KR100986292B1 - A replication factor of lactic acid bacteria and vector comprising the same - Google Patents

Abstract

The present invention relates to a novel lactic acid bacteria vector, specifically, a replication factor having a nucleotide sequence of SEQ ID NO: 1, a replicon comprising a nucleotide sequence of SEQ ID NO: 1, a plasmid having a nucleotide sequence of SEQ ID NO: 2, and It relates to a shuttle vector prepared by connecting a plasmid having the nucleotide sequence of SEQ ID NO: 2 and E. coli derived vector. The plasmid of the present invention is a theta-type vector isolated from the kimchi lactic acid bacterium, Leukonostock Citrium, which is stably introduced into and replicates the kimchi lactic acid bacteria including the leukonostock citrium, and thus the desired external gene and usefulness in kimchi lactic acid bacteria. Since it can be used as a backbone vector that can be used for gene expression, it is expected to be applied to various industries.

Kimchi Lactobacillus, Leukotostock Citrium, Plasmid, Theta Vector, Shuttle Vector

Description

Kimchi Lactobacillus Replication Factor and Vector Containing the Same {A REPLICATION FACTOR OF LACTIC ACID BACTERIA AND VECTOR COMPRISING THE SAME}

The present invention relates to an E. coli-lactic acid shuttle vector plasmid prepared by linking the plasmid isolated from kimchi lactic acid bacteria, specifically, leuconosstock citrium, and the plasmid and E. coli derived vector.

Recently, research has been actively conducted to mass produce high value-added substances beneficial to the human body using microorganisms, and among them, a gene expression system using lactic acid bacteria has emerged as a new expression system.

The gene expression system using the lactic acid bacteria is to develop lactic acid bacteria having a new function in addition to the original characteristics of lactic acid bacteria, the introduction of the foreign gene safely into the lactic acid bacteria, that is, the research for transformation.

Conjugation method or protoplast method, which was used early in the transformation of lactic acid bacteria, has not only low transfection efficiency but also has a problem of time consuming and poor reproducibility. In order to solve this problem, an electropoartion using a plasmid has been proposed. In order to perform the transformation using the electroporation method, an essential element is a carrier capable of delivering a foreign gene, that is, a vector.

In general, the vector present in lactic acid bacteria is a circular bacterial plasmid. The circular bacterial plasmid uses two methods, rolling-circle replication (RCR) and theta, as a method used for propagation. RCR-type plasmids, which are widely found in Gram-positive bacteria, produce a single-stranded intermediate during replication, resulting in large segregation instability, generation of polymeric plasmid multimers, and insertion of DNA. In this case, there is a problem that the replication stability decreases due to a problem such as a decrease in the copy number, so that it can be more stably replicated by double strand growth than a single-stranded replication intermediate. The development of theta type plasmids is required.

Kimchi is a traditional Korean fermented food made from Chinese cabbage or radish as a main ingredient and fermented with various spices such as garlic, ginger, green onion, red pepper powder, and salted fish. During fermentation, various low-molecular substances are produced by various microorganisms such as lactic acid bacteria. It is reported to not only have a unique taste and aroma, but also have various functionalities. Therefore, much research is being conducted to isolate new lactic acid bacteria having useful functions from kimchi.

Leuconostoc among the lactic acid bacteria sp.) is a type of heterofermentive lactic acid bacteria. In addition, leuconosstock is a lactic acid bacterium that plays an important role in the fermentation of vegetables, including sauerkraut, pickle and silage, and in particular, leuconosstock is a dominant bacterium that appears in the early-medium term fermentation of kimchi. As a result, it is reported that organic acids and carbon dioxide produced during fermentation of kimchi influence the characteristic taste of kimchi. In particular, Leuconostoc citreum ( Luconostoc citreum ) is reported as a bacteria that are separated from the raw material of kimchi, such as cabbage and garlic.

Until recently, many Leukotostock genera have been isolated from kimchi, some of which contain microorganisms of one or more plasmids of various sizes, and studies have been carried out to characterize them.

Most of the plasmids isolated in the leukonostock are related to RCR-type plasmids, and studies on non-RCR-type plasmids are very limited. Studies on non-RCR plasmids include Leuconostoc mesenteroides ssp. pTXL1 and Leuconostoc isolated from mesenteroides Y110 Only pFMBL1 isolated from mesenteroides SY2 and the like, and sequencing and replication mechanisms of plasmids derived from leukonostock citrium, Leu . This is a cryptic plasmid pIH01 of 1.8 kb, an RCR plasmid isolated from citreum IH3.

As is well known, most lactic acid bacteria plasmid vectors are Lactococcus , Lactobacillus , Leuconostoc , Pediococcus and Streptococcus isolated from dairy fermented foods such as milk or cheese. the lactic acid bacteria fermented milk products, such as origin (Streptococcus) and Bifidobacterium (Bifidobacterium), most of them having in particular a plasmid is a plasmid RCR type. As described above, the RCR plasmid has a disadvantage in that replication stability is poor.

Accordingly, in order to overcome this problem, it is possible to insert large DNA inserts among kimchi lactic acid bacteria vectors and to develop structurally stable theta plasmids or theta type vectors.

However, theta-type plasmid is a host-narrow range vector having a narrow host range, and it is difficult to match a host suitable for expression of a protein using the developed vector. Therefore, while developing a useful theta-type plasmid, the development of the expression host must be made together. In particular, as described above, the lactic acid bacteria vector relates to lactic acid bacteria isolated from fermented food products such as milk or cheese. Since the research on kimchi lactic acid bacteria has been limited, the development of theta type plasmid that can use kimchi lactic acid bacteria as a host is almost impossible. There is no situation. Currently, studies on theta-type plasmids that can utilize kimchi lactic acid bacteria as expression host microorganisms have been carried out by Leu . Leu reported to be capable of expressing heterologous genes in citreum . pSJEaga (a derivative of theta type pFMBL1), a theta-type plasmid derived from mesentroides, and its host Leu . The only study on citreum KCTC3526 (ATCC49370) is available.

Our traditional fermented food is consumed every day in almost every process in Korea, and is widely used in various kimchi lactic acid bacteria including leukonostock citrium, which is a kind of dominant species from the early fermentation of kimchi, which is recently recognized for its various functionalities. There is a need for development of theta-type plasmid and vector system using the same, which can be used as a shuttle vector that can be used together with a vector available in E. coli for efficient replication and use.

An object of the present invention is to provide a replication factor usable for theta plasmid.

It is also an object of the present invention to provide a replicon comprising the replication factor.

Moreover, an object of this invention is to provide the theta type | mold plasmid which can be used for kimchi lactic acid bacteria.

In addition, an object of the present invention is to provide a shuttle vector prepared by connecting the theta-type plasmid available to kimchi lactic acid bacteria and E. coli derived vector.

In order to achieve the above object, the present invention provides a replication factor having a nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a replicon comprising a nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a plasmid having the nucleotide sequence of SEQ ID NO: 2.

The present invention also provides a shuttle vector prepared by connecting a plasmid having the nucleotide sequence of SEQ ID NO: 2 and E. coli derived vector.

The present invention also provides a transformant transformed with the shuttle vector.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention, while studying a gene expression system that can safely introduce and express foreign genes to kimchi lactic acid bacteria isolated from kimchi, which is a traditional Korean food, a large number of dominant species in the early fermentation of kimchi Isolate pCC3, a setter type plasmid capable of stably replicating and expressing genes from citrum ( Leuconostoc citreum ), and analyzing the replication form of the plasmid pCC3, and then shuttle vector recombined with E. coli derived vector (shuttle vector) was prepared, and the shuttle vector was introduced into a strain of leuconosstock citrium, a lactic acid bacterium host cell suitable for transformation using the shuttle vector, thereby confirming that transformation was stably possible and completed the present invention.

In the present invention, "plasmid" has the same meaning as is commonly known in the technical field to which the present invention belongs, that is to say cyclic non-chromosomal elements present in bacteria. Preparation of Plasmids, Plasmids One of ordinary skill in the art to which the present invention pertains for the transfer and binding of DNA, transformation, and the like may use known methods, which are described in Sambrook, J. et al. , 'Molecular Cloning: A Laboratory Manual, Second Edition', Cold Spring Harbor Laboratory Press (1989).

In the present invention, "replicon" refers to a genetic material that is continuously replicated by the action of a replication factor called a initiator, a replication control factor or a replication initiation protein (rep protein). In a unit structure, the replication may proceed continuously starting at an origin of replication (Ori) or a replicator, etc. The replicon may be a chromosomal DNA of a bacterial species, a DNA or RNA of a virus or a plasmid in an autonomous cytoplasm. , Preferably a plasmid.

The present invention provides a plasmid isolated from Leuconostoc citreum .

The plasmid is a novel plasmid isolated from leukonostock citrium that has not been reported so far, named pCC3. The plasmid pCC3 is 3,338bp, G + C content is 33.2%, and there are three types of ORFs (ORF1, ORF2 and ORF3), which are expected to be expressed according to the size of the expressed amino acid, and seven plaindromic seuqence. (Inverted repeat: IRI- Ⅶ) and 7 direct repeats (DRI- Ⅶ) may exist.

The plasmid may be preferably a plasmid having a nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of SEQ ID NO: 2 may include a lactic acid bacterium having a nucleotide sequence of SEQ ID NO: 1, preferably, a replication factor derived from leukonostock citrium. The plasmid having the nucleotide sequence of SEQ ID NO: 2 was named pCC3.

ORF1 of the pCC3 has a molecular weight of 23,417 Da, and has not been identified with a ribosomal binding protein (RBS) or a -35 / -10 box sequence found at the start of transcription of various genes. It was determined that there was no sequence association and it was not confirmed exactly what function the gene was.

The ORF2 of pCC3 has a molecular weight of 22,272 Da and 100% homology with the DNA-binding protein gene of Leuconostoc citreum pCB42 (Luconostoc citreum pCB42), on top of which -35 / -10 box sequence was identified.

ORF3 of pCC3 has a molecular weight of 15,654 Da and is a setter plasmid, leuconosstock mesenteroides pTXL1 ( Leuconostoc). mesenteroides pTXL1) and flow Pocono stock mesen teroyi des pFMBL1 replication initiation protein (rep protein of (Leuconostoc mesenteroides pFMBL1): replication initiation protein) and were respectively show a low homology of about 62% and 61%. Above the ORF3, there was an AGAG sequence expected to be RBS, and between the two IRs (IRIII and IV) and the repeated DR IV was a sequence estimated as promoter (-35 / -10 box). The ORF3 has been identified as a gene encoding a protein essential for replication of the plasmid, and preferably may be a polynucleotide having a sequence of SEQ ID NO: 3. In addition, the replication initiation protein encoded by ORF3 may preferably be a polypeptide having the amino acid sequence of SEQ ID NO: 4.

In addition, in relation to the replication of the plasmid pCC3, it was confirmed that ORF1 and ORF2 had no influence on the progress of replication of the plasmid, and ORF3 was determined as the replication initiating protein to determine whether or not pCC3 was replicated.

In addition, the present invention relates to a vector including a multiple cloning site (MCS) and a selection marker, in addition to the plasmid having the nucleotide sequence of SEQ ID NO: 2. The vector may be a cloning vector. In addition, the cloning vector may further include one or more selected from the group consisting of a replication origin and a promoter of E. coli, in addition to a plasmid having a nucleotide sequence of SEQ ID NO: 2, and a selection marker.

The cloning vector is capable of stably replicating and expressing foreign genes in kimchi lactic acid bacteria, especially leukonostock citrium, one of the strains known as predominant species in kimchi when kimchi is properly ripened, thus transforming or transforming kimchi lactic acid bacteria or kimchi lactic acid bacteria. It is very useful to manufacture a gene expression system using, and can be usefully used for industrial improvement of probiotics.

The MCS refers to a DNA fragment having various restriction enzyme sites, and the MCS is recognized and cleaved by a specific restriction enzyme, thereby allowing insertion of a target gene into the cleaved MCS site. The MCS may be used without limitation as long as it is known to those skilled in the art to which the present invention belongs, various vectors known in the art belonging to the present invention having MCS, preferably E. coli vectors, One example may be derived from pUC18 or pUC19.

In addition, the selection marker (selection marker) means a gene that can select whether the gene is cloned, that is, whether the vector is inserted into the host cell or transformed, and for drug resistance, nutritional requirements, cytotoxic agents, etc. It may be a gene that confers a selectable phenotype, such as resistance or expression of surface proteins. The selection marker may preferably be an antibiotic resistance gene, a chromogenic enzyme gene or a luminescent / fluorescent gene. In addition, various genes known as selection markers in the art to which the present invention pertains may be used. The selection marker may be derived from various vectors known in the art to which the present invention pertains, preferably for E. coli vectors, for example pUC18 or pUC19.

