PATENT DOCUMENT

Abstract:
The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve production yield of said DNA molecules in fermentation culture.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application Ser. No. 61/205,561, entitled “DNA Plasmids With Improved Copy Number” which was filed Jan. 21, 2009, the entire contents of which are incorporated herein by reference. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was supported in part with government support under Grant No. 2 R44 GM072141-02, awarded by the National Institutes of Health. The government has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules such as plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof, and more particularly to vector modifications that improve production yield of said DNA molecules in fermentation culture. Such recombinant DNA molecules are useful in biotechnology, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines. 
     BACKGROUND OF THE INVENTION 
       E. coli  plasmids have long been an important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (e.g., gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines. Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to the patient. 
     Therapeutic plasmids often contain a pMB1, ColE1 or pBR322 derived replication origin. Common high copy number derivatives have mutations affecting copy number regulation, such as rop (Repressor of primer gene) deletion, with a second site mutation that increases copy number (e.g. pMB1 pUC G to A point mutation, or ColE1 pMM1). Higher temperature (42° C.) can be employed to induce selective plasmid amplification with pUC and pMM1 replication origins. 
     Published Application WO2006023546 (Carnes, A E and Williams, J A) disclose methods for fed-batch fermentation, in which plasmid-containing  E. coli  cells were grown at a reduced temperature during part of the fed-batch phase, during which growth rate was restricted, followed by a temperature up-shift and continued growth at elevated temperature in order to accumulate plasmid; the temperature shift at restricted growth rate improved yield and purity of plasmid. Other fermentation processes for plasmid production are described in Carnes A. E.  BioProcess Intl  2005; 3:36-44, which is incorporated herein by reference in its entirety. 
     There is a significant need for methods that increase production yield of plasmid DNA. High specific yields are very desirable since increased plasmid yield per gram of bacteria, or increased plasmid relative to genomic DNA, lead directly to higher final product purities. Further improvements in yield or increases in the percentage plasmid per total DNA would further decrease production cost, improve purity and simplify removal of genomic DNA (gDNA). 
     SUMMARY OF THE INVENTION 
     The present invention relates generally to methods of increasing production yield of covalently closed super-coiled plasmid DNA. 
     One object of the invention is to provide improved copy number plasmid vectors. Yet another object of the invention is to provide methods for improving plasmid copy number. 
     According to one object of the invention, a method of increasing production yield of covalently closed super-coiled plasmid DNA comprises modifying the plasmid DNA to add one or more components selected from the group consisting of an SV40 enhancer, PAS-BH region, and PAS; transforming the modified plasmid DNA into a bacterial cell line rendered competent for transformation; and isolating the resultant transformed bacterial cells. 
     According to another object of the invention, said methods of plasmid modification improve plasmid yield in subsequent shake flask and or fermentation culture. 
     According to yet another object of the invention, a method of increasing production yield of covalently closed super-coiled plasmid DNA comprises modifying the plasmid DNA to remove one or more transcriptional terminators; transforming the modified plasmid DNA into a bacterial cell line rendered competent for transformation; and isolating the resultant transformed bacterial cells. 
     It is another object of the present invention to provide compositions of matter for construction of a vector. 
     According to one object of the invention, a composition for construction of a vector comprises an SV40 enhancer with at least 90% sequence identity to the sequence set forth as SEQ ID NO: 2, and a plasmid DNA replicon, wherein said SV40 enhancer is operably linked to said plasmid DNA replicon. According to another object of the invention, said SV40 enhancer improves plasmid yield in subsequent shake flask and or fermentation culture. According to still another object of the invention, said vector has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     Yet another object of the invention provides a composition for construction of a vector, comprising a PAS-BH site with at least 90% sequence identity to the sequence set forth as SEQ ID NO: 3, and a plasmid DNA replicon, wherein said PAS-BH site is operably linked to said plasmid DNA replicon. According to another object of the invention, said PAS-BH site improves plasmid yield in subsequent shake flask and or fermentation culture. Furthermore, another object of the invention provides that said vector has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     Still another object of the invention provides a composition for construction of a vector, comprising a PAS-BH region with at least 90% sequence identity to the sequence set forth as SEQ ID NO: 4, and a plasmid DNA replicon, wherein said PAS-BH region is operably linked to said plasmid DNA replicon. According to another object of the invention, said PAS-BH region improves plasmid yield in subsequent shake flask and or fermentation culture. Furthermore, another object of the invention provides that said vector has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     Further objects and advantages of the invention will become apparent from a consideration of the drawings and ensuing description. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  depicts pNTCUltra1 PAS-BH; 
         FIG. 2  depicts NTC8685; and 
         FIG. 3  depicts SV40-CMV enhancer. 
     
