagctc catgtcaaca agaataaaac gcgttttcgg gtttacctct tccagataca 2940 gctcatctgc aatgcattaa tgcattgact gcaacctagt aacgcctttc aggctccggc 3000 gaagagaaga atagcttagc agagctattt tcattttcgg gagacgagat caagcagatc 3060 aacggtcgtc aagagaccta cgagactgag gaatccgctc ttggctccac gcgactatat 3120 atttgtctct aattgtactt tgacatgctc ctcttcttta ctctgatagc ttgactatga 3180 aaattccgtc accagcccct gggttcgcaa agataattgc atgtttcttc cttgaactct 3240 caagcctaca ggacacacat tcatcgtagg tataaacctc gaaatcagtt cctactaaga 3300 tggtatacaa tagtaaccat gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc 3360 cggccgaaac ttttttacaa ctctcctatg agtcgtttac ccagaatgca caggtacact 3420 tgtttagagg taatccttct ttctagaagt cctcgtgtac tgtgtaagcg cccactccac 3480 atctccactc gatggtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg 3540 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 3600 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 3660 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg 3720 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 3780 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3840 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3900 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 3960 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 4020 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 4080 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 4140 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 4200 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 4260 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 4320 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 4380 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 4440 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 4500 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 4560 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 4620 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 4680 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 4740 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 4800 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 4860 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 4920 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 4980 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 5040 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 5100 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 5160 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 5220 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5280 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5340 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 5400 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 5460 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5520 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 5580 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5640 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5700 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 5760 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5820 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 5880 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 5940 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 6000 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 6060 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 6120 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 6180 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 6240 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 6300 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctcca 6360 ccgcggtggc ggccgccctt gtatctctac acacaggctc aaatcaataa gaagaacggt 6420 tcgtcttttt cgtttatatc ttgcatcgtc ccaaagctat tggcgggata ttctgtttgc 6480 agttggctga cttgaagtaa tctctgcaga tctttcgaca ctgaaatacg tcgagcctgc 6540 tccgcttgga agcggcgagg agcctcgtcc tgtcacaact accaacatgg agtacgataa 6600 gggccagttc cgccagctca ttaagagcca gttcatgggc gttggcatga tggccgtcat 6660 gcatctgtac ttcaagtaca ccaacgctct tctgatccag tcgatcatcc gctgaaggcg 6720 ctttcgaatc tggttaagat ccacgtcttc gggaagccag cgactggtga cctccagcgt 6780 ccctttaagg ctgccaacag ctttctcagc cagggccagc ccaagaccga caaggcctcc 6840 ctccagaacg ccgagaagaa ctggaggggt ggtgtcaagg aggagtaagc tccttattga 6900 agtcggagga cggagcggtg tcaagaggat 6930 <210> 61 <211> 6977 <212> DNA <213> Artificial Sequence <220> <223> pB-E2cast <400> 61 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgctctag aactagtgcg gccgcccttg tatctctaca cacaggctca aatcaataag 2280 aagaacggtt cgtctttttc gtttatatct tgcatcgtcc caaagctatt ggcgggatat 2340 tctgtttgca gttggctgac ttgaagtaat ctctgcagat ctttcgacac tgaaatacgt 2400 cgagcctgct ccgcttggaa gcggcgagga gcctcgtcct gtcacaacta ccaacatgga 2460 gtacgataag ggccagttcc gccagctcat taagagccag ttcatgggcg ttggcatgat 2520 ggccgtcatg catctgtact tcaagtacac caacgctctt ctgatccagt cgatcatccg 2580 ctgaaggcgc tttcgaatct ggttaagatc cacgtcttcg ggaagccagc gactggtgac 2640 ctccagcgtc cctttaaggc tgccaacagc tttctcagcc agggccagcc caagaccgac 2700 aaggcctccc tccagaacgc cgagaagaac tggaggggtg gtgtcaagga ggagtaagct 2760 ccttattgaa gtcggaggac ggagcggtgt caagaggata ttcttcgact ctgtattata 2820 gataagatga tgaggaattg gaggtagcat agcttcattt ggatttgctt tccaggctga 2880 gactctagct tggagcatag agggtccttt ggctttcaat attctcaagt atctcgagtt 2940 tgaacttatt ccctgtgaac cttttattca ccaatgagca ttggaatgaa catgaatctg 3000 aggactgcaa tcgccatgag gttttcgaaa tacatccgga tgtcgaaggc ttggggcacc 3060 tgcgttggtt gaatttagaa cgtggcacta ttgatcatcc gatagctctg caaagggcgt 3120 tgcacaatgc aagtcaaacg ttgctagcag ttccaggtgg aatgttatga tgagcattgt 3180 attaaatcag gagatatagc atgatctcta gttagctcac cacaaaagtc agacggcgta 3240 accaaaagtc acacaacaca agctgtaagg atttcggcac ggctacggaa gacggagaag 3300 ccaccttcag tggactcgag taccatttaa ttctatttgt gtttgatcga gacctaatac 3360 agcccctaca acgaccatca aagtcgtata gctaccagtg aggaagtgga ctcaaatcga 3420 cttcagcaac atctcctgga taaactttaa gcctaaacta tacagaataa