The antibiotic resistance gene may be an erythromycin resistance gene, neomycin resistance gene, hygromycin resistance gene, ampicillin resistance gene, kanamycin resistance gene, chloramphenicol acetyl transferase gene, and the like, but is not limited thereto. May be a gene.

The chromogenic enzyme gene may be a β-galactosidase gene or a β-glucuronidase (GUS) gene and the like, but is not limited thereto. The light emitting gene may be a luciferase gene or the like, and the fluorescent gene may be a BFP gene, a CFP gene, a GFP gene, a YFP gene, an RFP gene, or the like, but is not limited thereto.

The present invention also relates to a replication factor having the nucleotide sequence of SEQ ID NO: 1. The replication factor may be a lactic acid bacteria-derived plasmid, preferably a leuconosstock citrium-derived plasmid, more preferably a replication factor contained in the pCC3.

The replication factor of pCC3 may include a sequence estimated as a promoter (-35 / -10 box) between ORF3 of SEQ ID NO: 3, two IRs (IRIII and IV) of the upper base sequence, and DRIV which is repeated. Preferably, it may be a replication factor having a nucleotide sequence of SEQ ID NO: 1. The replication factor is a replication factor of the plasmid isolated from the leukonostock citrium, the replication factor may be used as a replication factor of the plasmid of kimchi lactic acid bacteria, including the leukonostock citrium, shuttle vector using the same or recombinant plasmid using the same. have.

In the case of the plasmid containing only the sequence of ORF3 identified as the replication initiation protein, the inventor of the present invention confirmed that the plasmid was not replicated, and thus the replication including the sequence estimated as its promoter together with the sequence of ORF3. The factor was determined.

The present invention also relates to a replicon comprising the replication factor.

The replication of the pCC3 plasmid does not proceed when the sequence includes only the sequence of ORF3 as described above, and the sequence between the sequence of ORF3 and the two IRs (IRIII and IV), which are base sequences existing on the upper portion of the ORF3, and the repeated DR IV. It may proceed if it contains a sequence estimated to be a promoter included in (promoter).

Therefore, the replicon includes a sequence estimated as a promoter included between the sequence of the ORF3 and two IRs (IRIII and IV), which are nucleotide sequences existing on the upper portion of the ORF3, and the repeated DR IV. It may include a replication factor, the replication factor may be preferably a replication factor having a nucleotide sequence of SEQ ID NO: 1.

It was confirmed that the replicon including the replication factor can be safely cloned and expressed by transforming the leukonostock citrium belonging to the kimchi lactic acid bacteria.

In addition, the replicon is characterized in that it comprises the replication factor, together with the plasmid present in the kimchi lactic acid bacteria containing leuconosstock citrium, preferably the plasmid of leuconosstock citrium, more preferably The plasmid of pCC3 or part thereof may be further included, or may further include a plasmid or part thereof capable of replicating in E. coli .

More specifically, the replicon may be a vector including an MCS and a selection marker, in addition to a replication factor having the nucleotide sequence of SEQ ID NO: 1. In addition, the replicon may be a plasmid having the nucleotide sequence of SEQ ID NO: 2.

The vector is capable of reliably copying and expressing a foreign gene in the kimchi lactic acid bacterium, especially in the early stage of fermentation of kimchi, in a large amount of dominant species, leukonostock citrium, and the expression of a gene using a transformant or kimchi lactic acid bacteria of kimchi lactic acid bacteria. Since the system can be manufactured, it is very useful, and industrially, it can also be useful for improving probiotics.

The present invention also relates to a shuttle vector prepared by linking a plasmid having the nucleotide sequence of SEQ ID NO: 2 with an E. coli derived vector.

The shuttle vector is a replication factor having a nucleotide sequence of SEQ ID NO: 1; MCS; And E. coli-derived vectors including a selection marker and an E. coli replication origin and a promoter, the shuttle vector has both a replication origin of Lactobacillus and an origin of replication of E. coli, thereby allowing mutual replication in Lactobacillus and E. coli. Therefore, the shuttle vector can replicate and express the gene of interest in lactic acid bacteria and Escherichia coli.

The shuttle vector of the present invention may be prepared by directly inserting the nucleotide sequence of the origin of replication of E. coli into the plasmid having the nucleotide sequence of SEQ ID NO: 2, or by connecting the plasmid having the nucleotide sequence of SEQ ID NO: 2 and the E. coli derived vector. Can be. The shuttle vector may be a shuttle vector capable of replicating both Escherichia coli and lactic acid bacteria.

The E. coli vector refers to E. coli-derived vectors or vectors capable of replicating in E. coli, E. coli-derived transformation vectors known to those skilled in the art, for example, pUC18 or pUC19, It is not limited to this.

The origin of replication of E. coli may be a replication origin contained in E. coli-derived transformation vector known to those skilled in the art. Since the base sequence of the origin of replication of E. coli is known to those of ordinary skill in the art to which the present invention belongs, those of ordinary skill in the art to which the present invention belongs to the known origin of replication of E. coli Can easily be inserted into the shuttle vector.

In order to insert the origin of replication of E. coli or to bind to E. coli-derived vector, the plasmid having the nucleotide sequence of SEQ ID NO: 2 was cut with a restriction enzyme to prepare a linear plasmid DNA, and then the E. coli derived from the same restriction enzyme. It can be prepared by linking a site containing the origin of replication of E. coli obtained by cutting with the vector or the restriction enzyme transformed by genetic engineering method (eg ligation).

Preparing a recombinant shuttle vector by directly inserting the nucleotide sequence of the origin of replication of E. coli into the plasmid having the nucleotide sequence of SEQ ID NO: 2 or connecting the plasmid having the nucleotide sequence of SEQ ID NO: 2 with the E. coli derived vector; The transformation method using the shuttle vector may be performed by a method known to those skilled in the art to which the present invention belongs, such as US Pat. No. 5,688,683 described with respect to the lactic acid bacteria-derived shuttle vector.

The shuttle vector, more specifically, the shuttle vector for expression in kimchi lactic acid bacteria and Escherichia coli is for replication and / or expression of the target gene in lactic acid bacteria and Escherichia coli.

The gene of interest may be inserted into the plasmid having the nucleotide sequence of SEQ ID NO: 2 or the shuttle vector prepared by connecting the plasmid having the nucleotide sequence of SEQ ID NO: 2 with the E. coli derived vector. Therefore, the present invention provides a recombinant vector in which the target gene is inserted into the plasmid or shuttle vector.

In the present invention, the target gene refers to a gene to be cloned in lactic acid bacteria or a gene to be expressed in a specific host cell.

The gene of interest may be inserted into the MCS present in the plasmid or shuttle vector of the present invention. For example, the target gene may be PCR amplified with a primer containing a restriction enzyme sequence so as to be inserted into the MCS of each vector, and then inserted into the MCS of a cloning vector or shuttle vector cut with the restriction enzyme and cut with the restriction enzyme. .

The gene of interest may be operably linked to a promoter. By “operably linked” is meant that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment. That is, the gene of interest may be linked so that its expression can be controlled by a promoter in the vector.

The gene of interest means all genes capable of transforming lactic acid bacteria, and more specifically, may be genes capable of enhancing activity, function, and / or usefulness as probiotics of lactic acid bacteria corresponding to host cells of transformation.

In particular, since the plasmid of the present invention is isolated from kimchi lactic acid bacteria isolated from kimchi, the plasmid of the present invention and the vectors prepared using the same are used for foods containing kimchi, preferably for enhancing human immunity, promoting growth, and preventing diseases. The target gene included in the functional food including the food which can be performed and which can impart or improve such functionality can be used as the target gene of the present invention.

Examples of medical, industrially useful object genes that can be inserted into the replicon, plasmid or shuttle vector of the invention, specifically into the MCS region of the replicon, plasmid or shuttle vector, include hormones, cytokines, enzymes, coagulation factors, There are genes encoding transport proteins, receptors, regulatory proteins, structural proteins, transcription factors, antigens, antibodies and the like.

In addition, the replicon, plasmid or shuttle vector of the present invention may further include an expression control sequence in addition to the promoter to control the expression of the gene of interest. "Expression control sequence" refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a particular host cell. In addition, the expression control sequence may include any operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence for controlling termination of transcription and translation, in addition to a promoter for initiating transcription. Include.

The shuttle vector may be specifically a shuttle vector prepared by connecting the plasmid having the nucleotide sequence of SEQ ID NO: 2 and pUC18. The shuttle vector may be capable of mutual replication in Escherichia coli and leukonostock citrium, may have a cleavage map as described in FIG. 8, preferably may have a nucleotide sequence of SEQ ID NO: 11.

In addition, the shuttle vector may further include an erythromycin resistance gene as a selection marker, preferably may have a nucleotide sequence of SEQ ID NO: 12.

In order to improve the transformation and replication efficiency of the kimchi lactic acid bacteria, specifically, leuconosstock citrium, it is preferable to minimize the size of DNA that can be replicated in leuconosstock citrium, and when the size of the DNA is small, Even after transformation, it is not only stable in kimchi lactic acid bacteria, specifically, leukonostock citrium, but also has excellent transformation efficiency.

Therefore, in order to reduce the size of the shuttle vector, the present invention may be a shuttle vector prepared by removing the ampicillin resistance gene contained in pUC18 as a selection marker, preferably a shuttle vector having a nucleotide sequence of SEQ ID NO: 13 Can be.

In addition, in order to further reduce the size of the shuttle vector, in the plasmid having the nucleotide sequence of SEQ ID NO: 2 may be a shuttle vector from which the sequence of ORF1 does not affect the replication of the gene, preferably the base of SEQ ID NO: 14 It may be a shuttle vector having a sequence, the size of the shuttle vector may be 4,910bp.

In addition, in order to further reduce the size of the shuttle vector, in the plasmid having the nucleotide sequence of SEQ ID NO: 2 may be a shuttle vector removing the sequence of ORF1 and ORF2 that does not affect the replication of the gene, preferably SEQ ID NO: 15 It may be a shuttle vector having a base sequence of, the size of the shuttle vector may be 3,612bp.

In addition, the present invention may be a recombinant vector in which the target gene is inserted into the shuttle vector. The gene of interest may preferably be inserted into the MCS region of the shuttle vector.

The present invention also provides a transformant transformed from a host cell with the vector, shuttle vector or recombinant shuttle vector inserted with the target gene in the shuttle vector.

The host cell of the transformant may be a bacterium, and the bacterium may be E. coli or lactic acid bacteria. The Escherichia coli may be a common Escherichia coli used in the preparation of a transformant, and the lactic acid bacteria may be a kimchi lactic acid bacterium, preferably, a strain of the genus Ryukonostok, more preferably, a strain of Ryukonostok citrium, for example Conostock Citrium GJ7 (KFCC 11322) or Leukonostock Citrium C16.

The transformed bacteria can be usefully used for mass production of proteins expressed from genes of interest. Thus, the transformed bacteria can be cultured to obtain the desired protein mass produced from the culture.

The present invention also relates to a method for producing a transformant comprising introducing the recombinant vector into a bacterium.

The bacterium is preferably E. coli or lactic acid bacteria.

The step of introducing the recombinant vector into the bacteria is a method known in the art, such as, but not limited to, a heat shock method (heat shock method, calcium phosphate precipitation method, electroporation, gene gun ( gene gun) and other known methods for introducing DNA into cells.

As described above, the replicon or the plasmid according to the present invention can be stably replicated and expressed in kimchi lactic acid bacteria, specifically leukonostock citrium, including the known strain, leukonostock citrium C16, Since the shuttle vector can be stably expressed in both Escherichia coli and lactic acid bacteria, it is isolated from kimchi, our native food, and enables the development of a gene expression system using lactic acid bacteria and probiotics using the same. It can be used in food and medicine, so the industrial use value will be very large.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1 Screening and Identification of Strains

About 100 kinds of kimchi lactic acid bacteria isolated from kimchi were inoculated in 5 ml of MRS (Difco aboratories, Detroit, Mich., U.S.A.) liquid medium and incubated at 30 ° C. for 12 hours to separate plasmid DNA.