    
    
     Table 1: SV40 enhancer and PAS-BH improve plasmid yields 
     Table 2: Antibiotic-free vector expression and manufacture 
     Table 3: NTC8682, NTC8482, NTC7682, NTC7482, NTC8685, NTC8485, NTC7685, and NTC7485 vectors. 
     SEQ ID NO:1: pNTCUltra1 PAS-BH
     SEQ ID NO:2: SV40 enhancer   SEQ ID NO:3: PAS-BH   SEQ ID NO:4: pBR322 origin region between ROP and PAS-BL (pBR322 2067-2351)   SEQ ID NO:5: Apal-Kpnl dual terminator   SEQ ID NO:6: NTC8685   SEQ ID NO:7: NTC8485   SEQ ID NO:8: NTC8682   SEQ ID NO:9: NTC8482   SEQ ID NO:10: NTC7685   SEQ ID NO:11: NTC7485   SEQ ID NO:12: NTC7682   SEQ ID NO:13: NTC7482   SEQ ID NO:14: NTC8482, NTC8682, NTC7482, NTC7682 optimized TPA-SalI secretion signal
 
Definition of Terms
   AF: Antibiotic-free   bp: basepairs   ccc: Covalently Closed Circular   DNA replicon: A genetic element that can replicate under its own control; examples include plasmids, cosmids, bacterial artificial chromosomes (BACs), bacteriophages, viral vectors and hybrids thereof.     E. coli: Escherichia coli , a gram negative bacteria   g: Gram, kg for kilogram   kan: Kanamycin   kanR: Kanamycin Resistance gene   kozak sequence: Optimized sequence of consensus DNA sequence gccRccATG (R=G or A) immediately upstream of an ATG start codon that ensures efficient tranlation initiation. A SalI site (GTCGAC) immediately upstream of the ATG start codon (GTCGACATG) is an effective Kozak sequence   OD 600 : optical density at 600 nm   PAS: Primosomal assembly site   PAS-BH: Primosomal assembly site on the heavy (leading) strand (SEQ ID NO:3)   PAS-BH region: pBR322 origin region between ROP and PAS-BL (approximately pBR322 2067-2351; SEQ ID NO:4)   PAS-BH: Primosomal assembly site on the light (lagging) strand   PCR: Polymerase Chain Reaction   Plasmid: An extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA.   Primosomal assembly site: DNA sequence that binds priA, which, in turn, recruits the remaining proteins to form the preprimosome [priB, dnaT, recruits dnaB (delivered by dnaC)], which then also recruits primase (dnaG), which then, finally, makes a short RNA substrate for DNA polymerase I   pUC origin: pBR322-derived origin, with G to A transition that increases copy number at elevated temperature   pUC plasmid: Plasmid containing the pUC origin   SV40 enhancer: Region containing the 72 by and optionally the 21 by repeats (SEQ ID NO:2; see  FIG. 3 )   Transcription terminator: A DNA sequence that marks the end of a gene or operon for transcription. This may be an intrinsic transcription terminator or a Rho-dependent transcriptional terminator. For an intrinsic terminator, such as the trpA terminator, a hairpin structure forms within the transcript that disrupts the mRNA-DNA-RNA polymerase ternary complex. The dual terminator in pNTCUltra1-term+ contains two intrinsic terminators, the fd gene VIII terminator and the tonB bidirectional terminator. Alternatively, Rho-dependent transcriptional terminators require Rho factor, an RNA helicase protein complex, to disrupt the nascent mRNA-DNA-RNA polymerase ternary complex.   vector: A gene delivery vehicle, including viral (e.g. alphavirus, poxvirus, lentivirus, retrovirus, adenovirus, adenovirus related virus, etc.) and nonviral (e.g. plasmid, midge, transcriptionally active PCR fragment, minicircles, bacteriophage, etc.) vectors. These are well known in the art and are included herein by reference.   