gataggtgga 3480 gagcttatac cgagctccca aatctgtcca gatcatggtt gaccggtgcc tggatcttcc 3540 tatagaatca tccttattcg ttgacctagc tgattctgga gtgacccaga gggtcatgac 3600 ttgagcctaa aatccgccgc ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc 3660 tgtacagtga ccggtgactc tttctggcat gcggagagac ggacggacgc agagagaagg 3720 gctgagtaat aagccactgg ccagacagct ctggcggctc tgaggtgcag tggatgatta 3780 ttaatccggg accggccgcc cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg 3840 accaagaatc tattgcatca tcggagaata tggagcttca tcgaatcacc ggcagtaagc 3900 gaaggagaat gtgaagccag gggtgtatag ccgtcggcga aatagcatgc cattaaccta 3960 ggtacagaag tccaattgct tccgatctgg taaaagattc acgagatagt accttctccg 4020 aagtaggtag agcgagtacc cggcgcgtaa gctccctaat tggcgaattc ccatccggca 4080 tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc aggtgtatga aaccggaaag 4140 gccgctcagg agctggccag cggcgcagac cgggaacaca agctggcagt cgacccatcc 4200 ggtgctctgc actcgacctg ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc 4260 tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa catttgttgc 4320 catattttcc tgctttcccc accagctgct cttttctttt ctctttcttt tcccatcttc 4380 agtatattca tcttcccata tgcatccaag aacctttatt tcccctaagt aagtactttg 4440 ctacatccat actccatcct tcccatccct tattcctttg aacctttcag ttcgagcttt 4500 cccacttcat cgcagcttga ctaacagcta ccccgcttga gcagacatca ccatgaccga 4560 cagcccgatc ttgaccgtcg acctgtcccg ctggcgggcc ggtggcgcgg acgcggctgc 4620 cgtgtgcgcc gccgtcgacg agtcgctcca gaaggccggc ttcctgctgg tgaccggcca 4680 cgggatcgac ccggtgctcc ccgcggagct gcgggcggca gcccgcgcct tcttcgcgct 4740 gccggccgag gtgaagcaga agtacgcggt cggggtcggg ggccggggct ggatcgggcc 4800 cggcatggag gccaacgcct acgccgaggg taccgagacc cccccggacc tgaaggagac 4860 ctataactcg ggggcgcaaa cgccggtcgg cgtaccggag gtggacgact actggttcgc 4920 acccgacgtc tggcccattg aagcgccgcg gctcaaggag ctcttcacgg catggaccgc 4980 acagtgcaag acactcagcg acgacctcct ggcgctgatg gccgcctccc tcggcttcgc 5040 cgccgcggac aaccccttcc gcgacctggc tggcaacgcc acctggacct ccaacatcaa 5100 ccactacccg gcgatggccg tcgtgggcga gcccgagccg gggcagtacc ggatcggccc 5160 gcacaccgac ttcggcacgg tgaccgtcct cgaccgcgaa cccggttccg gcggcctgca 5220 ggtctacagc gaagacgggg gatgggctga cgcgccgtac gaccccgcgg ccctcacggt 5280 caacatcggg gacctcctcg agtactggtc cggtggtcgg tggccggccg ggcgccaccg 5340 ggtgctgccg ccccagccgg acgcccccga ggaggacctg atgtccctga tcttcttcta 5400 tgaactggac catgacgccg tcgtcacgcc gatcggtaag cccctggggc gccgcgccgg 5460 cctggagcct gtggtctccg gcgagttcat ccgggagcgc ctggacgcga tcaccgtcgg 5520 ctgataaaag cttatcgata ccgtcgacct cgagacttaa cgttactgaa atcatcaaac 5580 agcttgacga atctggatat aagatcgttg gtgtcgatgt cagctccgga gttgagacaa 5640 atggtgttca ggatctcgat aagatacgtt catttgtcca agcagcaaag agtgccttct 5700 agtgatttaa tagctccatg tcaacaagaa taaaacgcgt tttcgggttt acctcttcca 5760 gatacagctc atctgcaatg cattaatgca ttgactgcaa cctagtaacg cctttcaggc 5820 tccggcgaag agaagaatag cttagcagag ctattttcat tttcgggaga cgagatcaag 5880 cagatcaacg gtcgtcaaga gacctacgag actgaggaat ccgctcttgg ctccacgcga 5940 ctatatattt gtctctaatt gtactttgac atgctcctct tctttactct gatagcttga 6000 ctatgaaaat tccgtcacca gcccctgggt tcgcaaagat aattgcatgt ttcttccttg 6060 aactctcaag cctacaggac acacattcat cgtaggtata aacctcgaaa tcagttccta 6120 ctaagatggt atacaatagt aaccatgcat ggttgcctag tgaatgctcc gtaacaccca 6180 atacgccggc cgaaactttt ttacaactct cctatgagtc gtttacccag aatgcacagg 6240 tacacttgtt tagaggtaat ccttctttct agaagtcctc gtgtactgtg taagcgccca 6300 ctccacatct ccactcgatg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct 6360 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 6420 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 6480 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 6540 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6600 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6660 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6720 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6780 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6840 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6900 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 6960 taacgcttac aatttag 6977 <210> 62 <211> 6404 <212> DNA <213> Artificial Sequence <220> <223> pB-E3cast <400> 62 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgcgttta aacccttgta tctctacaca caggctcaaa tcaataagaa gaacggttcg 2280 tctttttcgt ttatatcttg catcgtccca aagctattgg cgggatattc tgtttgcagt 2340 tggctgactt gaagtaatct ctgcagatct ttcgacactg aaatacgtcg agcctgctcc 2400 gcttggaagc ggcgaggagc ctcgtcctgt cacaactacc aacatggagt acgataaggg 2460 ccagttccgc cagctcatta agagccagtt catgggcgtt ggcatgatgg ccgtcatgca 2520 tctgtacttc aagtacacca acgctcttct gatccagtcg atcatccgct gaaggcgctt 2580 tcgaatctgg ttaagatcca cgtcttcggg aagccagcga ctggtgacct ccagcgtccc 2640 tttaaggctg ccaacagctt tctcagccag ggccagccca agaccgacaa ggcctccctc 2700 cagaacgccg agaagaactg gaggggtggt gtcaaggagg agtaagctcc ttattgaagt 2760 cggaggacgg agcggtgtca agaggatatt cttcgactct gtattataga taagatgatg 2820 aggaattgga ggtagcatag cttcatttgg atttgctttc caggctgaga ctctagcttg 2880 gagcatagag ggtcctttgg ctttcaatat tctcaagtat ctcgagtttg aacttattcc 2940 ctgtgaacct tttattcacc aatgagcatt ggaatgaaca tgaatctgag gactgcaatc 3000 gccatgaggt tttcgaaata catccggatg tcgaaggctt ggggcacctg cgttggttga 3060 atttagaacg tggcactatt gatcatccga tagctctgca aagggcgttg cacaatgcaa 3120 gtcaaacgtt gctagcagtt ccaggtggaa tgttatgatg agcattgtat taaatcagga 3180 gatatagcat gatctctagt tagctcacca caaaagtcag acggcgtaac caaaagtcac 3240 acaacacaag ctgtaaggat ttcggcacgg ctacggaaga cggagaagcc accttcagtg 3300 gactcgagta ccatttaatt ctatttgtgt ttgatcgaga cctaatacag cccctacaac 3360 gaccatcaaa gtcgtatagc taccagtgag gaagtggact caaatcgact tcagcaacat 3420 ctcctggata aactttaagc ctaaactata cagaataaga taggtggaga gcttataccg 3480 agctcccaaa tctgtccaga tcatggttga ccggtgcctg gatcttccta tagaatcatc 3540 cttattcgtt gacctagctg attctggagt gacccagagg gtcatgactt gagcctaaaa 3600 