The separated plasmid DNA was electrophoresed on a 0.8% agarose (Cambrex BioScience Rockland, Inc., ME, USA) gel to confirm the presence and size of the plasmid DNA. As a result, strain C4 with a small size (about 3.3kb or less) plasmid was selected and Leuconostoc as plasmid free strain without plasmid. citreum GJ7 (patent: 0528641, KFCC 11322), Leuconostoc GJ2 kimchi (Patent Application: 10-2006-0041059) and the final selection was to develop a host strain for the development of vector NJ2, DC3, C12, C16 and HJ5 newly separated, she was also shown in 2 the results.

2 is Lane1 is GJ2 strain, Lane2 is C16 strain, Lane3 is C12 strain, Lane4 is GJ7 strain, Lane5 is HJ5 strain, Lane6 is NJ26 strain, Lane7 is DC3 strain, Lane8 is C4 strain to be.

Isolation and sequencing of 16S ribosomal RNA from the final selection strain, C4 and C16 were Leuconostoc citreum , DC3 and HJ5 are Lactobacillus sakei and C12 were identified as Leuconostoc mesenteroides , and these strains were named Leuconostoc citreum C4, Leuconostoc citreum C16, Lactobacillus sakei DC3, Lactobacillus sakei HJ5, and Leuconostoc mesenteroides C12.

<Example 2> Leu. citreum And analysis of plasmid pCC3 from

Example 2-1. Leu. Isolation of pCC3 from citreum C4

Leu isolated in Example 1 above . citreum C4 was incubated at 30 ° C. for 12 hours using MRS liquid medium. Centrifugation (9,500 × g, 3min) of 5ml of the lactic acid bacteria culture medium to remove the culture supernatant, and the remaining pellet was suspended in 200μl of 25% sucrose solution to which lysozyme (30mg / ml) and mutanolysin (350 unit / ml) were added. Treatment for 15 minutes at. After the treatment was completed, the supernatant was removed by centrifugation (9,500 × g, 3 min) and plasmids were separated using a QIAprepSpin miniprep kit (QIAGEN Inc., USA).

Example 2-2. Analysis of pCC3

The isolated plasmid pCC3 was digested with various restriction enzymes, and was cleaved by EcoR V, Hind III, Acc I and Kpn I. In the E. coli vector processing a pCC3 treated with Kpn Ⅰ restriction enzymes to form a single cut with the same restriction enzymes pUC18 was inserted using T4 DNA ligase (TaKaRa), using Bigdye terminator cycle sequencing ready reaction kit (BigDye) The base sequence was determined. DNA and protein homology analysis and ORF analysis were analyzed using NCBI (http://www.ncbi.nlm.nih.gov/) program and DNASIS (HITACHI software Engineering Co. Janpan) program. 4 to 7 and Tables 1 to 3 are shown.

As a result of nucleotide sequence analysis of pCC3 using the NCBI program and the DNASIS program, the total base sequence was 3,338bp, the G + C content was 33.2%, and three ORFs (open reading frames) were expected to be expressed. The ORF was named ORF1, ORF2 and ORF3 depending on the size of the amino acid expressed. In addition, seven plaindromic sequences (Inverted repeat: IRI-VII) and seven direct repeats (DRⅠ-VII) were identified.

As a result of comparing the homology of the ORFs of pCC3 with the known proteins, it is not clear to date whether the ORF1 is a function of exactly 23,417 Da and its molecular weight. No found ribosomal binding protein (RBS) or -35 / -10 box sequence was identified. The ORF2 showed a 100% homology with the DNA-binding protein gene of Leuconostoc citreum plasmid pCB42 with a molecular weight of 22,272 Da, and identified the GGAA sequence and -35 / -10 box sequence expected by RBS.

The ORF3 had a molecular weight of 15,654 Da and Leuconostoc , theta replication plasmid, as shown in Table 2 below. The homologous replication protein (rep protein: replication initiation protein) of mesenteroides pTXL1 and Leuconostoc mesenteroides pFBL1 was approximately 62% and 61%, respectively. On top of ORF3 there was an AGAG sequence expected to be RBS, and between the two IRs (IRIII, IV) and the repeated DRIV was a promoter (-35 / -10 box) sequence. The above features are seen in plasmids with theta replication mechanism, and pCC3 was also expected to replicate by theta mechanism.

In addition, multiple alignment with the rep protein of theta replication plasmids was performed, and the results are shown in FIG. 7.

As shown in FIG. 7, the rep protein of pCC3 showed a closer relationship with the plasmid rep protein derived from Leuconostoc than the plasmid rep protein derived from Lactococcus or Lactobacillus .

From the above results, Leu . citreum Plasmid pCC3 from C4 was not known until now in new Leu . Theta replication plasmid from citreum was identified.

< Example  3> LAB - E. coli  Shuttle Vector

Based on the pCC3 sequencing results of Example 2, a shuttle vector replicated in E. coli and lactic acid bacteria was produced by the method described in FIG. 5.

More specifically, first, cutting the pCC3 using Kpn Ⅰ, and then inserted into the same restriction enzyme in place of the E. coli vector pUC18 was constructed pUCC3 (6,024bp) of SEQ ID NO: 11. PCR was performed to add the erythromycin resistance gene to the pUCC3. The Erythromycin resistance gene is a previously reported pIL2530α (Kim et al ., 1995, J Microbiol Biotechnol, 5: 257-263), and amplified by polymerase chain reaction (PCR) using primers Em-F and Em-R shown in Table 4 below.

designation 5 ’→ 3’ SEQ ID NO: Em-f   5′-TAGACGCGTATAAGACTTAGAAGCAAAC-3 ′ 9 Em-r   5′-TCTATGCATGTGTAACTTTCCAAATTTAC-3 ′ 10

The erythromycin resistance gene (911 bp) amplified by the PCR was inserted at the SmaI site in pUCC3E to construct pUCC3E (6,935 bp) of SEQ ID NO: 12.

To minimize the size of the shuttle vector, pUCC3E was digested with Eco0109Ⅰ-Eam1105Ⅰ, the ampicillin resistant gene (980 bp) was removed, the Klenowfragment was treated, end-filled, and blunt-end ligation to construct pUCC3E1 (5959 bp) of SEQ ID NO: 13 It was.

In addition, in order to determine the minimum replication factor that can be replicated in pCC3, as shown in Figure 9, a total of four types of shuttle vector in which one ORF is sequentially deleted from three ORF.

pUCC3E1-① removed ORF1 site (1,048bp) using restriction enzyme Xcm I- Kpn I, and then end-filled the remaining sequence site (2,290bp) by processing Klenowfragment and then inserted pCC3 of pUCC3E1.

pUCC3E1-②, like pUCC3E1-①, end-filled the 1,656bp DNA frgment containing ORF2 by inserting the restriction enzyme, Acc II, to confirm the replication of the ORF2 site and inserting it into pCC3 site of pUCC3E1.

pUCC3E1-③ was put up the remaining portion (1,911bp) except handles BstP Ⅰ- Kpn Ⅰ by ORF2 pCC3 in place of pUCC3E1.

Finally, to confirm the replication of the site including ORF3 and its specific base sequence, 991bp of DNA fragment was end-filled using Bpm I- Xcm I and put into pCC3 site of pUCC3E1 to pUCC3E1-④ Was constructed.

Example 4 LAB- E. coli Transformation of the shuttle vector

In order to investigate the replication of the E. coli -LAB shuttle vector pUCC3E1 prepared in Example 3, E. coli TG1 was used as an E. coli host strain, and Lactococcus lactis MG1363 (Gasson), a strain for lactic acid bacteria, was used. , MJ, 1983, J Bacteriol, 154: 1-9) and Leuconostoc citreum C4, Leuconostoc , a plasmid free strain isolated from Example 1 above. citreum C16, Lactobacillus sakei DC3, Lactobacillus sakei HJ5, Leuconostoc kimchi GJ2, Leuconostoc mesenteroides C12 was used.

Example 4-1. Transformation with E. coli TG1

E. coli -LAB shuttle vector pUCC3E1 was transformed into E. coli TG1 as a host cell using the CaCl ₂ method (Sambrook et al. al ., Molecular cloning (1989).

Example 4-2. Transformation into Lactococcus lactis MG1363 Strain

The transformation of E. coli -LAB shuttle vector pUCC3E1 to L. lactis MG1363 was carried out using the method reported by Holo and Nes (Holo, H and IF Nes, Appl. Environ.Microbiol, 60: 1401-1413 (1989)). It was.

Example 4-3. Transformation into Isolated Plasmid Free Strains

To transform into strain GJ7, GJ2, NJ2, DC3, C12, C16, HJ5, lactic acid bacteria precultured in MRS liquid medium containing 1% glucose was inoculated 1% in 100 ml of MRS liquid medium containing 1% glucose. Incubation was carried out at 30 ° C. until the absorbance of 600 nm was about 0.4 to 0.6.

After centrifugation of the culture (9,500 × g, 5 min, 4 ℃), the supernatant was discarded and the cells were washed twice with 10 ml of cold 10 mM MgCl ₂ solution, and then washed once with 10 ml of 0.5 M sucroese, 10% glycerol solution. The washed cells were suspended in 0.5 ml of 0.5 M sucroese, 10% glycerol solution. 10 μl (1 μg) of pUCC3E1 was added to 50 μl of the suspension, mixed, transferred to a 2 mm electroporation cuvette, and transformed into 2.5kv, 25μF, and 200Ω.

1 ml of fresh MRS medium was added for 3 hours at 30 ° C, and then plated on MRS plate medium containing erythromycin added at 5 μg / ml, and cultured at 30 ° C for 3 days to confirm colony formation. 5 is shown.

As shown in Table 5, pUCC3E1 was able to replicate in E. coli TG1 Escherichia coli host cells, the same Leu . Although replication was possible in citreum GJ7 and C16, Leu. kimchi , Leu. Not only mesenteroides but also Lactococcus or Lactobacillus were not transformed. This is a disadvantage of theta plasmid. In particular, although theta plasmid is structurally stable and can be inserted into a large size insert, it is difficult to find an appropriate host for its expression even when the host range is narrow and developed as a shuttle vector. There is this.

However, the shuttle vector pUCC3E1 developed in this study was able to build a host-vector system by developing a host suitable for transformation of the vector.

In addition, four vector vectors (pUCC3E1-①, pUCC3E1-②, pUCC3E1-③, pUCC3E1-④) constructed to determine the minimum replication factors that can be replicated in pCC3 are Leu . citreum C16 was transformed, and the results are shown in FIG. 9.

As shown in FIG. 9, ORF1 and ORF2 were not related to the replication of pCC3, and it was confirmed that ORF3 is a gene related to replication. In addition, in the case of pUCC3E1-③, Leu. The transfection was not performed in citreum C16, but in the case of including the upper nucleotide sequence of ORF3 gene in pUCC3E1-④. Therefore, OFR3 and its base sequence were found to be important factors in the replication of pCC3.

Example 5 Stability of pUCC3E1 in Transformants

Leu transformed with the pUCC3E1 shuttle vector in Example 4 . The stability of the shuttle vector in citreum C16 was confirmed.

Specifically, Leu containing pUCC3E1 . Citreum C16 cells were incubated in late MRS medium with erythromycin added at 5 μg / ml until the late exponential phase, and then inoculated in MRS liquid medium containing no antibiotics at 100-fold dilution and incubated at 30 ° C. After 24 hours of culture, the cells were inoculated in MRS liquid medium containing no antibiotics at 100-fold dilution conditions. Repeating this process, the culture solution was diluted 10-fold with fresh MRS liquid medium, and then the diluted culture solution was plated on MRS solid medium or solid medium containing antibiotics, respectively, to measure the number of cononyms generated. The stability (%) of cells containing pUCC3E1 was measured as shown in Equation 1 below.

[Calculation 1]

Leu . The generation time of citreum C16 was measured as 1.25 hours when incubated at 30 ℃ in MRS medium. Leu of pUCC3E1 . The stability results in citreum C16 are as described in Table 6 below.

As shown in Table 6, the 100 generation time is equal to 5.2 days, and the stability of pUCC3E1 was 94.2% on the 5th day of passage and 80.1% on the 9th day of passage.

The replication stability of the plasmid is most often required to obtain antibiotics that can cause economic and stability problems in the industrial use of microorganisms, as confirmed above, shuttle vector pUCC3E1 is Leu . Excellent transfection efficiency and high replication stability in citreum C16, unlike the previously studied vectors, sufficient replication is possible even in a medium without antibiotics, thus ensuring stability even when used in fermentation or bioconversion processes. The advantage is that you can.

< Example  6> ssDNA intermediate  Confirm

The presence of ssDNA intermediate was verified to confirm the replication mechanism of the pUCC3E1.