     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The invention relates generally to plasmid DNA compositions and methods to improve plasmid copy number. The invention can be practiced to improve the copy number of vectors such as eukaryotic expression plasmids useful for gene therapy, genetic immunization and or interferon therapy. It is to be understood that all references cited herein are incorporated by reference in their entirety. 
     According to one preferred embodiment, the present invention provides for method of increasing production yield of covalently closed super-coiled plasmid DNA, which comprises modifying the plasmid DNA to add one or more components selected from the group consisting of an SV40 enhancer, PAS-BH region, and PAS; transforming the modified plasmid DNA into a bacterial cell line rendered competent for transformation; and isolating the resultant transformed bacterial cells. 
     According to another preferred embodiment, a method of increasing production yield of covalently closed super-coiled plasmid DNA comprises modifying the plasmid DNA to remove one or more transcriptional terminators; transforming the modified plasmid DNA into a bacterial cell line rendered competent for transformation; and isolating the resultant transformed bacterial cells. 
     In one preferred embodiment, a SV40 enhancer is incorporated into a plasmid DNA backbone to improve plasmid copy number. In another preferred embodiment, a fragment of the pBR322 origin containing a primosomal assembly site is incorporated into a plasmid DNA backbone to improve plasmid copy number. In yet another preferred embodiment, a SV40 enhancer and a fragment of the pBR322 origin containing a primosomal assembly site are both incorporated into a plasmid DNA backbone to improve plasmid copy number. 
     The methods of plasmid modification of the present invention have been surprisingly found to improve plasmid yield in subsequent shake flask and or fermentation culture. 
     Plasmid copy number is preferably increased by employing specific constructs or compositions incorporated in a vector. According to one preferred embodiment, the present invention provides a composition for construction of a vector, comprising an SV40 enhancer with at least 90% sequence identity to the sequence set forth as SEQ ID NO: 2, and a plasmid DNA replicon, wherein the SV40 enhancer is operably linked to the plasmid DNA replicon. It has been surprisingly found that this SV40 enhancer improves plasmid yield in subsequent shake flask and or fermentation culture. According to another preferred embodiment, the vector has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     As used herein, the term “sequence identity” refers to the degree of identity between any given query sequence, e.g., SEQ ID NO: 2, and a subject sequence. A subject sequence may, for example, have at least 90 percent, at least 95 percent, or at least 99 percent sequence identity to a given query sequence. To determine percent sequence identity, a query sequence (e.g., a nucleic acid sequence) is aligned to one or more subject sequences using any suitable sequence alignment program that is well known in the art, for instance, the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid sequences to be carried out across their entire length (global alignment). Chema et al.,  Nucleic Acids Res.,  31(13):3497-500 (2003). In a preferred method, the sequence alignment program (e.g., ClustalW) calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair-wise alignments of nucleic acid sequences, suitable default parameters can be selected that are appropriate for the particular alignment program. The output is a sequence alignment that reflects the relationship between sequences. To further determine percent identity of a subject nucleic acid sequence to a query sequence, the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. 
     According to another preferred embodiment, the present invention provides a composition for construction of a vector, comprising a PAS-BH site with at least 90% sequence identity to the sequence set forth as SEQ ID NO: 3, and a plasmid DNA replicon, wherein the PAS-BH site is operably linked to the plasmid DNA replicon. It has been surprisingly found that this PAS-BH site improves plasmid yield in subsequent shake flask and or fermentation culture. According to another preferred embodiment, the vector has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     According to another preferred embodiment, the present invention provides a composition for construction of a vector, comprising a PAS-BH region with at least 90% sequence identity to the sequence set forth as SEQ ID NO: 4, and a plasmid DNA replicon, wherein said PAS-BH region is operably linked to said plasmid DNA replicon. It has been surprisingly found that this PAS-BH region improves plasmid yield in subsequent shake flask and or fermentation culture. According to another preferred embodiment, the vector preferably has at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13. 
     Turning now to the drawings,  FIG. 1 . shows an annotated map of the pNTCUltra1 PAS-BH vector with the locations of the PAS-BH primosomal assembly site, SV40 enhancer and other key elements indicated. 
       FIG. 2  shows an annotated map of the antibiotic-free NTC8685-EGFP vector with the locations of the PAS-BH primosomal assembly site, SV40 enhancer and other key elements indicated. 
       FIG. 3  shows an annotated map of the SV40-CMV enhancer region in the pNTCUltra1 PAS-BH and NTC8682, NTC8482, NTC7682, NTC7482, NTC8685, NTC8485, NTC7685, and NTC7485 vector backbones with locations of the SV40 enhancer 72 by and 21 by repeats (SEQ ID NO:2), the start of the CMV enhancer and the CMV upstream region deletion in the NTC8685, NTC8682, NTC7685, NTC7682 vectors shown. Coordinates −674 and −562 in the CMV upstream region are relative to the CMV transcription start site. 
     The invention also relates to compositions and methods for producing high copy plasmids using the gram negative bacterium  E. coli  as a production host. The present invention provides sequences that, when introduced into a vector backbone, increase plasmid copy number. 
     We disclose herein the surprising observation that a SV40 enhancer can be utilized as a plasmid copy number enhancer. We further disclose the surprising observation that plasmid copy number is improved by addition of a previously considered nonfunctional region of the pBR322 origin. This region is not present in pUC plasmids. 
     As described herein, plasmid copy number is increased by addition of an SV40 enhancer to the plasmid backbone. In yet another preferred embodiment, the SV40 enhancer is inserted upstream of the eukaryotic promoter. In yet another preferred embodiment, the SV40 enhancer is included as a chimeric SV40-CMV promoter. In yet another preferred embodiment, the SV40 enhancer comprises one or more copies of the 72 by enhancer. 
     In yet another preferred embodiment, plasmid copy number is increased by addition of part or all of the pBR322 origin from between the ROP and PAS-BL site to the plasmid backbone. In yet another preferred embodiment, the pBR322 origin region is inserted upstream of the eukaryotic promoter. In yet another preferred embodiment, the origin region is included as an extended pUC origin. In yet another preferred embodiment, the pBR322 origin region contains the PAS-BH site. In yet another preferred embodiment, the additional origin region is from pBR322 coordinates 2067-2351 (GenBank Accession #: J01749). 
     In another preferred embodiment, plasmid copy number is increased by removal of transcriptional terminator sequences from the plasmid backbone. 
     EXAMPLES 
     The methods of the invention are further illustrated by the following examples. These are provided by way of illustration and are not intended in any way to limit the scope of the invention. 
     Example 1 
     Development of kanR Plasmid Vectors with Improved Copy Number 
     NTCUItra1=6544 by NTC7164-hmPA-EGFP (SV40-CMV promoter, ubiquitin targeting vector, anthrax protective antigen). The SV40-CMV promoter and ubiquitin targeting vectors are disclosed in Williams, J A 2006 World Patent Application WO2006078979 and included herein by reference in their entirety. 
     The pNTCUltra1-term+ plasmid contains a 100 by ApaI-KpnI dual terminator containing the fd gene VIII terminator-tonB bidirectional terminator of the following sequence: 
     SEQ ID NO:5: 100 by ApaI-KpnI dual terminator 
     
       
         
               
             
           
               
                 G GGCCCACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAC   
               
               
                   
               
               
                 
                   TGCAAACAAATAGTCAAAAGCCTCCGGTCGGAGGCTTTTGACTTTGGTAC 
                 
               
               
                   
               
               
                 C. 
               