tccgccgcct ccaccatttg tagaaaaatg tgacgaactc gtgagctctg tacagtgacc 3660 ggtgactctt tctggcatgc ggagagacgg acggacgcag agagaagggc tgagtaataa 3720 gccactggcc agacagctct ggcggctctg aggtgcagtg gatgattatt aatccgggac 3780 cggccgcccc tccgccccga agtggaaagg ctggtgtgcc cctcgttgac caagaatcta 3840 ttgcatcatc ggagaatatg gagcttcatc gaatcaccgg cagtaagcga aggagaatgt 3900 gaagccaggg gtgtatagcc gtcggcgaaa tagcatgcca ttaacctagg tacagaagtc 3960 caattgcttc cgatctggta aaagattcac gagatagtac cttctccgaa gtaggtagag 4020 cgagtacccg gcgcgtaagc tccctaattg gcgaattccc atccggcatc tgtagggcgt 4080 ccaaatatcg tgcctctcct gctttgccag gtgtatgaaa ccggaaaggc cgctcaggag 4140 ctggccagcg gcgcagaccg ggaacacaag ctggcagtcg acccatccgg tgctctgcac 4200 tcgacctgct gaggtccctc agtccctggt aggcagcttt gccccgtctg tccgcccggt 4260 gtgtcggcgg ggttgacaag gtcgttgcgt cagtccaaca tttgttgcca tattttcctg 4320 ctttccccac cagctgctct tttcttttct ctttcttttc ccatcttcag tatattcatc 4380 ttcccatatg catccaagaa cctttatttc ccctaagtaa gtactttgct acatccatac 4440 tccatccttc ccatccctta ttcctttgaa cctttcagtt cgagctttcc cacttcatcg 4500 cagcttgact aacagctacc ccgcttgagc agacatcacc atgacggaca tcggtgaacc 4560 ggagcctggt cagtaccgga tcggcccgca cacggacttc ggcaccgtca cgatcctgga 4620 ccgggagccg ggagccggag ggctgcaggt gtactccgag cagggcggct gggccgacgc 4680 cccgtacgac ccgtcggccc tgacggtcaa catcggagac ctgctcgagt actggtcggg 4740 gggcaggtgg ccggcgggcc gccaccgggt gctcccgcca caggcggacg ctccggagga 4800 agacctggtg tctctcatct tcttctacga actcgaccac gacgccgtcg tgaccccgct 4860 cgaccccccg gtcggaaaga tcgccggccg gccgccggtc atcggcggcg atttcatccg 4920 ggagaggctg gacgccataa ccgtcggctg ataaaagctt atcgataccg tcgacctcga 4980 gacttaacgt tactgaaatc atcaaacagc ttgacgaatc tggatataag atcgttggtg 5040 tcgatgtcag ctccggagtt gagacaaatg gtgttcagga tctcgataag atacgttcat 5100 ttgtccaagc agcaaagagt gccttctagt gatttaatag ctccatgtca acaagaataa 5160 aacgcgtttt cgggtttacc tcttccagat acagctcatc tgcaatgcat taatgcattg 5220 actgcaacct agtaacgcct ttcaggctcc ggcgaagaga agaatagctt agcagagcta 5280 ttttcatttt cgggagacga gatcaagcag atcaacggtc gtcaagagac ctacgagact 5340 gaggaatccg ctcttggctc cacgcgacta tatatttgtc tctaattgta ctttgacatg 5400 ctcctcttct ttactctgat agcttgacta tgaaaattcc gtcaccagcc cctgggttcg 5460 caaagataat tgcatgtttc ttccttgaac tctcaagcct acaggacaca cattcatcgt 5520 aggtataaac ctcgaaatca gttcctacta agatggtata caatagtaac catgcatggt 5580 tgcctagtga atgctccgta acacccaata cgccggccga aactttttta caactctcct 5640 atgagtcgtt tacccagaat gcacaggtac acttgtttag aggtaatcct tctttctaga 5700 agtcctcgtg tactgtgtaa gcgcccactc cacatctcca ctcgatggta cccaattcgc 5760 cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg 5820 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 5880 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 5940 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 6000 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 6060 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 6120 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 6180 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 6240 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 6300 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 6360 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttag 6404 <210> 63 <211> 7059 <212> DNA <213> Artificial Sequence <220> <223> pB-E4cast <400> 63 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgcccttg tatctctaca cacaggctca aatcaataag aagaacggtt cgtctttttc 2280 gtttatatct tgcatcgtcc caaagctatt ggcgggatat tctgtttgca gttggctgac 2340 ttgaagtaat ctctgcagat ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa 2400 gcggcgagga gcctcgtcct gtcacaacta ccaacatgga gtacgataag ggccagttcc 2460 gccagctcat taagagccag ttcatgggcg ttggcatgat ggccgtcatg catctgtact 2520 tcaagtacac caacgctctt ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct 2580 ggttaagatc cacgtcttcg ggaagccagc gactggtgac ctccagcgtc cctttaaggc 2640 tgccaacagc tttctcagcc agggccagcc caagaccgac aaggcctccc tccagaacgc 2700 cgagaagaac tggaggggtg gtgtcaagga ggagtaagct ccttattgaa gtcggaggac 2760 ggagcggtgt caagaggata ttcttcgact ctgtattata gataagatga tgaggaattg 2820 gaggtagcat agcttcattt ggatttgctt tccaggctga gactctagct tggagcatag 2880 agggtccttt ggctttcaat attctcaagt atctcgagtt tgaacttatt ccctgtgaac 2940 cttttattca ccaatgagca ttggaatgaa catgaatctg aggactgcaa tcgccatgag 3000 gttttcgaaa tacatccgga tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa 3060 cgtggcacta ttgatcatcc gatagctctg caaagggcgt tgcacaatgc aagtcaaacg 3120 ttgctagcag ttccaggtgg aatgttatga tgagcattgt attaaatcag gagatatagc 3180 atgatctcta gttagctcac cacaaaagtc agacggcgta accaaaagtc acacaacaca 3240 agctgtaagg atttcggcac ggctacggaa gacggagaag ccaccttcag tggactcgag 3300 taccatttaa ttctatttgt gtttgatcga gacctaatac agcccctaca acgaccatca 3360 aagtcgtata gctaccagtg aggaagtgga ctcaaatcga cttcagcaac atctcctgga 3420 taaactttaa gcctaaacta tacagaataa gataggtgga gagcttatac cgagctccca 3480 aatctgtcca gatcatggtt gaccggtgcc tggatcttcc tatagaatca tccttattcg 3540 ttgacctagc tgattctgga gtgacccaga gggtcatgac ttgagcctaa aatccgccgc 3600 ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc 3660 tttctggcat gcggagagac ggacggacgc agagagaagg gctgagtaat aagccactgg 3720 ccagacagct ctggcggctc tgaggtgcag tggatgatta ttaatccggg accggccgcc 3780 cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg accaagaatc tattgcatca 3840 tcggagaata tggagcttca tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag 3900 gggtgtatag ccgtcggcga aatagcatgc cattaaccta ggtacagaag tccaattgct 3960 tccgatctgg taaaagattc acgagatagt accttctccg aagtaggtag agcgagtacc 4020 cggcgcgtaa gctccctaat tggcgaattc ccatccggca tctgtagggc gtccaaatat 4080 cgtgcctctc ctgctttgcc aggtgtatga aaccggaaag gccgctcagg agctggccag 4140 cggcgcagac cgggaacaca agctggcagt cgacccatcc ggtgctctgc actcgacctg 4200 ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc tgtccgcccg gtgtgtcggc 4260 ggggttgaca aggtcgttgc gtcagtccaa