Leu with pUCC3E1 . citreum C16 was incubated at 30 ° C. in MRS liquid medium containing erythromycin (5 μg / ml) until the OD ₆₀₀ was 0.6-0.8. Whole-cell lysates were prepared according to the methods of Noirot-Gros and Ehrich, and treated with S1 nuclease and electrophoresed in 0.8% agarose gel. After the electrophoresis, the agarose gel was capillaly transferred using a 10 × SSC buffer solution to a nylon membrane (Amersham) without undergoing a denaturation step. Labeling was carried out using a DNA probe cut pCC3 with Kpn I and DIG high prime DNA labeling and detection starter kit I (Roche). As a control, pCW4 identified as RCR plasmid (Park et) al ., Plasmid, 52: 84-88 (2004)), and the results are shown in FIG. 10.

As shown in FIG. 10, in (A) pCW4, the fast migrating band disappeared by S1 nuclease treatment, so that ssDNA could be confirmed, but (B) ssDNA could not be confirmed in pUCC3E1. Thus, pCC3 was identified as a plasmid with theta replication mechanism.

1 is a diagram showing a phylogenetic relationship between leukonostock citrium and other flexible microorganisms according to an embodiment of the present invention.

Figure 2 is an electrophoresis picture confirming the size of the plasmid of lactic acid bacteria isolated from kimchi according to an embodiment of the present invention.

Figure 3 is a diagram showing a cleavage map of the plasmid pCC3 isolated from leukonostock citrium C4 according to an embodiment of the present invention.

4 is a diagram showing the nucleotide sequence of the plasmid pCC3 according to an embodiment of the present invention.

5 is a diagram showing the amino acid sequence of the ORF of the plasmid pCC3 according to an embodiment of the present invention.

6 is a diagram showing the entire nucleotide sequence of the plasmid pCC3 according to an embodiment of the present invention and its putative function.

7 is a diagram comparing the replication initiation protein of the plasmid pCC3 with another theta type plasmid according to an embodiment of the present invention. In FIG. 7, all base sequences in the same column are the same position, and conserved substitution; It means semi-conserved substitution.

8 is a diagram showing a manufacturing process and a cleavage map of the shuttle vector pUCC3E1 according to an embodiment of the present invention.

FIG. 9 is a diagram illustrating a cleavage map for fabricating a replicon including a portion of an ORF of pCC3 in order to secure a minimum replica replicon of pCC3 according to an embodiment of the present invention.

10 is to compare the generation of ssDNA intermediates in pUCC3E1 and pCW4 according to an embodiment of the present invention, + means that the endonuclease S1 treatment,-means no endonuclease S1, SS Is the site for single stranded DNA.

FIG. 11 is a diagram showing a 16S rDNA sequence of Leukonostock Citrium C16 according to an embodiment of the present invention. FIG.