             
          
         
       
     
     ApaI (GGGCCC) and KpnI (GGTACC) DNA sequence is shown. The underlined dual terminator sequence is not present in pNTCUltra1. 
     pNTCUltra1-PAS-BH=6826 by NTCUItra1 derivative contains the pUC19 replication origin with an additional 285 by of pBR322 origin (starting at the Pvull site immediately downstream of the ROP gene; pBR322 coordinates 2067-2351; SEQ ID NO:4) that includes the 48 by PAS-BH site (SEQ ID NO:3). 
     pNTCUltra1 (-SV40) has the SV40 enhancer deleted (by removal of the 324 by ApaI-XbaI term-SV40 enhancer region from pNTCUltra1-term+; this region contains two copies of the SV40 72 by repeat). 
     pNTCUltra4-PAS-BH=5895 by vector substitutes the PAS-BH containing region of the pBR322 origin for the SV40 enhancer upstream of CMV promoter. 
     pNTCUltra4=the PAS-BH containing region of the pBR322 origin is deleted from pNTCUltra4-PAS-BH 
     Example 2 
     Plasmid Fermentation and Analytical Evaluation 
     Fermentation: Fermentations were performed using proprietary fed-batch media (NTC3019) in New Brunswick BioFlo 110 bioreactors as described (Carnes and Williams, Supra, 2006). The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 μg/mL kanamycin. The plates were grown at 30° C.; cells were resuspended in media, and used to provide approximately 0.1% inoculums for the fermentations. 
     Fermentations were performed with  E. coli  strain DH5α [F-φ80dlacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17(rk−, mk+) phoA supE44 λthi-1 gyrA96 relA1]. Antibiotic-free RNA-OUT plasmid fermentations were performed in DH5α strains containing chromosomally integrated pCAH63-CAT RNA-IN-SacB (P5/6 6/6) as disclosed in Williams, J A 2008 World Patent Application WO2008153733. SacB ( Bacillus subtilis  levansucrase) is a counterselectable marker which is lethal to  E. coli  cells in the presence of sucrose. Translation of SacB from the RNA-IN-SacB transcript is inhibited by plasmid encoded RNA-OUT. This facilitates plasmid selection in the presence of sucrose, by inhibition of SacB mediated lethality. 
     Analytical Methods: Culture samples were taken at key points and at regular intervals during all fermentations. Samples were analyzed immediately for biomass (OD 600 ) and for plasmid yield. Plasmid yield was determined by quantification of plasmid obtained from Qiagen Spin Miniprep Kit preparations as described (Cames and Williams, Supra, 2006). Briefly, cells were alkaline lysed, clarified, plasmid was column purified, and eluted prior to quantification. Agarose gel electrophoresis analysis (AGE) was performed on 0.8-1% Tris/acetate/EDTA (TAE) gels as described in Cames and Williams, supra, 2006. 
     Example 3 
     Plasmid Vectors Containing PAS-BH and/or the SV40 Enhancer have Improved Copy Number 
     In the process of developing high yielding vector backbones, sequences that dramatically and surprisingly affect yield were observed, and are described below. 
     Positioning of Prokaryotic Terminator Sequences 
     A pNTCUltra1 plasmid, with a dual terminator sequence inserted upstream of the SV40 enhancer (pNTCUltra1-term+), was inexplicably a poor producer. The slow growth, poor induction and low overall yield of 119 mg/L at harvest (2.5 mg plasmid/L/OD 600 ) was in contrast to the control pNTCUltra1 plasmid without the terminator region (Table 1: RF157, 1930 mg/L, 17.3 mg plasmid/L/OD 600 ). Improving plasmid copy number by removing transcription terminators was surprising and unexpected. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 SV40 enhancer and PAS-BH improve plasmid yields 
               
             
          
           
               
                   
                   
                   
                   
                 Fermentation 
               
               
                   
                   
                   
                 Fermentation 
                 specific yield 
               
               
                 Fermentation† 
                 Vector 
                 Modification 
                 yield (mg/L) 
                 (mg/L/OD 600 ) 
               
               
                   
               
             
          
           
               
                 RF157 
                 pNTCUltra1 
                 SV40-CMV 
                 1930 
                 17.3 
               
               
                 RF158 
                 pNTCUltra1 (−SV40) 
                 CMV 
                 1050 
                 9.9 
               
               
                 RF160 
                 pNTCUltra1-PAS-BH 
                 SV40-CMV 
                 2210 
                 22.9 
               
               
                 RF167++ 
                   
                 +PAS-BH origin 
                  2220++ 
                 25.3++ 
               
               
                 RF154 
                 pNTCUltra4-PAS-BH 
                 PAS-BH-CMV 
                 1230 
                 12.3 
               
               
                 RF161 
                 pNTCUltra4 
                 CMV 
                  800 
                 10.2 
               
               
                 RF164 
                 gWIZ-D 
                 PAS-BH-CMV 
                 1210 
                 14.4 
               
               
                   
               
               
                 †All fermentations induced at 50 OD 600  for 10.5 h at 42° C., 1 h 25° C. hold 
               
               
                 ++12 h induction, final yield was 51 mg plasmid/g DCW 
               
             
          
         
       