catttgttgc catattttcc tgctttcccc 4320 accagctgct cttttctttt ctctttcttt tcccatcttc agtatattca tcttcccata 4380 tgcatccaag aacctttatt tcccctaagt aagtactttg ctacatccat actccatcct 4440 tcccatccct tattcctttg aacctttcag ttcgagcttt cccacttcat cgcagcttga 4500 ctaacagcta ccccgcttga gcagacatca ccatggccct gccgcccgtc atgctgagga 4560 acacgcgggt ctccccccgg atgcgcatct actcccgggt gatgtcggag ctgtcgctgc 4620 ctatcctgga cctctcgcag ctggacgccg gcccggaagc cgccgcccgg ttccgggacg 4680 acctgagggc ggccacccac gacgtcgggt tcttctacct caccggcacg ggcgtcagtc 4740 ccgagctcga agcgcgcctg caccgcgccg ctctcgactt cttcgcgctg ccggaggcgg 4800 agaagctggc gatcgagaac atcaactcgc cgcacttccg gggctacact cgggtgggtg 4860 gggagcgcac ccagggcgag gtcgactggc gggagcagat cgacatcggc cccgagcgcg 4920 acgccgtaga tggtggcccg gcctacaatc ggctgatcgg gccgaacctg tggcccgcgg 4980 cccagccgga gctgaaggaa gtggtggcgg agtggcattc cacgctcagc gaggtcagcc 5040 ggcgcctcct ccgcgcgtgg gcggagaccc tgggggcgga gcctagctac ttcgacgagc 5100 acttcggaga gccctccacc ctcatcaaga tcgtccggta ccccggcacc cacgagcccg 5160 agccgcagca gggtgtcggg gcgcataaag actcaggtgt gctcacgctc ctgtgggtgg 5220 aacccggcaa gggtggcctg caggtggagc gggacggaac gtgggttgac gccccctccg 5280 tgcccggtgc gttcgtcgtc aacatcggcg aactcctgga gtacgccacc gggggctacc 5340 tgaaggcgac caaccaccgg gtggtctccc cgaaggcccc cgacgagagg atctccatcc 5400 ccttcttctt caatccggcc ctcgataagc ggctgccgct gatcgagctg cccgcgcccc 5460 tcgcggccga ggcgaccggg gtgaccgacg acccgcgtaa cccgatccac gccctgtacg 5520 gcgagaacgc cctgaagtca cggctgcgtg cccaccccga cgtggccgcc atccaccacg 5580 cggacctggt aggcgcgagc gcgtgataaa agcttatcga taccgtcgac ctcgagactt 5640 aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 5700 gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 5760 caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 5820 gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 5880 aacctagtaa cgcctttcag gctccggcga agagaagaat agcttagcag agctattttc 5940 attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 6000 atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 6060 cttctttact ctgatagctt gactatgaaa attccgtcac cagcccctgg gttcgcaaag 6120 ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 6180 taaacctcga aatcagttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 6240 agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 6300 tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 6360 tcgtgtactg tgtaagcgcc cactccacat ctccactcga tggtacccaa ttcgccctat 6420 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac 6480 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 6540 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 6600 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 6660 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 6720 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 6780 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 6840 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 6900 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 6960 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 7020 aacgcgaatt ttaacaaaat attaacgctt acaatttag 7059 <210> 64 <211> 6987 <212> DNA <213> Artificial Sequence <220> <223> pB-E5cast <400> 64 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgcccttg tatctctaca cacaggctca aatcaataag aagaacggtt cgtctttttc 2280 gtttatatct tgcatcgtcc caaagctatt ggcgggatat tctgtttgca gttggctgac 2340 ttgaagtaat ctctgcagat ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa 2400 gcggcgagga gcctcgtcct gtcacaacta ccaacatgga gtacgataag ggccagttcc 2460 gccagctcat taagagccag ttcatgggcg ttggcatgat ggccgtcatg catctgtact 2520 tcaagtacac caacgctctt ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct 2580 ggttaagatc cacgtcttcg ggaagccagc gactggtgac ctccagcgtc cctttaaggc 2640 tgccaacagc tttctcagcc agggccagcc caagaccgac aaggcctccc tccagaacgc 2700 cgagaagaac tggaggggtg gtgtcaagga ggagtaagct ccttattgaa gtcggaggac 2760 ggagcggtgt caagaggata ttcttcgact ctgtattata gataagatga tgaggaattg 2820 gaggtagcat agcttcattt ggatttgctt tccaggctga gactctagct tggagcatag 2880 agggtccttt ggctttcaat attctcaagt atctcgagtt tgaacttatt ccctgtgaac 2940 cttttattca ccaatgagca ttggaatgaa catgaatctg aggactgcaa tcgccatgag 3000 gttttcgaaa tacatccgga tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa 3060 cgtggcacta ttgatcatcc gatagctctg caaagggcgt tgcacaatgc aagtcaaacg 3120 ttgctagcag ttccaggtgg aatgttatga tgagcattgt attaaatcag gagatatagc 3180 atgatctcta gttagctcac cacaaaagtc agacggcgta accaaaagtc acacaacaca 3240 agctgtaagg atttcggcac ggctacggaa gacggagaag ccaccttcag tggactcgag 3300 taccatttaa ttctatttgt gtttgatcga gacctaatac agcccctaca acgaccatca 3360 aagtcgtata gctaccagtg aggaagtgga ctcaaatcga cttcagcaac atctcctgga 3420 taaactttaa gcctaaacta tacagaataa gataggtgga gagcttatac cgagctccca 3480 aatctgtcca gatcatggtt gaccggtgcc tggatcttcc tatagaatca tccttattcg 3540 ttgacctagc tgattctgga gtgacccaga gggtcatgac ttgagcctaa aatccgccgc 3600 ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc 3660 tttctggcat gcggagagac ggacggacgc agagagaagg gctgagtaat aagccactgg 3720 ccagacagct ctggcggctc tgaggtgcag tggatgatta ttaatccggg accggccgcc 3780 cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg accaagaatc tattgcatca 3840 tcggagaata tggagcttca tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag 3900 gggtgtatag ccgtcggcga aatagcatgc cattaaccta ggtacagaag tccaattgct 3960 tccgatctgg taaaagattc acgagatagt accttctccg aagtaggtag agcgagtacc 4020 cggcgcgtaa gctccctaat tggcgaattc ccatccggca tctgtagggc gtccaaatat 4080 cgtgcctctc ctgctttgcc aggtgtatga aaccggaaag gccgctcagg agctggccag 4140 cggcgcagac cgggaacaca agctggcagt cgacccatcc ggtgctctgc actcgacctg 4200 ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc tgtccgcccg gtgtgtcggc 4260 ggggttgaca aggtcgttgc gtcagtccaa catttgttgc catattttcc tgctttcccc 4320 accagctgct cttttctttt ctctttcttt tcccatcttc agtatattca tcttcccata 4380 tgcatccaag aacctttatt tcccctaagt