<110> KIM, IN CHEOL <120> A REPLICATION FACTOR OF LACTIC ACID BACTERIA AND VECTOR          COMPRISING THE SAME <130> DPP20081997KR <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 663 <212> DNA <213> Replication factor_pCC3 <400> 1 gccattgcag aggtctaatt ctgtaatggc aaaaccctga tagttgtaat atctaggtca 60 ccaagtccta acccttgcag gggttgaggt ctttttattt gtatttaaat gttgatatga 120 gtatagcatt aagaactata catgacaata aacaaacaaa taatcttatg ttttaataat 180 taaacgacca acttaaaacg tcaacaatat agtaaactta aaacgtaaac tttacatcgc 240 ttaagtttac gtttggagag actgatgaca cacgaatttg ataccattat cgcgatagca 300 gacgaactag aaatatctcg tcaagcctta aatagaaagg ctaaacgcct taatatagat 360 ttatctaaaa agtcattcac cgataaagaa tggcaacttt tagtgtcaaa caaacgtaaa 420 cccaaaaagt caacttcaag taactatgtt gacactttta cagcgcaaca actagctgaa 480 aaagatgatt taattaatta tttaaaatca caaattaaag aaaaagataa acaaattgat 540 catgcgcaac aattacaatt aattgctgaa caaagattaa cagagacaaa taaaacccta 600 attgagtatc aagaaaaaga aaatcagcca aagaaaggat tctggcaaag gttatttaaa 660 tag 663 <210> 2 <211> 3338 <212> DNA <213> pCC3 plasmid <400> 2 ctaccgcgat tgtgagagct agaccaattt atttgagcta aattcacaaa ccccaatccc 60 ataaaaagcg ccaaaaccta ttcgaataag gtttggcgct tttcacttgg cataagatgg 120 attatgttaa gttcatatat gactactatg atttgttagc taaaatataa tattgctata 180 cctgtaccaa taaaaaagag atctagtgcg ataattatta ttttacctac tttgttgtaa 240 gggttaacac ttataccata gccaaaagct ttcggcaccc atagtggagg atttggcatt 300 ccttcataat tttgataaac aaccttatcg aatttgacag taacatgaat taaaatgata 360 gacagcatta aaataaatac tgtaacaaac tttgttaagc ttttattgct agttactaaa 420 ggtataatta ctactgccat aagtacaata agcacagtca atacaatcaa taagacagaa 480 tacttcttat tagccttttt cgtgttaatc aattcataat ccttttgtac tttattcatc 540 aattctgtat ctcctttgat gatatccaaa gaaaagttat aatagttgct taattctata 600 agcatgggaa tatctggaaa attttttcca ttttcccaat ttgacaaaga ttgcctagtg 660 ataaagagat tatctgcaac tttttgctgt gtttcatctt ttttctttcg ttgttcaact 720 aaaatagtag atagttccat atttgttccc ttcctacgcc attatattac tttgacaaca 780 aatttatttc tagcaaatat ttcttgcatt gttcatttag caatgataac aagggcaatc 840 agtttgattt gagcagagaa agccctaagt atttaggact tttaagattg ttcaaggtat 900 aattcagaat catatcgctt gtatcaacca cgtatatcgc tctaatttct cttctatcgt 960 ctgcccaata gaaaccctta cttttataag tcaataaggt tagaacacgt cagacaccgc 1020 ttaaatcgct ttatatgcga tttaaaccat atagtgtgct aattctatat gccacttctt 1080 tgccaattac ttgcgggcga gcctacccat aatcgaattg ttcattcccc acaatctcgc 1140 tttgattgct ttggacgtgg tttagacgac ctttcagccc tcttgacgga tacacgcctg 1200 ttccgtatta gttgttagga taacgtccga caacgattga acagacgtaa aaagcgtatc 1260 ctcgtgctta gttaggctgg aggatacgct ttcatagtcg tttatagttc ttttttgagt 1320 aaagctgttt actctgagtc tataagtttg tataatagta agtattctaa gccattgcag 1380 aggtctaatt ctgtaatggc aaaaccctga tagttgtaat atctaggtca ccaagtccta 1440 acccttgcag gggttgaggt ctttttattt gtatttaaat gttgatatga gtatagcatt 1500 aagaactata catgacaata aacaaacaaa taatcttatg ttttaataat taaacgacca 1560 acttaaaacg tcaacaatat agtaaactta aaacgtaaac tttacatcgc ttaagtttac 1620 gtttggagag actgatgaca cacgaatttg ataccattat cgcgatagca gacgaactag 1680 aaatatctcg tcaagcctta aatagaaagg ctaaacgcct taatatagat ttatctaaaa 1740 agtcattcac cgataaagaa tggcaacttt tagtgtcaaa caaacgtaaa cccaaaaagt 1800 caacttcaag taactatgtt gacactttta cagcgcaaca actagctgaa aaagatgatt 1860 taattaatta tttaaaatca caaattaaag aaaaagataa acaaattgat catgcgcaac 1920 aattacaatt aattgctgaa caaagattaa cagagacaaa taaaacccta attgagtatc 1980 aagaaaaaga aaatcagcca aagaaaggat tctggcaaag gttatttaaa tagttttcta 2040 atagtacttt tatagcgtcc gttttgtttg ttagtatttt tcgttcatca accgtccgct 2100 tatcaaaatt gattttaatt aaaaaaggga ttgggaattt cccaaacaaa aacatatcaa 2160 tttagatata tttttacctc atgtgtgacc acacatagta tgctcgcaac aagaactttt 2220 gttgtttttc aagttgtcga taacagcgac ttctgataca tatttttgag cgcaaacaac 2280 tccattttag aagtggagtg taagtgcgca ttctgataaa attggcaagc aatttcatca 2340 ctttgaaaaa gtgatgtgta agtgcgcatt aaactcattt tatgtcgttt cgctgacgga 2400 ttattttgct tattagtata attaggctag agacacattt aaatgcaata aggagcttaa 2460 attatggctg ataaaaatat ttttgaaaag ttgttttggg aagctgaaaa tacgaactta 2520 caggttttaa tggataatgc tttaaacgaa aaagatcccg acaagaaaaa agttttacat 2580 gcaatttatg aaaatacttg ctgtcagata actgtcataa gactgtcgta tactttattt 2640 caccaaagca ttatattgaa attatgaata aaacaaatgt atcacaactt agaaaacaac 2700 atggcttaac gcaacaagag ctagcagata aatcttacgt tactattcga acagtccaac 2760 gattagaagc aggcgaagat gtcagcttaa gtagtttaag tgccatagca aatgcgctat 2820 ctgtccctat aagcggcctt tttgatgata tagagcaaaa agaaaaagag caagaaattt 2880 tagattactc acaacaacag gtttctcaac tatatcgacg aaaaaacgag tcagccgttt 2940 taagtcttat actaatagcc atagatatat cagtgttatc attttatggc tactggattg 3000 gtcaacaacc tgaaaatgag caagctatat tcggaatcat ttggttatta cttttgttct 3060 tgttaatagc aggatcatta tatgtttatc gtattatagg agtcgaaagg ttagataaaa 3120 agtatcctaa aacagtgggc cttgttgaaa aaaaacaaaa ggaaacggaa aagatagaca 3180 acatttgggc ctttttagct aattattggt gggtaatttt tccaatcggt ggtttcttgt 3240 cttggcttat cccaacactc acaaatgctt ttaggtagtt cgtccactca taaatcacac 3300 atgagctaag ttttcacaaa tacaaaaagc ctaggtac 3338 <210> 3 <211> 399 <212> DNA <213> ORF 3_pCC3 <400> 3 atgacacacg aatttgatac cattatcgcg atagcagacg aactagaaat atctcgtcaa 60 gccttaaata gaaaggctaa acgccttaat atagatttat ctaaaaagtc attcaccgat 120 aaagaatggc aacttttagt gtcaaacaaa cgtaaaccca aaaagtcaac ttcaagtaac 180 tatgttgaca cttttacagc gcaacaacta gctgaaaaag atgatttaat taattattta 240 aaatcacaaa ttaaagaaaa agataaacaa attgatcatg cgcaacaatt acaattaatt 300 gctgaacaaa gattaacaga gacaaataaa accctaattg agtatcaaga aaaagaaaat 360 cagccaaaga aaggattctg gcaaaggtta tttaaatag 399 <210> 4 <211> 132 <212> PRT <213> ORF3_pCC3 <400> 4 Met Thr His Glu Phe Asp Thr Ile Ile Ala Ile Ala Asp Glu Leu Glu   1 5 10 15 Ile Ser Arg Gln Ala Leu Asn Arg Lys Ala Lys Arg Leu Asn Ile Asp              20 25 30 Leu Ser Lys Lys Ser Phe Thr Asp Lys Glu Trp Gln Leu Leu Val Ser          35 40 45 Asn Lys Arg Lys Pro Lys Lys Ser Thr Ser Ser Asn Tyr Val Asp Thr      50 55 60 Phe Thr Ala Gln Gln Leu Ala Glu Lys Asp Asp Leu Ile Asn Tyr Leu  65 70 75 80 Lys Ser Gln Ile Lys Glu Lys Asp Lys Gln Ile Asp His Ala Gln Gln                  85 90 95 Leu Gln Leu Ile Ala Glu Gln Arg Leu Thr Glu Thr Asn Lys Thr Leu             100 105 110 Ile Glu Tyr Gln Glu Lys Glu Asn Gln Pro Lys Lys Gly Phe Trp Gln         115 120 125 Arg Leu Phe Lys     130 <210> 5 <211> 615 <212> DNA <213> ORF1_pCC3 <400> 5 atgaataaaa caaatgtatc acaacttaga aaacaacatg gcttaacgca acaagagcta 60 gcagataaat cttacgttac tattcgaaca gtccaacgat tagaagcagg cgaagatgtc 120 agcttaagta gtttaagtgc catagcaaat gcgctatctg tccctataag cggccttttt 180 gatgatatag agcaaaaaga aaaagagcaa gaaattttag attactcaca acaacaggtt 240 tctcaactat atcgacgaaa aaacgagtca gccgttttaa gtcttatact aatagccata 300 gatatatcag tgttatcatt ttatggctac tggattggtc aacaacctga aaatgagcaa 360 gctatattcg gaatcatttg gttattactt ttgttcttgt taatagcagg atcattatat 420 gtttatcgta ttataggagt cgaaaggtta gataaaaagt atcctaaaac agtgggcctt 480 gttgaaaaaa aacaaaagga aacggaaaag atagacaaca tttgggcctt tttagctaat 540 tattggtggg taatttttcc aatcggtggt ttcttgtctt ggcttatccc aacactcaca 600 aatgctttta ggtag 615 <210> 6 <211> 204 <212> PRT <213> ORF1_pCC3_amino acid <400> 6 Met Asn Lys Thr Asn Val Ser Gln Leu Arg Lys Gln His Gly Leu Thr   1 5 10 15 Gln Gln Glu Leu Ala Asp Lys Ser Tyr Val Thr Ile Arg Thr Val Gln              20 25 30 Arg Leu Glu Ala Gly Glu Asp Val Ser Leu Ser Ser Leu Ser Ala Ile          35 40 45 Ala Asn Ala Leu Ser Val Pro Ile Ser Gly Leu Phe Asp Asp Ile Glu      50 55 60 Gln Lys Glu Lys Glu Gln Glu Ile Leu Asp Tyr Ser Gln Gln Gln Val  65 70 75 80 Ser Gln Leu Tyr Arg Arg Lys Asn Glu Ser Ala Val Leu Ser Leu Ile                  85 90 95 Leu Ile Ala Ile Asp Ile Ser Val Leu Ser Phe Tyr Gly Tyr Trp Ile             100 105 110 Gly Gln Gln Pro Glu Asn Glu Gln Ala Ile Phe Gly Ile Ile Trp Leu         115 120 125 Leu Leu Leu Phe Leu Leu Ile Ala Gly Ser Leu Tyr Val Tyr Arg Ile     130 135 140 Ile Gly Val Glu Arg Leu Asp Lys Lys Tyr Pro Lys Thr Val Gly Leu 145 150 155 160 Val Glu Lys Lys Gln Lys Glu Thr Glu Lys Ile Asp Asn Ile Trp Ala                 165 170 175 Phe Leu Ala Asn Tyr Trp Trp Val Ile Phe Pro Ile Gly Phe Leu             180 185 190 Ser Trp Leu Ile Pro Thr Leu Thr Asn Ala Phe Arg         195 200 <210> 7 <211> 585 <212> DNA <213> ORF2_pCC3 <400> 7 ttagctaaaa tataatattg ctatacctgt accaataaaa aagagatcta gtgcgataat 60 tattatttta cctactttgt tgtaagggtt aacacttata ccatagccaa aagctttcgg 120 cacccatagt ggaggatttg gcattccttc ataattttga taaacaacct tatcgaattt 180 gacagtaaca tgaattaaaa tgatagacag cattaaaata aatactgtaa caaactttgt 240 taagctttta ttgctagtta ctaaaggtat aattactact gccataagta caataagcac 300 agtcaataca atcaataaga cagaatactt cttattagcc tttttcgtgt taatcaattc 360 ataatccttt tgtactttat tcatcaattc tgtatctcct ttgatgatat ccaaagaaaa 420 gttataatag ttgcttaatt ctataagcat gggaatatct ggaaaatttt ttccattttc 480 ccaatttgac aaagattgcc tagtgataaa gagattatct gcaacttttt gctgtgtttc 540 atcttttttc tttcgttgtt caactaaaat agtagatagt tccat 585 <210> 8 <211> 194 <212> PRT <213> ORF2_pCC3_amino acid <400> 8 Met Glu Leu Ser Thr Ile Leu Val Glu Gln Arg Lys Lys Lys Asp Glu   1 5 10 15 Thr Gln Gln Lys Val Ala Asp Asn Leu Phe Ile Thr Arg Gln Ser Leu              20 25 30 Ser Asn Trp Glu Asn Gly Lys Asn Phe Pro Asp Ile Pro Met Leu Ile          35 40 45 Glu Leu Ser Asn Tyr Tyr Asn Phe Ser Leu Asp Ile Ile Lys Gly Asp      50 55 60 Thr Glu Leu Met Asn Lys Val Gln Lys Asp Tyr Glu Leu Ile Asn Thr  65 70 75 80 Lys Lys Ala Asn Lys Lys Tyr Ser Val Leu Leu Ile Val Leu Thr Val                  85 90 95 Leu Ile Val Leu Met Ala Val Val Ile Ile Pro Leu Val Thr Ser Asn             100 105 110 Lys Ser Leu Thr Lys Phe Val Thr Val Phe Ile Leu Met Leu Ser Ile         115 120 125 Ile Leu Ile His Val Thr Val Lys Phe Asp Lys Val Val Tyr Gln Asn     130 135 140 Tyr Glu Gly Met Pro Asn Pro Pro Leu Trp Val Pro Lys Ala Phe Gly 145 150 155 160 Tyr Gly Ile Ser Val Asn Pro Tyr Asn Lys Val Gly Lys Ile Ile Ile                 165 170 175 Ile Ala Leu Asp Leu Phe Phe Ile Gly Thr Gly Ile Ala Ile Leu Tyr             180 185 190 Phe ser         <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Em-F <400> 9 tagacgcgta taagacttag aagcaaac 28 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Em-R <400> 10 tctatgcatg tgtaactttc caaatttac 29 <210> 11 <211> 6024 <212> DNA <213> pUCC3 plasmid <400> 11 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420 actctagagg atccccgggt accatggatc cgaaaaacat aaacactttt gaatcgagta 480 cacactaaat actcacctgc ttgatggatt ttcgtaaaca ctcacaaccc tattcggttc 540 tgttctttgg tggctaacct ttttaatggg tggttattaa tcgatttttc cgggtttaca 600 acagatagaa aaggcaaagg aaaacaaaaa aaagttgttc cgggtgacaa aatcctatga 660 aaaatagatt ggaaagctga ggatattatg ctatttgtat attactagga cgataattgt 720 tcttgttttc attattggtt tactaaggct tatatcgaac gagtaaaagt ccaacaactg 780 gttaggtcat cggtatttta ctattgtgac tatatagata