     
     SV40 Enhancer (SEQ ID NO:2) 
     Surprisingly, the pNTCUltra1 vector yielded approximately twice the production yield of gWiz-derived (gWIZ-D) plasmids (Genlantis, San Diego Calif.) (Table 1, RF157 versus RF164). The pNTCUltra improvement compared to gWiz was not insert-specific, and yields in these ranges were obtained with a variety of insert sequences within these vector backbones. These vectors are both eukaryotic expression vectors containing the same elements in different orientations; pUC origin, a kanR selectable marker, and a eukaryotic expression cassette comprising the CMV promoter-gene of interest-eukaryotic terminator. Deletion analysis surprisingly demonstrated that the SV40 enhancer in pNTCUltra1 (SEQ ID NO:2) was critical for improved productivity (Table 1; RF157 versus RF158). The use of the SV40 enhancer to improve plasmid copy number was surprising and unexpected. 
     pBR322 Origin Region 2067-2351 (SEQ ID NO:4) 
     Therapeutic plasmids such as VR1012 (Vical) and derivative gWIZ (Gene Therapy Systems), pPJV7563 (PowderMed/Pfizer), V1JNS (Merck), pVAX1 (Invitrogen), pLL14 (Merial), and pNTCUltra1 (Nature Technology Corporation) were all constructed using origin regions derived from high copy pUC plasmids. To increase copy number, these vectors all deleted the Repressor of primer (ROP) gene. The ROP deletion also includes a leading strand primosomal assembly site (PAS-BH) present in pBR322 (Marians K J, Soeller W, Zipursky S L. 1982.  J. Biol. Chem.  257: 5656-5662). This site is presumed nonfunctional, since 1) the ROP deletion including PAS-BH dramatically increases copy number and 2) Deletion of PAS-BH does not dramatically affect plasmid replication in vivo (Masai H, Arai K I. 1989. J. Bacteriol. 171: 2975-2980). A more precise deletion of the ROP gene (ROP only deletion, includes pBR322 region 2067-2351 compared to the pUC origin ROP +PAS-BH deletion) was constructed (pNTCUltra1 PAS-BH;  FIG. 1 ; SEQ ID NO:1) and evaluated for plasmid copy number. Surprisingly, extension of the pUC promoter to include the deleted 285 by region of pBR322 (SEQ ID NO:4) including PAS-BH (SEQ ID NO:3) further improved productivity (Table 1; RF160 versus RF157) up to 2220 mg/L and 25.3 mg/L/OD 600 (51 mg/gDCW) with a 12 hr induction (Table 1; RF167). While not limiting the application of the invention, improved yields may be due to PAS-BH mediated enhanced primosomal assembly on the leading strand when it is within the DNA polymerase I replication region. Alternatively, another functional region unrelated to PAS-BH or primosomal assembly may be present in this region. Interestingly, yield improvement was also observed in plasmids containing the PAS-BH site 3 kb downstream of the origin (Table 1 RF154, pNTCUltra4 PAS-BH-CMV versus RF161, CMV). 
     pNTCUltra1-PAS-BH and 1VTC7482-41H-HA 
     In summary, a vector backbone, pNTCUltra1, was identified that is twice the productivity of previously characterized high-yielding vectors such as pVAX1 and gWiz. Productivity enhancement was surprisingly determined to be due to a SV40 enhancer present in the pNTCUltra1 vector. Productivity was further improved by inclusion of a leading-strand primosomal assembly site in the vector, downstream of the replication origin (pNTCUltra1-PAS-BH;  FIG. 1 ). This enhancement is also observed with NTC7482-41H-HA compared to gWIZ (Table 2). NTC7482-41H-HA has the same vector backbone as pNTCUltra1, but different transgene sequences. This demonstrates that the enhancement is not affected by alterations to the eukaryotic portion of the vector. 
     Example 4 
     Antibiotic-free Plasmid Vectors Containing PAS-BH and/or the SV40 Enhancer have Improved Copy Number 
     The NTC8385, NTC8485 and NTC8685 plasmids are antibiotic-free vectors that contain a short regulatory RNA (RNA-OUT) in place of the antibiotic resistance marker (kanR). The creation and application of RNA-OUT based antibiotic-free vectors is disclosed in Williams, Supra, 2008 and are included herein by reference. 
     NTC8485 (SEQ ID NO:7) has the equivalent SV40 and PAS-BH backbone as pNTCUltra1-PAS-BH and NTC7482-41H-HA. NTC8685 ( FIG. 2 ; SEQ ID NO:6) has the equivalent SV40 and PAS-BH backbone as pNTCUltra1-PAS-BH and NTC7482-41H-HA and additionally incorporates a deletion of sequences between the SV40 enhancer and the CMV promoter ( FIG. 3 ). This deletion is the only difference between NTC8485 and NTC8685. NTC8385 (Williams, Supra, 2008) is an equivalent antibiotic-free vector without the SV40 and PAS-BH regions. The fermentation yield of NTC8485 (RF248) and NTC8685 (RF252) was significantly improved relative to NTC8385 (RF247; Table 2). This was observed as both an increase in specific yield (a measure of plasmid amount per cell) and specific yield/size (a measure of plasmid copy number per cell) (Table 2). The improved yield was not at the expense of eukaryotic cell expression which is highest with the NTC8685 backbone (Table 2). This also demonstrates that improved copy number is not dependent on the kanR gene since it was observed with kanR and antibiotic-free SV40 and PAS-BH backbones. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Antibiotic-free vector expression and manufacture 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Specific 
                 Ferm 
                   
               
               
                   
                   
                   
                   
                 Ferm 
                 yield 
                 Spec 
               
               
                   