aagtactttg ctacatccat actccatcct 4440 tcccatccct tattcctttg aacctttcag ttcgagcttt cccacttcat cgcagcttga 4500 ctaacagcta ccccgcttga gcagacatca ccatgtcaag cgccatcatc gccgaaatcc 4560 cggtgatcga cctgggccca ttcggagacg gggacgcgcc gggggcggcg gaagtggccg 4620 cggcattcga ccgggcctgc agggacctgg ggttcgtggt cgtcgtgggg cacggcgtgc 4680 cccaggccgt catcgacgcc gcgcaccgga gtgcccgcgc gttctttgac ctggacctcg 4740 ctacccgcga gcggtacgcg gccccgcccg gcgggttcgt cgggtaccgc gggctcggag 4800 ccgaggggct gtcttacagt ctggaccagg aggctgcgcc ggacttcaag gagacatata 4860 ccgtcggacg catcgatcgc ggagacgagc cttacttcac gtcccagctg gggcggatgt 4920 acatgccgga ccctacgtgg ccggcggagg tccccgagtt cgcagcgtcg tgggccgagt 4980 tctatcgcca gatggaccgg gtcgcctggc gcctgatgcg cgcgttcgcc accgcgctgg 5040 ccctcccccg cgagttcttc gacgacaaga ttgatcggaa catctcgtgc ctgcgcgcgc 5100 tgaactaccc ccacccgtcc acaccccccc agcccggcca gctgcgcgcg ggcgcccaca 5160 cggactacgg ctccctcacg ctgctcagca tggccgacgc gcccgggggc ctcgaggtgc 5220 agcgggacga cgggtcgtgg gcgccggtcc gggtcccggc agacgccttc gtgatgaacg 5280 tgggcgacct tatggcgcag tggaccaacg accgttggcg ctcgtcgatg caccgcgtga 5340 cgaatccccc cccggacgcg atcggcgaca cgcggcggca gagcctggcg ttcttccacc 5400 agcccaacta tgacgccgag gtcatcccgc tcgcgtcctg ctgcggcccg gccaaccccc 5460 cgaagtacgc ccgcaccacg agcggcgagc acctgttcat gaagatgacc aaggcgaaga 5520 acctgaacgt ctgataaaag cttatcgata ccgtcgacct cgagacttaa cgttactgaa 5580 atcatcaaac agcttgacga atctggatat aagatcgttg gtgtcgatgt cagctccgga 5640 gttgagacaa atggtgttca ggatctcgat aagatacgtt catttgtcca agcagcaaag 5700 agtgccttct agtgatttaa tagctccatg tcaacaagaa taaaacgcgt tttcgggttt 5760 acctcttcca gatacagctc atctgcaatg cattaatgca ttgactgcaa cctagtaacg 5820 cctttcaggc tccggcgaag agaagaatag cttagcagag ctattttcat tttcgggaga 5880 cgagatcaag cagatcaacg gtcgtcaaga gacctacgag actgaggaat ccgctcttgg 5940 ctccacgcga ctatatattt gtctctaatt gtactttgac atgctcctct tctttactct 6000 gatagcttga ctatgaaaat tccgtcacca gcccctgggt tcgcaaagat aattgcatgt 6060 ttcttccttg aactctcaag cctacaggac acacattcat cgtaggtata aacctcgaaa 6120 tcagttccta ctaagatggt atacaatagt aaccatgcat ggttgcctag tgaatgctcc 6180 gtaacaccca atacgccggc cgaaactttt ttacaactct cctatgagtc gtttacccag 6240 aatgcacagg tacacttgtt tagaggtaat ccttctttct agaagtcctc gtgtactgtg 6300 taagcgccca ctccacatct ccactcgatg gtacccaatt cgccctatag tgagtcgtat 6360 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 6420 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 6480 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt 6540 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 6600 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 6660 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 6720 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 6780 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 6840 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 6900 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 6960 aacaaaatat taacgcttac aatttag 6987 <210> 65 <211> 6999 <212> DNA <213> Artificial Sequence <220> <223> pB-E6cast <400> 65 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgcccttg tatctctaca cacaggctca aatcaataag aagaacggtt cgtctttttc 2280 gtttatatct tgcatcgtcc caaagctatt ggcgggatat tctgtttgca gttggctgac 2340 ttgaagtaat ctctgcagat ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa 2400 gcggcgagga gcctcgtcct gtcacaacta ccaacatgga gtacgataag ggccagttcc 2460 gccagctcat taagagccag ttcatgggcg ttggcatgat ggccgtcatg catctgtact 2520 tcaagtacac caacgctctt ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct 2580 ggttaagatc cacgtcttcg ggaagccagc gactggtgac ctccagcgtc cctttaaggc 2640 tgccaacagc tttctcagcc agggccagcc caagaccgac aaggcctccc tccagaacgc 2700 cgagaagaac tggaggggtg gtgtcaagga ggagtaagct ccttattgaa gtcggaggac 2760 ggagcggtgt caagaggata ttcttcgact ctgtattata gataagatga tgaggaattg 2820 gaggtagcat agcttcattt ggatttgctt tccaggctga gactctagct tggagcatag 2880 agggtccttt ggctttcaat attctcaagt atctcgagtt tgaacttatt ccctgtgaac 2940 cttttattca ccaatgagca ttggaatgaa catgaatctg aggactgcaa tcgccatgag 3000 gttttcgaaa tacatccgga tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa 3060 cgtggcacta ttgatcatcc gatagctctg caaagggcgt tgcacaatgc aagtcaaacg 3120 ttgctagcag ttccaggtgg aatgttatga tgagcattgt attaaatcag gagatatagc 3180 atgatctcta gttagctcac cacaaaagtc agacggcgta accaaaagtc acacaacaca 3240 agctgtaagg atttcggcac ggctacggaa gacggagaag ccaccttcag tggactcgag 3300 taccatttaa ttctatttgt gtttgatcga gacctaatac agcccctaca acgaccatca 3360 aagtcgtata gctaccagtg aggaagtgga ctcaaatcga cttcagcaac atctcctgga 3420 taaactttaa gcctaaacta tacagaataa gataggtgga gagcttatac cgagctccca 3480 aatctgtcca gatcatggtt gaccggtgcc tggatcttcc tatagaatca tccttattcg 3540 ttgacctagc tgattctgga gtgacccaga gggtcatgac ttgagcctaa aatccgccgc 3600 ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc 3660 tttctggcat gcggagagac ggacggacgc agagagaagg gctgagtaat aagccactgg 3720 ccagacagct ctggcggctc tgaggtgcag tggatgatta ttaatccggg accggccgcc 3780 cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg accaagaatc tattgcatca 3840 tcggagaata tggagcttca tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag 3900 gggtgtatag ccgtcggcga aatagcatgc cattaaccta ggtacagaag tccaattgct 3960 tccgatctgg taaaagattc acgagatagt accttctccg aagtaggtag agcgagtacc 4020 cggcgcgtaa gctccctaat tggcgaattc ccatccggca tctgtagggc gtccaaatat 4080 cgtgcctctc ctgctttgcc aggtgtatga aaccggaaag gccgctcagg agctggccag 4140 cggcgcagac cgggaacaca agctggcagt cgacccatcc ggtgctctgc actcgacctg 4200 ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc tgtccgcccg gtgtgtcggc 4260 ggggttgaca aggtcgttgc gtcagtccaa catttgttgc catattttcc tgctttcccc 4320 accagctgct cttttctttt ctctttcttt tcccatcttc agtatattca tcttcccata 4380 tgcatccaag aacctttatt tcccctaagt aagtactttg ctacatccat actccatcct 4440 tcccatccct tattcctttg aacctttcag ttcgagcttt cccacttcat cgcagcttga 4500 ctaacagcta ccccgcttga gcagacatca