ccgataatca tattctgaat 840 tttgccgact gagcaaaaaa gcagctatat caactctttg gacaacaaca ctcattagat 900 tttaaagaac gagaaaaaga aaaacgagat atagtagttt ttccggcgaa tatccctgtc 960 tatcgcgtaa acgataccgt gaatttgatg aattcgactg tagaagcgga cgaagattag 1020 caacctgaca agcttatcat tgcattctaa atagacgatc gagaacaacg caattcggta 1080 caacaaaaga ttcaacacta tgtaaacaaa ataagtatta aagttatatt acgaaaccac 1140 tttatttcat atgctgtcag aatactgtca atagactgtc gttcataaaa gtatttaacg 1200 tacattttga aaaaagaaca gccctagaaa aagcaaattt cgtaataggt aattttggac 1260 attcaagcat aaaagtcgaa gggttttgtt gaaaagtttt tataaaaata gtcggtatta 1320 aattcgagga ataacgtaaa tttacacaga gatcggatta atatgattat tcgttttatt 1380 aggcagtcgc tttgctgtat tttactcaaa ttacgcgtga atgtgtagtg aaaaagtttc 1440 actactttaa cgaacggtta aaatagtctt acgcgtgaat gtgaggtgaa gattttacct 1500 caacaaacgc gagtttttat acatagtctt cagcgacaat agctgttgaa ctttttgttg 1560 ttttcaagaa caacgctcgt atgatacaca ccagtgtgta ctccattttt atatagattt 1620 aactatacaa aaacaaaccc tttaagggtt agggaaaaaa ttaattttag ttaaaactat 1680 tcgcctgcca actacttgct ttttatgatt gtttgttttg cctgcgatat tttcatgata 1740 atcttttgat aaatttattg gaaacggtct taggaaagaa accgactaaa agaaaaagaa 1800 ctatgagtta atcccaaaat aaacagagac aattagaaac aagtcgttaa ttaacattaa 1860 caacgcgtac tagttaaaca aatagaaaaa gaaattaaac actaaaattt attaattaat 1920 ttagtagaaa aagtcgatca acaacgcgac attttcacag ttgtatcaat gaacttcaac 1980 tgaaaaaccc aaatgcaaac aaactgtgat tttcaacggt aagaaatagc cacttactga 2040 aaaatctatt tagatataat tccgcaaatc ggaaagataa attccgaact gctctataaa 2100 gatcaagcag acgatagcgc tattaccata gtttaagcac acagtagtca gagaggtttg 2160 catttgaatt cgctacattt caaatgcaaa attcaaatga tataacaact gcaaaattca 2220 accagcaaat taataatttt gtattctaat aaacaaacaa ataacagtac atatcaagaa 2280 ttacgatatg agtatagttg taaatttatg tttatttttc tggagttggg gacgttccca 2340 atcctgaacc actggatcta taatgttgat agtcccaaaa cggtaatgtc ttaatctgga 2400 gacgttaccg aatcttatga atgataatat gtttgaatat ctgagtctca tttgtcgaaa 2460 tgagtttttt cttgatattt gctgatactt tcgcatagga ggtcggattg attcgtgctc 2520 ctatgcgaaa aatgcagaca agttagcaac agcctgcaat aggattgttg attatgcctt 2580 gtccgcacat aggcagttct cccgactttc cagcagattt ggtgcaggtt tcgttagttt 2640 cgctctaaca ccccttactt gttaagctaa tacccatccg agcgggcgtt cattaaccgt 2700 ttcttcaccg tatatcttaa tcgtgtgata taccaaattt agcgtatatt tcgctaaatt 2760 cgccacagac tgcacaagat tggaataact gaatattttc attcccaaag ataacccgtc 2820 tgctatcttc tctttaatct cgctatatgc accaactatg ttcgctatac taagacttaa 2880 tatggaactt gttagaattt tcaggattta tgaatcccga aagagacgag tttagtttga 2940 ctaacgggaa caatagtaac gatttacttg ttacgttctt tataaacgat ctttatttaa 3000 acaacagttt cattatatta ccgcatcctt cccttgttta taccttgata gatgataaaa 3060 tcaacttgtt gctttctttt ttctactttg tgtcgttttt caacgtctat tagagaaata 3120 gtgatccgtt agaaacagtt taaccctttt acctttttta aaaggtctat aagggtacga 3180 atatcttaat tcgttgataa tattgaaaag aaacctatag tagtttcctc tatgtcttaa 3240 ctacttattt catgttttcc taatacttaa ctaattgtgc tttttccgat tattcttcat 3300 aagacagaat aactaacata actgacacga ataacatgaa taccgtcatc attaatatgg 3360 aaatcattga tcgttatttt cgaattgttt caaacaatgt cataaataaa attacgacag 3420 atagtaaaat taagtacaat gacagtttaa gctattccaa caaatagttt taatacttcc 3480 ttacggttta ggaggtgata cccacggctt tcgaaaaccg ataccatatt cacaattggg 3540 aatgttgttt catccatttt attattaata gcgtgatcta gagaaaaaat aaccatgtcc 3600 atatcgttat aatataaaat cgattgttta gtatcatcag tatatacttg aattgtatta 3660 ggtagaatac ggttcacttt tcgcggtttg gaataagctt atccaaaacc gcgaaaaata 3720 ccctaacccc aaacacttaa atcgagttta tttaaccaga tcgagagtgt tagcgccatc 3780 cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 3840 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 3900 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 3960 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 4020 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 4080 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 4140 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 4200 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 4260 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 4320 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4380 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 4440 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4500 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 4560 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4620 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 4680 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 4740 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4800 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 4860 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 4920 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 4980 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 5040 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 5100 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 5160 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 5220 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 5280 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 5340 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 5400 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 5460 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 5520 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 5580 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 5640 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 5700 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 5760 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 5820 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 5880 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 5940 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 6000 ggcgtatcac gaggcccttt cgtc 6024 <210> 12 <211> 6935 <212> DNA <213> pUCC3E plasmid <400> 12 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420 actctagagg atcccctaga cgcgtataag acttagaagc aaacttaaga gtgtgttgat 480 agtgcagtat cttaaaattt tgtataatag gaattgaagt taaattagat gctaaaaatt 540 tgtaattaag aaggagtgat tacatgaaca aaaatataaa atattctcaa aactttttaa 600 cgagtgaaaa agtactcaac caaataataa aacaattgaa tttaaaagaa accgataccg 660 tttacgaaat tggaacaggt aaagggcatt taacgacgaa actggctaaa ataagtaaac 720 aggtaacgtc tattgaatta gacagtcatc tattcaactt atcgtcagaa aaattaaaac 780 tgaatactcg tgtcacttta attcaccaag atattctaca gtttcaattc cctaacaaac 840 agaggtataa aattgttggg agtattcctt accatttaag cacacaaatt attaaaaaag 900 tggtttttga aagccatgcg tctgacatct atctgattgt tgaagaagga ttctacaagc 960 gtaccttgga tattcaccga acactagggt tgctcttgca cactcaagtc tcgattcagc 1020 aattgcttaa gctgccagcg gaatgctttc atcctaaacc aaaagtaaac agtgtcttaa 1080 taaaacttac ccgccatacc acagatgttc cagataaata ttggaagcta tatacgtact 1140 ttgtttcaaa atgggtcaat cgagaatatc gtcaactgtt tactaaaaat cagtttcatc 1200 aagcaatgaa acacgccaaa gtaaacaatt taagtaccgt tacttatgag caagtattgt 1260 ctatttttaa tagttatcta ttatttaacg ggaggaaata attctatgag tcgcttttgt 1320 aaatttggaa agttacacat gcatagaggg taccatggat ccgaaaaaca taaacacttt 1380 tgaatcgagt acacactaaa tactcacctg cttgatggat tttcgtaaac actcacaacc 1440 ctattcggtt ctgttctttg gtggctaacc tttttaatgg gtggttatta atcgattttt 1500 ccgggtttac aacagataga aaaggcaaag gaaaacaaaa aaaagttgtt ccgggtgaca 1560 aaatcctatg aaaaatagat tggaaagctg aggatattat gctatttgta tattactagg 1620 acgataattg ttcttgtttt cattattggt ttactaaggc ttatatcgaa cgagtaaaag 1680 tccaacaact ggttaggtca tcggtatttt actattgtga ctatatagat accgataatc 1740 atattctgaa ttttgccgac tgagcaaaaa agcagctata tcaactcttt ggacaacaac 1800 actcattaga ttttaaagaa cgagaaaaag aaaaacgaga tatagtagtt tttccggcga 1860 atatccctgt ctatcgcgta aacgataccg tgaatttgat gaattcgact gtagaagcgg 1920 acgaagatta gcaacctgac aagcttatca ttgcattcta aatagacgat cgagaacaac 1980 gcaattcggt acaacaaaag attcaacact atgtaaacaa aataagtatt aaagttatat 2040 tacgaaacca ctttatttca tatgctgtca gaatactgtc aatagactgt cgttcataaa 2100 agtatttaac gtacattttg aaaaaagaac agccctagaa aaagcaaatt tcgtaatagg 2160 taattttgga cattcaagca taaaagtcga agggttttgt tgaaaagttt ttataaaaat 2220 agtcggtatt aaattcgagg aataacgtaa atttacacag agatcggatt aatatgatta 2280 ttcgttttat taggcagtcg ctttgctgta ttttactcaa attacgcgtg aatgtgtagt 2340 gaaaaagttt cactacttta acgaacggtt aaaatagtct tacgcgtgaa tgtgaggtga 2400 agattttacc tcaacaaacg cgagttttta tacatagtct tcagcgacaa tagctgttga 2460 actttttgtt gttttcaaga acaacgctcg tatgatacac accagtgtgt actccatttt 2520 tatatagatt taactataca aaaacaaacc ctttaagggt tagggaaaaa attaatttta 2580 gttaaaacta ttcgcctgcc aactacttgc tttttatgat tgtttgtttt gcctgcgata 2640 ttttcatgat aatcttttga taaatttatt ggaaacggtc ttaggaaaga aaccgactaa 2700 aagaaaaaga actatgagtt aatcccaaaa taaacagaga caattagaaa caagtcgtta 2760 attaacatta acaacgcgta ctagttaaac aaatagaaaa agaaattaaa cactaaaatt 2820 tattaattaa tttagtagaa aaagtcgatc aacaacgcga cattttcaca gttgtatcaa 2880 tgaacttcaa ctgaaaaacc caaatgcaaa caaactgtga ttttcaacgg taagaaatag 2940 ccacttactg aaaaatctat ttagatataa ttccgcaaat cggaaagata aattccgaac 3000 tgctctataa agatcaagca gacgatagcg ctattaccat agtttaagca cacagtagtc 3060 agagaggttt gcatttgaat tcgctacatt tcaaatgcaa aattcaaatg atataacaac 3120 tgcaaaattc aaccagcaaa ttaataattt tgtattctaa taaacaaaca aataacagta 3180 catatcaaga attacgatat gagtatagtt gtaaatttat gtttattttt ctggagttgg 3240 ggacgttccc aatcctgaac cactggatct ataatgttga tagtcccaaa acggtaatgt 3300 cttaatctgg agacgttacc gaatcttatg aatgataata tgtttgaata tctgagtctc 3360 atttgtcgaa atgagttttt tcttgatatt tgctgatact ttcgcatagg aggtcggatt 3420 gattcgtgct cctatgcgaa aaatgcagac aagttagcaa cagcctgcaa taggattgtt 3480 gattatgcct tgtccgcaca taggcagttc tcccgacttt ccagcagatt tggtgcaggt 3540 ttcgttagtt tcgctctaac accccttact tgttaagcta atacccatcc gagcgggcgt 3600 tcattaaccg tttcttcacc gtatatctta atcgtgtgat ataccaaatt tagcgtatat 3660 ttcgctaaat tcgccacaga ctgcacaaga ttggaataac tgaatatttt cattcccaaa 3720 gataacccgt ctgctatctt ctctttaatc tcgctatatg caccaactat gttcgctata 3780 ctaagactta atatggaact tgttagaatt ttcaggattt atgaatcccg aaagagacga 3840 gtttagtttg actaacggga acaatagtaa cgatttactt gttacgttct ttataaacga 3900 tctttattta aacaacagtt tcattatatt accgcatcct tcccttgttt ataccttgat 3960 agatgataaa atcaacttgt tgctttcttt tttctacttt gtgtcgtttt tcaacgtcta 4020 ttagagaaat agtgatccgt tagaaacagt ttaacccttt tacctttttt aaaaggtcta 4080 taagggtacg aatatcttaa ttcgttgata atattgaaaa gaaacctata gtagtttcct 4140 ctatgtctta actacttatt tcatgttttc ctaatactta actaattgtg ctttttccga 4200 ttattcttca taagacagaa taactaacat aactgacacg aataacatga ataccgtcat 4260 cattaatatg gaaatcattg atcgttattt tcgaattgtt tcaaacaatg tcataaataa 4320 aattacgaca gatagtaaaa ttaagtacaa tgacagttta agctattcca acaaatagtt 4380 ttaatacttc cttacggttt aggaggtgat acccacggct ttcgaaaacc gataccatat 4440 tcacaattgg gaatgttgtt tcatccattt tattattaat agcgtgatct agagaaaaaa 4500 taaccatgtc catatcgtta taatataaaa tcgattgttt agtatcatca gtatatactt 4560 gaattgtatt aggtagaata cggttcactt ttcgcggttt