                 Size 
                   
                   
                 Yield 
                 (mg/L/ 
                 Yield/ 
                 Expression 
               
               
                 Construct 
                 (kb) 
                 ID† 
                 Selection 
                 (mg/L) 
                 OD 600 ) 
                 size 
                 (HEK293) 
               
               
                   
               
             
          
           
               
                 gWIZ-EGFP 
                 5.75 
                 RF193 
                 Kan 
                 1370 
                 16.25 
                 2.8 
                 38,190 ± 9,329 
               
               
                 NTC7482-41H-HA 
                 6.21 
                 RF205 
                 Kan 
                 2200 
                 24.9 
                 4.0 
                 ND 
               
               
                 NTC8385 b   
                 3.59 
                 RF247 
                 Sucrose 
                 775 
                 9.11 
                 2.5 
                 47,808 ± 12,916 a   
               
               
                 NTC8485 b   
                 3.80 
                 RF248 
                 Sucrose 
                 1210 
                 16.2 
                 4.4 
                 52,141 ± 7,356 a   
               
               
                 NTC8685 b   
                 3.68 
                 RF252 
                 Sucrose 
                 1055 
                 12.7 
                 3.5 
                 60,418 ± 2,624 a   
               
               
                   
               
               
                 †All fermentations induced at 50 OD 600  for 10.5 h at 42° C., 1 h 25° C. hold 
               
               
                   a Expression from equivalent EGFP-encoding vector 
               
               
                   b NTC8385 and NTC8485 vector backbones were modified to contain the identical VA RNAI regulatory RNA as is present in NTC8685. All three vectors encode the same 585 bp transgene. 
               
             
          
         
       
     
     In summary, the novel SV40 enhancer and PAS-BH vector modifications resulted in dramatically higher increases in fermentation yields than previously defined yield improving modifications such as optimizing the orientation of the kanR and replication origin (Williams, Supra, 2006) or reduction of kanR gene expression (pLL14; Audonnet, J. 2008 World Patent Application WO2008136790). Indeed, the modifications do not require a kanR gene to improve copy number, and improved copy number was observed with the RNA-OUT RNA-based selectable marker as well. These modifications should therefore also improve plasmid copy number with a wide variety of alternative selection markers. 
     Example 5 
     NTC8682, NTC8482, NTC7682, NTC7482, NTC8685, NTC8485, NTC7685, and NTC7485 Vectors 
     A series of kanR and AF eukaryotic expression vectors incorporating the novel SV40 enhancer and PAS-BH vector modifications were made. The features of these vectors (NTC8682, NTC8482, NTC7682, NTC7482, NTC8685, NTC8485, NTC7685, and NTC7485) are summarized in Table 3. 
     NTC8682, NTC8482, NTC8685, and NTC8485 are antibiotic-free RNA-OUT versions of the kanR NTC7682, NTC7482, NTC7685, and NTC7485 equivalents. 
     NTC8482, NTC8485, NTC7482 and NTC7485 contain the full SV40-CMV enhancer region ( FIG. 3 ) while NTC8682, NTC8685, NTC7682 and NTC7685 all contain the CMV upstream region deletion between the SV40 enhancer and the CMV enhancer ( FIG. 3 ). 
     NTC8682, NTC8685, NTC7682 and NTC7685 additionally contain the expression enhancing Adenoviral serotype 5 VA RNAI regulatory RNA (VA1;  FIG. 2 ) while NTC8482, NTC8485, NTC7482 and NTC7485 do not. 
     NTC8482, NTC7682, NTC7482 and NTC8682 all express the secreted transgene product as TPA fusion proteins while, NTC8685, NTC8485, NTC7485and NTC7685 all express the native transgene product from a vector encoded ATG start codon. 
     The remainder of the vector sequences is identical between the different vectors. 
     These are just a few possible nonlimiting vector configurations. Many alternative vector configurations incorporating the novel SV40 enhancer and PAS-BH vector modifications may also be made, including but not limited to vectors with alternative selection markers, alternative promoters, alternative terminators, and different orientations of the various vector-encoded elements. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 NTC8682, NTC8482, NTC7682, NTC7482, NTC8685, 
               
               
                 NTC8485, NTC7685, and NTC7485 vectors 
               
             
          
           
               
                   
                   
                 VA RNAI 
                 Upstream 
                 Transgene 
               
               
                 Vector 
                 Selection 
                 present 
                 region deletion 
                 targeting 
               
               
                   
               
               
                 NTC8682 (SEQ ID NO: 8) 
                 Sucrose 
                 Yes 
                 Yes 
                 Secretion (TPA) 
               
               
                 NTC8482 (SEQ ID NO: 9) 
                 Sucrose 
                 No 
                 No 
                 Secretion (TPA) 
               
               
                 NTC7682 (SEQ ID NO: 12) 
                 Kanamycin 
                 Yes 
                 Yes 
                 Secretion (TPA) 
               
               
                 NTC7482 (SEQ ID NO: 13) 
                 Kanamycin 
                 No 
                 No 
                 Secretion (TPA) 
               
               
                 NTC8685 (SEQ ID NO: 6) 
                 Sucrose 
                 Yes 
                 Yes 
                 Native 
               
               
                 NTC8485 (SEQ ID NO: 7) 
                 Sucrose 
                 No 
                 No 
                 Native 
               
               
                 NTC7685 (SEQ ID NO: 10) 
                 Kanamycin 
                 Yes 
                 Yes 
                 Native 
               
               
                 NTC7485 (SEQ ID NO: 11) 
                 Kanamycin 
                 No 
                 No 
                 Native 
               
               
                   
               
             
          
         
       
     
     An example strategy for cloning into these vectors is outlined below. 
     