ccatgaccct ccagtacgtc cccatcatca 4560 acctggcgcc ctatttctcg ggcgagccgg acggcaaggc ggcggtcgcc caggcggtga 4620 accaggcgtg caaggacatc ggcttcctcg tcatcaccga gcaccagatc ccaaccgagc 4680 tgatcgaccg cgtctcgcgc ctcacgcggc aattcttcga tctgccgctc gccgaaaaga 4740 ggaaggtcga ccgcccttcg ccggagatgg tccgggggta ttccgccgtg gcggaggagt 4800 cgctgtccta ctcgctcgag gagtcggcgc cgggggacct gaaggagtcc ttctccatcg 4860 gcccgagcga cgtcccgaac gaggactact accacaacgc cgaagcgggg ccccacttcg 4920 cgccgaacgt gtggccggcc aacgaacggg tccctggctt ccaggaggcc tatcaggcct 4980 acttcggggc aatgtcccag ctcgcgcagt ccctcatgcg cttgttcgcc ctggccctcg 5040 agctcgacga atgcttcttc gacgaccgca ttgaccgcca catctccatg ttccggtcgc 5100 tctcctaccc ggacatcaag accgaggtcg aggccggcca gctgcgcgcg tccgcccaca 5160 ccgactacgg ctccctgacg atcgtacgcc ccgacaacgc cctgggaggc ctccaggttc 5220 ggaaccagca gggcgagtgg gtggacgttc cgtacgtgga gaacggcttc gtcgtgaaca 5280 tcggtgatct gatgatgcag tggaccaacg accagtggat cagcaccctg cacagggtcg 5340 tgaatccgcc catgacgagc gagcaggaca atcggcggca gtccctggtc ttcttccacc 5400 agccgaacta tgacaccctg atcgagtgcc tccccggctg cctgctgccg ggtaccacgc 5460 cccgccacgc ccccgtcacc agcggcgacc acctcctcgc caagtttgtc aagcagacga 5520 cgttcggagg gtccaaggtc gcctgataaa agcttatcga taccgtcgac ctcgagactt 5580 aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 5640 gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 5700 caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 5760 gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 5820 aacctagtaa cgcctttcag gctccggcga agagaagaat agcttagcag agctattttc 5880 attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 5940 atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 6000 cttctttact ctgatagctt gactatgaaa attccgtcac cagcccctgg gttcgcaaag 6060 ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 6120 taaacctcga aatcagttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 6180 agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 6240 tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 6300 tcgtgtactg tgtaagcgcc cactccacat ctccactcga tggtacccaa ttcgccctat 6360 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac 6420 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 6480 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 6540 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 6600 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 6660 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 6720 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 6780 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 6840 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 6900 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 6960 aacgcgaatt ttaacaaaat attaacgctt acaatttag 6999 <210> 66 <211> 6921 <212> DNA <213> Artificial Sequence <220> <223> pB-E7cast <400> 66 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgcccttg tatctctaca cacaggctca aatcaataag aagaacggtt cgtctttttc 2280 gtttatatct tgcatcgtcc caaagctatt ggcgggatat tctgtttgca gttggctgac 2340 ttgaagtaat ctctgcagat ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa 2400 gcggcgagga gcctcgtcct gtcacaacta ccaacatgga gtacgataag ggccagttcc 2460 gccagctcat taagagccag ttcatgggcg ttggcatgat ggccgtcatg catctgtact 2520 tcaagtacac caacgctctt ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct 2580 ggttaagatc cacgtcttcg ggaagccagc gactggtgac ctccagcgtc cctttaaggc 2640 tgccaacagc tttctcagcc agggccagcc caagaccgac aaggcctccc tccagaacgc 2700 cgagaagaac tggaggggtg gtgtcaagga ggagtaagct ccttattgaa gtcggaggac 2760 ggagcggtgt caagaggata ttcttcgact ctgtattata gataagatga tgaggaattg 2820 gaggtagcat agcttcattt ggatttgctt tccaggctga gactctagct tggagcatag 2880 agggtccttt ggctttcaat attctcaagt atctcgagtt tgaacttatt ccctgtgaac 2940 cttttattca ccaatgagca ttggaatgaa catgaatctg aggactgcaa tcgccatgag 3000 gttttcgaaa tacatccgga tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa 3060 cgtggcacta ttgatcatcc gatagctctg caaagggcgt tgcacaatgc aagtcaaacg 3120 ttgctagcag ttccaggtgg aatgttatga tgagcattgt attaaatcag gagatatagc 3180 atgatctcta gttagctcac cacaaaagtc agacggcgta accaaaagtc acacaacaca 3240 agctgtaagg atttcggcac ggctacggaa gacggagaag ccaccttcag tggactcgag 3300 taccatttaa ttctatttgt gtttgatcga gacctaatac agcccctaca acgaccatca 3360 aagtcgtata gctaccagtg aggaagtgga ctcaaatcga cttcagcaac atctcctgga 3420 taaactttaa gcctaaacta tacagaataa gataggtgga gagcttatac cgagctccca 3480 aatctgtcca gatcatggtt gaccggtgcc tggatcttcc tatagaatca tccttattcg 3540 ttgacctagc tgattctgga gtgacccaga gggtcatgac ttgagcctaa aatccgccgc 3600 ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc 3660 tttctggcat gcggagagac ggacggacgc agagagaagg gctgagtaat aagccactgg 3720 ccagacagct ctggcggctc tgaggtgcag tggatgatta ttaatccggg accggccgcc 3780 cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg accaagaatc tattgcatca 3840 tcggagaata tggagcttca tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag 3900 gggtgtatag ccgtcggcga aatagcatgc cattaaccta ggtacagaag tccaattgct 3960 tccgatctgg taaaagattc acgagatagt accttctccg aagtaggtag agcgagtacc 4020 cggcgcgtaa gctccctaat tggcgaattc ccatccggca tctgtagggc gtccaaatat 4080 cgtgcctctc ctgctttgcc aggtgtatga aaccggaaag gccgctcagg agctggccag 4140 cggcgcagac cgggaacaca agctggcagt cgacccatcc ggtgctctgc actcgacctg 4200 ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc tgtccgcccg gtgtgtcggc 4260 ggggttgaca aggtcgttgc gtcagtccaa catttgttgc catattttcc tgctttcccc 4320 accagctgct cttttctttt ctctttcttt tcccatcttc agtatattca tcttcccata 4380 tgcatccaag aacctttatt tcccctaagt aagtactttg ctacatccat actccatcct 4440 tcccatccct tattcctttg aacctttcag ttcgagcttt cccacttcat cgcagcttga 4500 ctaacagcta ccccgcttga gcagacatca ccatggacac caccgtgccg accttctccc 4560 tcgccgagct ccagcagggt ctgcaccagg acgagttccg gcggtgtctg cgcgacaagg 4620 ggctgttcta cctgaccgac tgcggcctaa cagacacgga gctcaagtcc gccaaggata 4680 tcgtcatcga ctttttcgag