ggaataagct tatccaaaac 4620 cgcgaaaaat accctaaccc caaacactta aatcgagttt atttaaccag atcgagagtg 4680 ttagcgccat ccgagctcga attcgtaatc atggtcatag ctgtttcctg tgtgaaattg 4740 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 4800 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 4860 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 4920 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 4980 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 5040 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 5100 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 5160 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 5220 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 5280 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 5340 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 5400 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 5460 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 5520 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 5580 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 5640 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 5700 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 5760 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 5820 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 5880 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 5940 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 6000 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 6060 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 6120 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 6180 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 6240 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 6300 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 6360 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 6420 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 6480 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 6540 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 6600 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 6660 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 6720 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 6780 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 6840 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 6900 ctataaaaat aggcgtatca cgaggccctt tcgtc 6935 <210> 13 <211> 5959 <212> DNA <213> pUCC3E1 plasmid <400> 13 ctgctttccc gcctcagtcc gttgatacct acttgcttta tctgtctagc gactctatcc 60 acggagtgac taattcgtaa ccattgacag tctggttcaa atgagtatat atgaaatcta 120 actaaatttt gaagtaaaaa ttaaattttc ctagatccac ttctaggaaa aactattaga 180 gtactggttt tagggaattg cactcaaaag caaggtgact cgcagtctgg ggcatctttt 240 ctagtttcct agaagaactc taggaaaaaa agacgcgcat tagacgacga acgtttgttt 300 ttttggtggc gatggtcgcc accaaacaaa cggcctagtt ctcgatggtt gagaaaaagg 360 cttccattga ccgaagtcgt ctcgcgtcta tggtttatga caggaagatc acatcggcat 420 caatccggtg gtgaagttct tgagacatcg tggcggatgt atggagcgag acgattagga 480 caatggtcac cgacgacggt caccgctatt cagcacagaa tggcccaacc tgagttctgc 540 tatcaatggc ctattccgcg tcgccagccc gacttgcccc ccaagcacgt gtgtcgggtc 600 gaacctcgct tgctggatgt ggcttgactc tatggatgtc gcactcgata ctctttcgcg 660 gtgcgaaggg cttccctctt tccgcctgtc cataggccat tcgccgtccc agccttgtcc 720 tctcgcgtgc tccctcgaag gtcccccttt gcggaccata gaaatatcag gacagcccaa 780 agcggtggag actgaactcg cagctaaaaa cactacgagc agtccccccg cctcggatac 840 ctttttgcgg tcgttgcgcc ggaaaaatgc caaggaccgg aaaacgaccg gaaaacgagt 900 gtacaagaaa ggacgcaata ggggactaag acacctattg gcataatggc ggaaactcac 960 tcgactatgg cgagcggcgt cggcttgctg gctcgcgtcg ctcagtcact cgctccttcg 1020 ccttctcgcg ggttatgcgt ttggcggaga ggggcgcgca accggctaag taattacgtc 1080 gaccgtgctg tccaaagggc tgacctttcg cccgtcactc gcgttgcgtt aattacactc 1140 aatcgagtga gtaatccgtg gggtccgaaa tgtgaaatac gaaggccgag catacaacac 1200 accttaacac tcgcctattg ttaaagtgtg tcctttgtcg atactggtac taatgcttaa 1260 gctcgagcct accgcgattg tgagagctag accaatttat ttgagctaaa ttcacaaacc 1320 ccaatcccat aaaaagcgcc aaaacctatt cgaataaggt ttggcgcttt tcacttggca 1380 taagatggat tatgttaagt tcatatatga ctactatgat ttgttagcta aaatataata 1440 ttgctatacc tgtaccaata aaaaagagat ctagtgcgat aattattatt ttacctactt 1500 tgttgtaagg gttaacactt ataccatagc caaaagcttt cggcacccat agtggaggat 1560 ttggcattcc ttcataattt tgataaacaa ccttatcgaa tttgacagta acatgaatta 1620 aaatgataga cagcattaaa ataaatactg taacaaactt tgttaagctt ttattgctag 1680 ttactaaagg tataattact actgccataa gtacaataag cacagtcaat acaatcaata 1740 agacagaata cttcttatta gcctttttcg tgttaatcaa ttcataatcc ttttgtactt 1800 tattcatcaa ttctgtatct cctttgatga tatccaaaga aaagttataa tagttgctta 1860 attctataag catgggaata tctggaaaat tttttccatt ttcccaattt gacaaagatt 1920 gcctagtgat aaagagatta tctgcaactt tttgctgtgt ttcatctttt ttctttcgtt 1980 gttcaactaa aatagtagat agttccatat ttgttccctt cctacgccat tatattactt 2040 tgacaacaaa tttatttcta gcaaatattt cttgcattgt tcatttagca atgataacaa 2100 gggcaatcag tttgatttga gcagagaaag ccctaagtat ttaggacttt taagattgtt 2160 caaggtataa ttcagaatca tatcgcttgt atcaaccacg tatatcgctc taatttctct 2220 tctatcgtct gcccaataga aacccttact tttataagtc aataaggtta gaacacgtca 2280 gacaccgctt aaatcgcttt atatgcgatt taaaccatat agtgtgctaa ttctatatgc 2340 cacttctttg ccaattactt gcgggcgagc ctacccataa tcgaattgtt cattccccac 2400 aatctcgctt tgattgcttt ggacgtggtt tagacgacct ttcagccctc ttgacggata 2460 cacgcctgtt ccgtattagt tgttaggata acgtccgaca acgattgaac agacgtaaaa 2520 agcgtatcct cgtgcttagt taggctggag gatacgcttt catagtcgtt tatagttctt 2580 ttttgagtaa agctgtttac tctgagtcta taagtttgta taatagtaag tattctaagc 2640 cattgcagag gtctaattct gtaatggcaa aaccctgata gttgtaatat ctaggtcacc 2700 aagtcctaac ccttgcaggg gttgaggtct ttttatttgt atttaaatgt tgatatgagt 2760 atagcattaa gaactataca tgacaataaa caaacaaata atcttatgtt ttaataatta 2820 aacgaccaac ttaaaacgtc aacaatatag taaacttaaa acgtaaactt tacatcgctt 2880 aagtttacgt ttggagagac tgatgacaca cgaatttgat accattatcg cgatagcaga 2940 cgaactagaa atatctcgtc aagccttaaa tagaaaggct aaacgcctta atatagattt 3000 atctaaaaag tcattcaccg ataaagaatg gcaactttta gtgtcaaaca aacgtaaacc 3060 caaaaagtca acttcaagta actatgttga cacttttaca gcgcaacaac tagctgaaaa 3120 agatgattta attaattatt taaaatcaca aattaaagaa aaagataaac aaattgatca 3180 tgcgcaacaa ttacaattaa ttgctgaaca aagattaaca gagacaaata aaaccctaat 3240 tgagtatcaa gaaaaagaaa atcagccaaa gaaaggattc tggcaaaggt tatttaaata 3300 gttttctaat agtactttta tagcgtccgt tttgtttgtt agtatttttc gttcatcaac 3360 cgtccgctta tcaaaattga ttttaattaa aaaagggatt gggaatttcc caaacaaaaa 3420 catatcaatt tagatatatt tttacctcat gtgtgaccac acatagtatg ctcgcaacaa 3480 gaacttttgt tgtttttcaa gttgtcgata acagcgactt ctgatacata tttttgagcg 3540 caaacaactc cattttagaa gtggagtgta agtgcgcatt ctgataaaat tggcaagcaa 3600 tttcatcact ttgaaaaagt gatgtgtaag tgcgcattaa actcatttta tgtcgtttcg 3660 ctgacggatt attttgctta ttagtataat taggctagag acacatttaa atgcaataag 3720 gagcttaaat tatggctgat aaaaatattt ttgaaaagtt gttttgggaa gctgaaaata 3780 cgaacttaca ggttttaatg gataatgctt taaacgaaaa agatcccgac aagaaaaaag 3840 ttttacatgc aatttatgaa aatacttgct gtcagataac tgtcataaga ctgtcgtata 3900 ctttatttca ccaaagcatt atattgaaat tatgaataaa acaaatgtat cacaacttag 3960 aaaacaacat ggcttaacgc aacaagagct agcagataaa tcttacgtta ctattcgaac 4020 agtccaacga ttagaagcag gcgaagatgt cagcttaagt agtttaagtg ccatagcaaa 4080 tgcgctatct gtccctataa gcggcctttt tgatgatata gagcaaaaag aaaaagagca 4140 agaaatttta gattactcac aacaacaggt ttctcaacta tatcgacgaa aaaacgagtc 4200 agccgtttta agtcttatac taatagccat agatatatca gtgttatcat tttatggcta 4260 ctggattggt caacaacctg aaaatgagca agctatattc ggaatcattt ggttattact 4320 tttgttcttg ttaatagcag gatcattata tgtttatcgt attataggag tcgaaaggtt 4380 agataaaaag tatcctaaaa cagtgggcct tgttgaaaaa aaacaaaagg aaacggaaaa 4440 gatagacaac atttgggcct ttttagctaa ttattggtgg gtaatttttc caatcggtgg 4500 tttcttgtct tggcttatcc caacactcac aaatgctttt aggtagttcg tccactcata 4560 aatcacacat gagctaagtt ttcacaaata caaaaagcct aggtaccatg ggagatacgt 4620 acacattgaa aggtttaaat gttttcgctg agtatcttaa taaaggaggg caatttatta 4680 tctattgata atttttatct gttatgaacg agtattcatt gccatgaatt taacaaatga 4740 aaccgcacaa agtaacgaac tactttgact aaaaatcatt tgtcaactgc tataagagct 4800 aactgggtaa aactttgttt catgcatata tcgaaggtta taaatagacc ttgtagacac 4860 cataccgccc attcaaaata attctgtgac aaatgaaaac caaatcctac tttcgtaagg 4920 cgaccgtcga attcgttaac gacttagctc tgaactcaca cgttctcgtt gggatcacaa 4980 gccacttata ggttccatgc gaacatctta ggaagaagtt gttagtctat ctacagtctg 5040 cgtaccgaaa gtttttggtg aaaaaattat taaacacacg aatttaccat tccttatgag 5100 ggttgttaaa atatggagac aaacaatccc ttaactttga catcttatag aaccacttaa 5160 tttcactgtg ctcataagtc aaaattaaaa agactgctat tcaacttatc tactgacaga 5220 ttaagttatc tgcaatggac aaatgaataa aatcggtcaa agcagcaatt tacgggaaat 5280 ggacaaggtt aaagcatttg ccatagccaa agaaaattta agttaacaaa ataataaacc 5340 aactcatgaa aaagtgagca atttttcaaa actcttataa aatataaaaa caagtacatt 5400 agtgaggaag aattaatgtt taaaaatcgt agattaaatt gaagttaagg ataatatgtt 5460 ttaaaattct atgacgtgat agttgtgtga gaattcaaac gaagattcag aatatgcgca 5520 gatcccctag gagatctcag ctggacgtcc gtacgttcga accgtgaccg gcagcaaaat 5580 gttgcagcac tgaccctttt gggaccgcaa tgggttgaat tagcggaacg tcgtgtaggg 5640 ggaaagcggt cgaccgcatt atcgcttctc cgggcgtggc tagcgggaag ggttgtcaac 5700 gcgtcggact taccgcttac cgcggactac gccataaaag aggaatgcgt agacacgcca 5760 taaagtgtgg cgtataccac gtgagagtca tgttagacga gactacggcg tatcaattcg 5820 gtcggggctg tgggcggttg tgggcgactg cgcgggactg cccgaacaga cgagggccgt 5880 aggcgaatgt ctgttcgaca ctggcagagg ccctcgacgt acacagtctc caaaagtggc 5940 agtagtggct ttgcgcgct 5959 <210> 14 <211> 4910 <212> DNA <213> pUCC3E1-1 plasmid <400> 14 ctgctttccc gcctcagtcc gttgatacct acttgcttta tctgtctagc gactctatcc 60 acggagtgac taattcgtaa ccattgacag tctggttcaa atgagtatat atgaaatcta 120 actaaatttt gaagtaaaaa ttaaattttc ctagatccac ttctaggaaa aactattaga 180 gtactggttt tagggaattg cactcaaaag caaggtgact cgcagtctgg ggcatctttt 240 ctagtttcct agaagaactc taggaaaaaa agacgcgcat tagacgacga acgtttgttt 300 ttttggtggc gatggtcgcc accaaacaaa cggcctagtt ctcgatggtt gagaaaaagg 360 cttccattga ccgaagtcgt ctcgcgtcta tggtttatga caggaagatc acatcggcat 420 caatccggtg gtgaagttct tgagacatcg tggcggatgt atggagcgag acgattagga 480 caatggtcac cgacgacggt caccgctatt cagcacagaa tggcccaacc tgagttctgc 540 tatcaatggc ctattccgcg tcgccagccc gacttgcccc ccaagcacgt gtgtcgggtc 600 gaacctcgct tgctggatgt ggcttgactc tatggatgtc gcactcgata ctctttcgcg 660 gtgcgaaggg cttccctctt tccgcctgtc cataggccat tcgccgtccc agccttgtcc 720 tctcgcgtgc tccctcgaag gtcccccttt gcggaccata gaaatatcag gacagcccaa 780 agcggtggag actgaactcg cagctaaaaa cactacgagc agtccccccg cctcggatac 840 ctttttgcgg tcgttgcgcc ggaaaaatgc caaggaccgg aaaacgaccg gaaaacgagt 900 gtacaagaaa ggacgcaata ggggactaag acacctattg gcataatggc ggaaactcac 960 tcgactatgg cgagcggcgt cggcttgctg gctcgcgtcg ctcagtcact cgctccttcg 1020 ccttctcgcg ggttatgcgt ttggcggaga ggggcgcgca accggctaag taattacgtc 