       
         
               
               
             
           
               
                   GTCGAC   ATG --------Gene of interest----Stop codon------ AGATCT   
                   
               
               
                 SalI                                                BglII 
               
             
          
         
       
     
     For the NTC8685, NTC8485, NTC7685, and NTC7485 vectors, the ATG start codon (double underlined) is immediately preceded by a unique SalI site. The SalI site is an effective Kozak sequence for translational initiation. In NTC8682, NTC8482, NTC7682, and NTC7482, the SalI site is downstream in frame with the optimized TPA secretion sequence (SEQ ID NO:14). The WA ATG start codon is double underlined and the SalI site single underlined. 
     
       
         
               
             
           
               
                 SEQ ID NO: 14: 
               
               
                   atg gatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagcagt 
               
               
                 cttcgtttcgcccagcggtaccggatcc gtcgac   
               
             
          
         
       
     
     For precise cloning, genes are copied by PCR amplification from clones, cDNA, or genomic DNA using primers with SalI (5′ end) and BglII (3′ end) sites. Alternatively, genes are synthesized chemically to be compatible with the unique SalI/BglII cloning sites in these vectors. 
     For NTC8685, NTC8485, NTC7685, and NTC7485, the start codon ATG must immediately follow the SalI site (GTCGACATG). For NTC8682, NTC8482, NTC7682, and NTC7482, the ATG is optional but the same reading frame must be retained. For all vectors one or two stop codon (preferably TAA or TGA) must be included prior to the BglII site. A PCR product designed for NTC8685, NTC8485, NTC7685, and NTC7485 vectors is compatible with, and can also be cloned into, the NTC8682, NTC8482, NTC7682, and NTC7482 vectors. 
     Thus, the reader will see that the improved copy number vectors of the invention provide for a rational approach to improve production yield of plasmids. 
     While the basis for yield improvement is unknown, optimal vectors are expected to have a higher copy number due to reduction of replication inhibiting factors and incorporation of replication promoting factors. Completion of replication can be confounded by many factors, including: sequences that cause inhibition of DNA polymerase III replication [such as protein-DNA complexes assembled on replication termini (Ter) or repressor binding sequences; stable RNA-DNA hybrids]; unusual DNA structures [e.g. internal-ribosome entry sites; Levy J. 2003 US Patent Application US2003180949]; or head on transcription-replication collisions. As well, yield may be improved by replication promoting factors that increase RNA primer synthesis rate and/or replication primer utilization. For example, optimization of the orientation of the selectable marker relative to the replication origin (Williams, Supra, 2006) or reducing the strength of the selectable marker promoter (Audonnet, Supra, 2008) may reduce marker mediated transcriptional interference of replication primer synthesis and/or reduce head on transcription-replication collisions. While not limiting the application of the invention, inclusion of a primosomal assembly site such as PAS-BH may be beneficial at a later replication step, through increasing the percent of replication primers that successfully initiate DNA polymerase HI mediated replication cycles. 
     While the above description contains many examples, these should not be construed as limitations on the scope of the invention, but rather should be viewed as an exemplification of preferred embodiments thereof. Many other variations are possible. For example, the selectable marker may be substituted with an alternative selectable marker. Likewise, the vectors may contain alternative promoters, terminators and/or transgenes to the examples provided herein. The orientation of the various vector-encoded elements may be changed relative to each other. The vectors may optionally contain additional functionalities, such as nuclear localizing sequences, the adenoviral VA RNAI , and/or immunostimulatory RNA elements as disclosed in Williams, Supra, 2008. Vectors containing combinations of SV40 enhancers and/or primosomal assembly sites could be rapidly created and screened for improved copy number. Alternatively, one or more primosomal assembly sites and/or SV40 enhancers could be inserted into different regions of a vector, in different orientations, and the resultant vectors screened for improved copy number. Alternatively, plasmid copy number may be improved by deletion of transcriptional terminators in existing vectors. 
     Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims. 
     
       
         
               
             
           
               
                 SEQ ID NO: 1: pNTCUltra1 PAS-BH (6826 bp) 
               
               
                 cgggcttttttttcttaggctgcctcgcgcgtttcggtgatgacggtgaa 
               
               
                   
               
               
                 aacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagc 
               
               
                   
               
               
                 ggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcg 
               
               
                   
               
               
                 ggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtat 
               
               
                   
               
               
                 actggcttaactatgcggcatcagagcagattgtactgagagtgcaccat 
               
               
                   
               
               
                 atgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcag 
               
               
                   
               
               
                 gcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggc 
               
               
                   
               
               
                 tgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccaca 
               
               
                   
               
               
                 gaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaa 
               
               
                   
               
               
                 ggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc 
               
               
                   
               
               
                 gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcaa 
               
               
                   
               
               
                 acccgacaggactataaagataccaggcgtttccccctggaagctccctc 
               
               
                   
               
               
                 gtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctt 
               
               
                   
               
               
                 tctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatc 
               
               
                   
               
               
                 tcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccc 
               
               
                   
               
               
                 cccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc 
               
               
                   