cacgggagcg aggccgagaa gcgggcggtc accagccccg 4740 tcccgacgat gcgccgcggc ttcaccggtc tggagtccga gtcgaccgcc caaatcacca 4800 acaccggatc ctactccgac tacagcatgt gttactccat ggggacggcc gacaacctct 4860 tcccctccgg ggatttcgag cgcatctgga cccagtactt cgaccgccag tacaccgcca 4920 gcagggccgt ggccagggag gtcctgcgcg ccaccgggac ggaaccggac ggcggggtgg 4980 aggccttcct ggactgcgaa ccgcttctgc ggttccggta cttcccccag gtccccgagc 5040 atcggtccgc cgaggagcag ccgctccgga tggcccccca ctacgacctg tccatggtga 5100 ccctcatcca gcagaccccg tgcgcaaacg ggttcgtgtc gctccaggcc gaggtgggcg 5160 gcgcgttcac agacctccct taccgacccg acgcggtgtt ggtcttctgc ggcgccatcg 5220 ccaccctcgt gacgggcggc caggtcaagg ccccccggca ccacgtggcc gccccgcggc 5280 gtgaccagat cgccggaagc agccgcacgt cgtccgtgtt cttcctgcgc ccgaacgcgg 5340 acttcacatt ctccgtcccc ctcgcgcggg agtgcggctt cgacgtctcc ctggacgggg 5400 agaccgccac gttccaggac tggatcggcg gcaactacgt caacatccgg cggacctcga 5460 aggcgtagta aaagcttatc gataccgtcg acctcgagac ttaacgttac tgaaatcatc 5520 aaacagcttg acgaatctgg atataagatc gttggtgtcg atgtcagctc cggagttgag 5580 acaaatggtg ttcaggatct cgataagata cgttcatttg tccaagcagc aaagagtgcc 5640 ttctagtgat ttaatagctc catgtcaaca agaataaaac gcgttttcgg gtttacctct 5700 tccagataca gctcatctgc aatgcattaa tgcattgact gcaacctagt aacgcctttc 5760 aggctccggc gaagagaaga atagcttagc agagctattt tcattttcgg gagacgagat 5820 caagcagatc aacggtcgtc aagagaccta cgagactgag gaatccgctc ttggctccac 5880 gcgactatat atttgtctct aattgtactt tgacatgctc ctcttcttta ctctgatagc 5940 ttgactatga aaattccgtc accagcccct gggttcgcaa agataattgc atgtttcttc 6000 cttgaactct caagcctaca ggacacacat tcatcgtagg tataaacctc gaaatcagtt 6060 cctactaaga tggtatacaa tagtaaccat gcatggttgc ctagtgaatg ctccgtaaca 6120 cccaatacgc cggccgaaac ttttttacaa ctctcctatg agtcgtttac ccagaatgca 6180 caggtacact tgtttagagg taatccttct ttctagaagt cctcgtgtac tgtgtaagcg 6240 cccactccac atctccactc gatggtaccc aattcgccct atagtgagtc gtattacgcg 6300 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 6360 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 6420 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc 6480 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6540 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6600 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6660 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6720 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6780 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 6840 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6900 atattaacgc ttacaattta g 6921 <210> 67 <211> 7059 <212> DNA <213> Artificial Sequence <220> <223> pB-E8cast <400> 67 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220 gccgcccttg tatctctaca cacaggctca aatcaataag aagaacggtt cgtctttttc 2280 gtttatatct tgcatcgtcc caaagctatt ggcgggatat tctgtttgca gttggctgac 2340 ttgaagtaat ctctgcagat ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa 2400 gcggcgagga gcctcgtcct gtcacaacta ccaacatgga gtacgataag ggccagttcc 2460 gccagctcat taagagccag ttcatgggcg ttggcatgat ggccgtcatg catctgtact 2520 tcaagtacac caacgctctt ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct 2580 ggttaagatc cacgtcttcg ggaagccagc gactggtgac ctccagcgtc cctttaaggc 2640 tgccaacagc tttctcagcc agggccagcc caagaccgac aaggcctccc tccagaacgc 2700 cgagaagaac tggaggggtg gtgtcaagga ggagtaagct ccttattgaa gtcggaggac 2760 ggagcggtgt caagaggata ttcttcgact ctgtattata gataagatga tgaggaattg 2820 gaggtagcat agcttcattt ggatttgctt tccaggctga gactctagct tggagcatag 2880 agggtccttt ggctttcaat attctcaagt atctcgagtt tgaacttatt ccctgtgaac 2940 cttttattca ccaatgagca ttggaatgaa catgaatctg aggactgcaa tcgccatgag 3000 gttttcgaaa tacatccgga tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa 3060 cgtggcacta ttgatcatcc gatagctctg caaagggcgt tgcacaatgc aagtcaaacg 3120 ttgctagcag ttccaggtgg aatgttatga tgagcattgt attaaatcag gagatatagc 3180 atgatctcta gttagctcac cacaaaagtc agacggcgta accaaaagtc acacaacaca 3240 agctgtaagg atttcggcac ggctacggaa gacggagaag ccaccttcag tggactcgag 3300 taccatttaa ttctatttgt gtttgatcga gacctaatac agcccctaca acgaccatca 3360 aagtcgtata gctaccagtg aggaagtgga ctcaaatcga cttcagcaac atctcctgga 3420 taaactttaa gcctaaacta tacagaataa gataggtgga gagcttatac cgagctccca 3480 aatctgtcca gatcatggtt gaccggtgcc tggatcttcc tatagaatca tccttattcg 3540 ttgacctagc tgattctgga gtgacccaga gggtcatgac ttgagcctaa aatccgccgc 3600 ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc 3660 tttctggcat gcggagagac ggacggacgc agagagaagg gctgagtaat aagccactgg 3720 ccagacagct ctggcggctc tgaggtgcag tggatgatta ttaatccggg accggccgcc 3780 cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg accaagaatc tattgcatca 3840 tcggagaata tggagcttca tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag 3900 gggtgtatag ccgtcggcga aatagcatgc cattaaccta ggtacagaag tccaattgct 3960 tccgatctgg taaaagattc acgagatagt accttctccg aagtaggtag agcgagtacc 4020 cggcgcgtaa gctccctaat tggcgaattc ccatccggca tctgtagggc gtccaaatat 4080 cgtgcctctc ctgctttgcc aggtgtatga aaccggaaag gccgctcagg agctggccag 4140 cggcgcagac cgggaacaca agctggcagt cgacccatcc ggtgctctgc actcgacctg 4200 ctgaggtccc tcagtccctg gtaggcagct ttgccccgtc tgtccgcccg gtgtgtcggc 4260 ggggttgaca aggtcgttgc gtcagtccaa catttgttgc catattttcc tgctttcccc 4320 accagctgct cttttctttt ctctttcttt tcccatcttc agtatattca tcttcccata 4380 tgcatccaag aacctttatt tcccctaagt aagtactttg ctacatccat actccatcct 4440 tcccatccct tattcctttg aacctttcag ttcgagcttt cccacttcat cgcagcttga 4500 ctaacagcta ccccgcttga gcagacatca ccatgcatcg cgcgggcggc agcgggctcg 4560 tccccagtgg ccgcatcgcc ggctattggc gatgcgagcg gccctggcgc atgcagcgcc 4620 gatcccgctg ctcgaccaag gccctggtga tgctggacac ccccctggcg aaggtgccga 4680 ccctcagtct cgcagaccag gcagtcgatc ccgaccgctt cgccgccgac ctgggcggtt 4740 cgttcaggcg gttcggcttc gccatcgtcg cggaccatgg catcccggac gacctcatcg 4800 cccgggcctg ggcgcagacc gaggccctgt tcgcccagcc ggacgccgac aagcggggct 4860 actacgtgga aggcgcgggc ggggcccgcg