1080 gaccgtgctg tccaaagggc tgacctttcg cccgtcactc gcgttgcgtt aattacactc 1140 aatcgagtga gtaatccgtg gggtccgaaa tgtgaaatac gaaggccgag catacaacac 1200 accttaacac tcgcctattg ttaaagtgtg tcctttgtcg atactggtac taatgcttaa 1260 gctcgagcct accgcgattg tgagagctag accaatttat ttgagctaaa ttcacaaacc 1320 ccaatcccat aaaaagcgcc aaaacctatt cgaataaggt ttggcgcttt tcacttggca 1380 taagatggat tatgttaagt tcatatatga ctactatgat ttgttagcta aaatataata 1440 ttgctatacc tgtaccaata aaaaagagat ctagtgcgat aattattatt ttacctactt 1500 tgttgtaagg gttaacactt ataccatagc caaaagcttt cggcacccat agtggaggat 1560 ttggcattcc ttcataattt tgataaacaa ccttatcgaa tttgacagta acatgaatta 1620 aaatgataga cagcattaaa ataaatactg taacaaactt tgttaagctt ttattgctag 1680 ttactaaagg tataattact actgccataa gtacaataag cacagtcaat acaatcaata 1740 agacagaata cttcttatta gcctttttcg tgttaatcaa ttcataatcc ttttgtactt 1800 tattcatcaa ttctgtatct cctttgatga tatccaaaga aaagttataa tagttgctta 1860 attctataag catgggaata tctggaaaat tttttccatt ttcccaattt gacaaagatt 1920 gcctagtgat aaagagatta tctgcaactt tttgctgtgt ttcatctttt ttctttcgtt 1980 gttcaactaa aatagtagat agttccatat ttgttccctt cctacgccat tatattactt 2040 tgacaacaaa tttatttcta gcaaatattt cttgcattgt tcatttagca atgataacaa 2100 gggcaatcag tttgatttga gcagagaaag ccctaagtat ttaggacttt taagattgtt 2160 caaggtataa ttcagaatca tatcgcttgt atcaaccacg tatatcgctc taatttctct 2220 tctatcgtct gcccaataga aacccttact tttataagtc aataaggtta gaacacgtca 2280 gacaccgctt aaatcgcttt atatgcgatt taaaccatat agtgtgctaa ttctatatgc 2340 cacttctttg ccaattactt gcgggcgagc ctacccataa tcgaattgtt cattccccac 2400 aatctcgctt tgattgcttt ggacgtggtt tagacgacct ttcagccctc ttgacggata 2460 cacgcctgtt ccgtattagt tgttaggata acgtccgaca acgattgaac agacgtaaaa 2520 agcgtatcct cgtgcttagt taggctggag gatacgcttt catagtcgtt tatagttctt 2580 ttttgagtaa agctgtttac tctgagtcta taagtttgta taatagtaag tattctaagc 2640 cattgcagag gtctaattct gtaatggcaa aaccctgata gttgtaatat ctaggtcacc 2700 aagtcctaac ccttgcaggg gttgaggtct ttttatttgt atttaaatgt tgatatgagt 2760 atagcattaa gaactataca tgacaataaa caaacaaata atcttatgtt ttaataatta 2820 aacgaccaac ttaaaacgtc aacaatatag taaacttaaa acgtaaactt tacatcgctt 2880 aagtttacgt ttggagagac tgatgacaca cgaatttgat accattatcg cgatagcaga 2940 cgaactagaa atatctcgtc aagccttaaa tagaaaggct aaacgcctta atatagattt 3000 atctaaaaag tcattcaccg ataaagaatg gcaactttta gtgtcaaaca aacgtaaacc 3060 caaaaagtca acttcaagta actatgttga cacttttaca gcgcaacaac tagctgaaaa 3120 agatgattta attaattatt taaaatcaca aattaaagaa aaagataaac aaattgatca 3180 tgcgcaacaa ttacaattaa ttgctgaaca aagattaaca gagacaaata aaaccctaat 3240 tgagtatcaa gaaaaagaaa atcagccaaa gaaaggattc tggcaaaggt tatttaaata 3300 gttttctaat agtactttta tagcgtccgt tttgtttgtt agtatttttc gttcatcaac 3360 cgtccgctta tcaaaattga ttttaattaa aaaagggatt gggaatttcc caaacaaaaa 3420 catatcaatt tagatatatt tttacctcat gtgtgaccac acatagtatg ctcgcaacaa 3480 gaacttttgt tgtttttcaa gttgtcgata acagcgactt ctgatacata tttttgagcg 3540 caaacaactc cattttacat gggagatacg tacacattga aaggtttaaa tgttttcgct 3600 gagtatctta ataaaggagg gcaatttatt atctattgat aatttttatc tgttatgaac 3660 gagtattcat tgccatgaat ttaacaaatg aaaccgcaca aagtaacgaa ctactttgac 3720 taaaaatcat ttgtcaactg ctataagagc taactgggta aaactttgtt tcatgcatat 3780 atcgaaggtt ataaatagac cttgtagaca ccataccgcc cattcaaaat aattctgtga 3840 caaatgaaaa ccaaatccta ctttcgtaag gcgaccgtcg aattcgttaa cgacttagct 3900 ctgaactcac acgttctcgt tgggatcaca agccacttat aggttccatg cgaacatctt 3960 aggaagaagt tgttagtcta tctacagtct gcgtaccgaa agtttttggt gaaaaaatta 4020 ttaaacacac gaatttacca ttccttatga gggttgttaa aatatggaga caaacaatcc 4080 cttaactttg acatcttata gaaccactta atttcactgt gctcataagt caaaattaaa 4140 aagactgcta ttcaacttat ctactgacag attaagttat ctgcaatgga caaatgaata 4200 aaatcggtca aagcagcaat ttacgggaaa tggacaaggt taaagcattt gccatagcca 4260 aagaaaattt aagttaacaa aataataaac caactcatga aaaagtgagc aatttttcaa 4320 aactcttata aaatataaaa acaagtacat tagtgaggaa gaattaatgt ttaaaaatcg 4380 tagattaaat tgaagttaag gataatatgt tttaaaattc tatgacgtga tagttgtgtg 4440 agaattcaaa cgaagattca gaatatgcgc agatccccta ggagatctca gctggacgtc 4500 cgtacgttcg aaccgtgacc ggcagcaaaa tgttgcagca ctgacccttt tgggaccgca 4560 atgggttgaa ttagcggaac gtcgtgtagg gggaaagcgg tcgaccgcat tatcgcttct 4620 ccgggcgtgg ctagcgggaa gggttgtcaa cgcgtcggac ttaccgctta ccgcggacta 4680 cgccataaaa gaggaatgcg tagacacgcc ataaagtgtg gcgtatacca cgtgagagtc 4740 atgttagacg agactacggc gtatcaattc ggtcggggct gtgggcggtt gtgggcgact 4800 gcgcgggact gcccgaacag acgagggccg taggcgaatg tctgttcgac actggcagag 4860 gccctcgacg tacacagtct ccaaaagtgg cagtagtggc tttgcgcgct 4910 <210> 15 <211> 3612 <212> DNA <213> pUCC3E1-4 plasmid <400> 15 ctgctttccc gcctcagtcc gttgatacct acttgcttta tctgtctagc gactctatcc 60 acggagtgac taattcgtaa ccattgacag tctggttcaa atgagtatat atgaaatcta 120 actaaatttt gaagtaaaaa ttaaattttc ctagatccac ttctaggaaa aactattaga 180 gtactggttt tagggaattg cactcaaaag caaggtgact cgcagtctgg ggcatctttt 240 ctagtttcct agaagaactc taggaaaaaa agacgcgcat tagacgacga acgtttgttt 300 ttttggtggc gatggtcgcc accaaacaaa cggcctagtt ctcgatggtt gagaaaaagg 360 cttccattga ccgaagtcgt ctcgcgtcta tggtttatga caggaagatc acatcggcat 420 caatccggtg gtgaagttct tgagacatcg tggcggatgt atggagcgag acgattagga 480 caatggtcac cgacgacggt caccgctatt cagcacagaa tggcccaacc tgagttctgc 540 tatcaatggc ctattccgcg tcgccagccc gacttgcccc ccaagcacgt gtgtcgggtc 600 gaacctcgct tgctggatgt ggcttgactc tatggatgtc gcactcgata ctctttcgcg 660 gtgcgaaggg cttccctctt tccgcctgtc cataggccat tcgccgtccc agccttgtcc 720 tctcgcgtgc tccctcgaag gtcccccttt gcggaccata gaaatatcag gacagcccaa 780 agcggtggag actgaactcg cagctaaaaa cactacgagc agtccccccg cctcggatac 840 ctttttgcgg tcgttgcgcc ggaaaaatgc caaggaccgg aaaacgaccg gaaaacgagt 900 gtacaagaaa ggacgcaata ggggactaag acacctattg gcataatggc ggaaactcac 960 tcgactatgg cgagcggcgt cggcttgctg gctcgcgtcg ctcagtcact cgctccttcg 1020 ccttctcgcg ggttatgcgt ttggcggaga ggggcgcgca accggctaag taattacgtc 1080 gaccgtgctg tccaaagggc tgacctttcg cccgtcactc gcgttgcgtt aattacactc 1140 aatcgagtga gtaatccgtg gggtccgaaa tgtgaaatac gaaggccgag catacaacac 1200 accttaacac tcgcctattg ttaaagtgtg tcctttgtcg atactggtac taatgcttaa 1260 gctcgagccg tttatagttc ttttttgagt aaagctgttt actctgagtc tataagtttg 1320 tataatagta agtattctaa gccattgcag aggtctaatt ctgtaatggc aaaaccctga 1380 tagttgtaat atctaggtca ccaagtccta acccttgcag gggttgaggt ctttttattt 1440 gtatttaaat gttgatatga gtatagcatt aagaactata catgacaata aacaaacaaa 1500 taatcttatg ttttaataat taaacgacca acttaaaacg tcaacaatat agtaaactta 1560 aaacgtaaac tttacatcgc ttaagtttac gtttggagag actgatgaca cacgaatttg 1620 ataccattat cgcgatagca gacgaactag aaatatctcg tcaagcctta aatagaaagg 1680 ctaaacgcct taatatagat ttatctaaaa agtcattcac cgataaagaa tggcaacttt 1740 tagtgtcaaa caaacgtaaa cccaaaaagt caacttcaag taactatgtt gacactttta 1800 cagcgcaaca actagctgaa aaagatgatt taattaatta tttaaaatca caaattaaag 1860 aaaaagataa acaaattgat catgcgcaac aattacaatt aattgctgaa caaagattaa 1920 cagagacaaa taaaacccta attgagtatc aagaaaaaga aaatcagcca aagaaaggat 1980 tctggcaaag gttatttaaa tagttttcta atagtacttt tatagcgtcc gttttgtttg 2040 ttagtatttt tcgttcatca accgtccgct tatcaaaatt gattttaatt aaaaaaggga 2100 ttgggaattt cccaaacaaa aacatatcaa tttagatata tttttacctc atgtgtgacc 2160 acacatagta tgctcgcaac aagaactttt gttgtttttc aagttgtcga taacagcgac 2220 ttctgataca tatttttgag cgcaaacaac tccattttac atgggagata cgtacacatt 2280 gaaaggttta aatgttttcg ctgagtatct taataaagga gggcaattta ttatctattg 2340 ataattttta tctgttatga acgagtattc attgccatga atttaacaaa tgaaaccgca 2400 caaagtaacg aactactttg actaaaaatc atttgtcaac tgctataaga gctaactggg 2460 taaaactttg tttcatgcat atatcgaagg ttataaatag accttgtaga caccataccg 2520 cccattcaaa ataattctgt gacaaatgaa aaccaaatcc tactttcgta aggcgaccgt 2580 cgaattcgtt aacgacttag ctctgaactc acacgttctc gttgggatca caagccactt 2640 ataggttcca tgcgaacatc ttaggaagaa gttgttagtc tatctacagt ctgcgtaccg 2700 aaagtttttg gtgaaaaaat tattaaacac acgaatttac cattccttat gagggttgtt 2760 aaaatatgga gacaaacaat cccttaactt tgacatctta tagaaccact taatttcact 2820 gtgctcataa gtcaaaatta aaaagactgc tattcaactt atctactgac agattaagtt 2880 atctgcaatg gacaaatgaa taaaatcggt caaagcagca atttacggga aatggacaag 2940 gttaaagcat ttgccatagc caaagaaaat ttaagttaac aaaataataa accaactcat 3000 gaaaaagtga gcaatttttc aaaactctta taaaatataa aaacaagtac attagtgagg 3060 aagaattaat gtttaaaaat cgtagattaa attgaagtta aggataatat gttttaaaat 3120 tctatgacgt gatagttgtg tgagaattca aacgaagatt cagaatatgc gcagatcccc 3180 taggagatct cagctggacg tccgtacgtt cgaaccgtga ccggcagcaa aatgttgcag 3240 cactgaccct tttgggaccg caatgggttg aattagcgga acgtcgtgta gggggaaagc 3300 ggtcgaccgc attatcgctt ctccgggcgt ggctagcggg aagggttgtc aacgcgtcgg 3360 acttaccgct taccgcggac tacgccataa aagaggaatg cgtagacacg ccataaagtg 3420 tggcgtatac cacgtgagag tcatgttaga cgagactacg gcgtatcaat tcggtcgggg 3480 ctgtgggcgg ttgtgggcga ctgcgcggga ctgcccgaac agacgagggc cgtaggcgaa 3540 tgtctgttcg acactggcag aggccctcga cgtacacagt ctccaaaagt ggcagtagtg 3600 gctttgcgcg ct 3612 <210> 16 <211> 1359 <212> DNA <213> Leu. citreum C16 16S rDNA <400> 16 gacccgggaa cgtattcacc gcggcgtgct gacccgcgat tactagcgat tccgacttcg 60 tgcagtcgag ttgcagactg cagtccgaac tgagacgtac tttaagagat tagctcacct 120 tcgcaggttg gcaactcgtt gtatacgcca ttgtagcacg tgtgtagccc aggtcataag 180 gggcatgatg atctgacgtc gtccccgcct tcctccggtt tgtcaccggc agtctcgcta 240 gagtgcccaa ctgaatgctg gcaactaaca ataagggttg cgctcgttgc gggacttaac 300 ccaacatctc acgacacgag ctgacgacga ccatgcacca cctgtcactt tgtctccgaa 360 gagaacactt ctatctctaa aagcttcaaa ggatgtcaag acctggtaag gttcttcgcg 420 ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg tccccgtcaa ttcctttgag 480 tttcaacctt gcggtcgtac tccccaggcg gaatacttaa tgcgttagct tcggcactaa 540 gaggcggaaa cctcctaaca cctagtattc gtcgtttacg gtgtggacta ccagggtatc 600 taatcctgtt tgctacccac actttcgagc ctcaacgtca gttgttgtcc agtaagccgc 660 cttcgccact ggtgttcttc catatatcta cgcattccac cgctacacat ggagttccac 720 ttacctctac aacactcaag ttaaccagtt tccaatgcca ttccggagtt gagctccggg 780 ctttcacatc agacttaatc aaccgtctgc gctcgcttta cgcccaataa atccggataa 840 cgctcgggac atacgtatta ccgcggctgc tggcacgtat ttagccgtcc ctttctggta 900 tggtaccgtc aaactaaaat cattccctat tttagcattt cttcccatac aacagtgctt 960 tacgacccga aagccttcat cacacacgcg gcgttgctcc atcaggcttg cgcccattgt 1020 ggaagattcc ctactgcagc ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg 1080 ccgatcagtc cctcaactcg gctatgcatc atcgtcttgg taagccttta ccccaccaac 1140 taactaatgc accgcggatc catctctagg tgacgccgta gcgcctttta acttgatatc 1200 atgcgatact aagttttatt cggtattagc atctgtttcc aaatgttatc cccagccttg 1260 aggcaggtta tccacgtgtt actcacccgt tcgccactcg cttgaaaggt gcaagcacct 1320 ctcgctgcgc gttcgacttg catgtattag gcacgccgc 1359  

Claims (11)

A replication factor having the nucleotide sequence of SEQ ID NO: 1. delete delete delete delete delete delete delete delete delete delete

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