gctacacccc gttcaagacc gagatcgcga 4920 aggacgccaa gcacgtcgac ctgaaggagt tctggcacgt ggggcgagag ctgcccacgg 4980 gccaccggta cgcgggtgag atggccccga acatctggcc cgcccagccc gatggcttca 5040 aggagacctt cgtcgagctg ttccgcgctt tcgacacggc gggcgacaag ctcctgagcg 5100 ccatcgcccg ggacctcggg ctggcgccgg actggttcga cctggcggtc aaggacggca 5160 actccgtcct ccgcctgctg cactaccccc cgatcccgat ggacgcggag ggcgtccgcg 5220 cgggagccca tgaggacatc aacctgatca cccttctcct gggtgccgag gaggctggac 5280 tggagctgct cgaccgcgac agcgggaatt ggctggccat caagcccccg gagggcgcga 5340 tggtagtgaa cgtcggggac atgctgcagc ggttgaccaa ccacgtcctg ccgtccacca 5400 cccaccgcgt cgtcaatccg cccccggagc gccgggccca ctcccgctac agcatgccct 5460 tctttctgca cccggccccc gacttcctga tcaagaccct gccccagacc gtcacctcct 5520 ccaacccgaa ccggtacccc accccgatca cggcgcacga ctacctccat gagcggcttg 5580 tggagatcgg tctcatcaag aagtgataaa agcttatcga taccgtcgac ctcgagactt 5640 aacgttactg aaatcatcaa acagcttgac gaatctggat ataagatcgt tggtgtcgat 5700 gtcagctccg gagttgagac aaatggtgtt caggatctcg ataagatacg ttcatttgtc 5760 caagcagcaa agagtgcctt ctagtgattt aatagctcca tgtcaacaag aataaaacgc 5820 gttttcgggt ttacctcttc cagatacagc tcatctgcaa tgcattaatg cattgactgc 5880 aacctagtaa cgcctttcag gctccggcga agagaagaat agcttagcag agctattttc 5940 attttcggga gacgagatca agcagatcaa cggtcgtcaa gagacctacg agactgagga 6000 atccgctctt ggctccacgc gactatatat ttgtctctaa ttgtactttg acatgctcct 6060 cttctttact ctgatagctt gactatgaaa attccgtcac cagcccctgg gttcgcaaag 6120 ataattgcat gtttcttcct tgaactctca agcctacagg acacacattc atcgtaggta 6180 taaacctcga aatcagttcc tactaagatg gtatacaata gtaaccatgc atggttgcct 6240 agtgaatgct ccgtaacacc caatacgccg gccgaaactt ttttacaact ctcctatgag 6300 tcgtttaccc agaatgcaca ggtacacttg tttagaggta atccttcttt ctagaagtcc 6360 tcgtgtactg tgtaagcgcc cactccacat ctccactcga tggtacccaa ttcgccctat 6420 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac 6480 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 6540 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 6600 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 6660 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 6720 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 6780 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 6840 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 6900 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 6960 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 7020 aacgcgaatt ttaacaaaat attaacgctt acaatttag 7059

Claims (17)

(a) preparing a CPC (cephalosporin C) highly productive Acremonium chrysogenum strain;
(b) deleting the CefEF and CefG genes of the strain; And
(c) DAOC (Deacetoxycephalosporin C) high productivity acremonium cream comprising transforming the strain of step (b) with an expression vector containing the CefE gene sequence of Mycobacterium abscessus Method for producing a sagenum strain.
(a1) preparing a CPC (cephalosporin C) highly productive acremonium chrysogenum strain;
(b1) deleting the CefEF and CefG genes of the strain;
(c1) Amycolatopsis lactamdurans , Gordonia rubripertincta , Microbacterium hydrocarbonoxydans , Nannocystis exedens , Capital Monas At least one microorganism selected from the group consisting of Pseudomonas synringae , Streptomyces clavuligerus , and Sphingomonas dokdonensis , and Mycobacterium abscessus Transforming the strain of step (b1) with each expression vector containing the CefE gene nucleotide sequence of; And
(d1) DAOC (Deacetoxycephalosporin C) high productivity acremonium chrysogenum strain production method comprising the step of protoplast fusion (protoplast fusion) of each of the transformed strains.
The method of claim 1, wherein the CefE gene of Mycobacterium abscessus consists of the nucleotide sequence of SEQ ID NO: 3.
The method of claim 2, wherein the CefE gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 8.
The method according to claim 1 or 2, wherein the CPC high productivity acremonium chrysogenum strain of step (a) or step (a1) is a strain having an accession number of KCTC13079BP.
The method of claim 1 or 2, wherein the CefEF and CefG genes of the step (b) or step (b1) are deleted, and the CPC high-productivity acremonium chrysogenum strain is A method, characterized in that it is a strain having an accession number of KCTC 13922BP.
The method of claim 1 or 2, wherein the CefE in step (c) or step (c1) is operably linked to a promoter consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9 to 13 Way.
The method of claim 1, wherein the expression vector comprises a nucleotide sequence of SEQ ID NO: 16.
The method of claim 2, wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14 to 21.
The method according to claim 1 or 2, wherein the DAOC-producing ability of the high-producing acremonium chrysogenum strain of the DAOC is 30 g/L or more.
Acremonium chrysogenum strain prepared according to the method of claim 1 or 2.
The strain according to claim 11, wherein the DAOC-producing ability of the strain is 30 g/L or more.
(i) culturing the strain of claim 11 under conditions capable of producing DAOC; And
(ii) a method for producing DAOC comprising the step of recovering DAOC from the medium.
The production method according to claim 13, wherein the culture in step (i) is a fermentation culture.
Consisting of the nucleotide sequence of SEQ ID NO: 3, a polynucleotide for high DAOC expression in acremonium chrysogenum strain.
Consisting of the nucleotide sequence of SEQ ID NO: 16, an expression vector for high DAOC expression in acremonium cresogenum strain.
(I) culturing the strain of claim 11 under conditions capable of producing DAOC;
(II) recovering DAOC from the medium; And
(III) 7-ADCA (7-aminodeacetoxycephalosporanic acid) production method comprising the step of treating CPC (Cephalosporin C) acylase on the recovered DAOC.

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