               
               
                 caacccggtaagacacgacttatcgccactggcagcagccactggtaaca 
               
               
                   
               
               
                 ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtag 
               
               
                   
               
               
                 tggccctactacggctacactagaagaacagtatttggtatctgcgctct 
               
               
                   
               
               
                 gctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca 
               
               
                   
               
               
                 aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagatt 
               
               
                   
               
               
                 acgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg 
               
               
                   
               
               
                 gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatga 
               
               
                   
               
               
                 gattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagt 
               
               
                   
               
               
                 tttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca 
               
               
                   
               
               
                 atgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcat 
               
               
                   
               
               
                 ccatagttgcctgactcctgcaaaccacgttgtgtctcaaaatctctgat 
               
               
                   
               
               
                 gttacattgcacaagataaaatatatcatcatgaacaataaaactgtctg 
               
               
                   
               
               
                 cttacataaacagtaatacaaggggtgttatgagccatattcaacgggaa 
               
               
                   
               
               
                 acgtcttgctcgaggccgcgattaaattccaacatggatgctgatttata 
               
               
                   
               
               
                 tgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatct 
               
               
                   
               
               
                 atcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggc 
               
               
                   
               
               
                 aaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactg 
               
               
                   
               
               
                 gctgacggaatttatgcctcttccgaccatcaagcattttatccgactcc 
               
               
                   
               
               
                 tgatgatgcatggttactcaccactgcgatccccgggaaaacagcattcc 
               
               
                   
               
               
                 aggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctg 
               
               
                   
               
               
                 gcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttt 
               
               
                   
               
               
                 taacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaata 
               
               
                   
               
               
                 acggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcct 
               
               
                   
               
               
                 gttgaacaagtctggaaagaaatgcataagcttttgccattctcaccgga 
               
               
                   
               
               
                 ttcagtcgtcactcatggtgatttctcacttgaaaccttatttttgacga 
               
               
                   
               
               
                 ggggaaattaataggttgtattgatgttggacgagtcggaatcgcagacc 
               
               
                   
               
               
                 gataccaggatcttgccatcctatggaactgcctcggtgagttttctcct 
               
               
                   
               
               
                 tcattacagaaacggctttttcaaaaatatggtattgataatcctgatat 
               
               
                   
               
               
                 gaataaattgcagtttcatttgatgctcgatgagtttttctaagccctga 
               
               
                   
               
               
                 tcactgtggaatgtgtgtcagttagggtgtggaaagtccccaggctcccc 
               
               
                   
               
               
                 agcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggt 
               
               
                   
               
               
                 gtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcat 
               
               
                   
               
               
                 ctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcc 
               
               
                   
               
               
                 cctaactccgcccaggatccgctctagatggccattgcatacgttgtatc 
               
               
                   
               
               
                 catatcataatatgtacatttatattggctcatgtccaacattaccgcca 
               
               
                   
               
               
                 tgttgacattgattattgactagttattaatagtaatcaattacggggtc 
               
               
                   
               
               
                 attagttcatagcccatatatggagttccgcgttacataacttacggtaa 
               
               
                   
               
               
                 atggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaata 
               
               
                   
               
               
                 atgacgtatgttcccatagtaacgccaatagggactttccattgacgtca 
               
               
                   
               
               
                 atgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgt 
               
               
                   
               
               
                 atcatatgccaagtacgccccctattgacgtcaatgacggtaaatggccc 
               
               
                   
               
               
                 gcctggcattatgcccagtacatgaccttatgggactttcctacttggca 
               
               
                   
               
               
                 gtacatctacgtattagtcatcgctattaccatggtgatgcggttttggc 
               
               
                   
               
               
                 agtacatcaatgggcgtggatagcggtttgactcacggggatttccaagt 
               
               
                   
               
               
                 ctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacg 
               
               
                   
               
               
                 ggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcg 
               
               
                   
               
               
                 gtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaac 
               
               
                   
               
               
                 cgtcagacacttgctaaagacaataacaaatttacttgcaatcccccaaa 
               
               
                   
               
               
                 acagacaggtaagtatcctttttacagcacaacttaatgagacagataga 
               
               
                   
               
               
                 aactggtcttgtagaaacagagagtcgcctgcttttctgccaggtgctga 
               
               
                   
               
               
                 cttctctcccctgggctttttctttttctcaggttgaaaagaagaagacg 
               
               
                   
               
               
                 aagaagacgaagaagacaaagccgccaccatgcagatcttcgtgaagacc 
               
               
                   
               
               
                 ctgacgggcaagaccaccactcttggggtcgagcccagtgacaccatcga 
               
               
                   
               
               
                 gaatgtcaaggccaagatccaagacaaggaaggcatcccacctgaccagc 
               
               
                   
               
               
                 agaggctgatattcgcgggcaaacagctggaggatggccgcaccctgtcc 
               
               
                   
               
               
                 gactacaacatccagaaagagtccaccttgcacctggtgctgcgtctgcg 
               
               
                   
               
               
                 cggtgccgctatggaggtgaagcaggagaaccggctgctcaacgaaagcg 
               
               
                   
               
               
                 aaagcagcagccagggcctgctcggctactacttcagcgacctgaacttc 
               
               
                   
               
               
                 caggctcccatggtggtgaccagcagcaccaccggcgatctgagcatccc 
               
               
                   
               
               
                 cagcagcgagctggagaacatccccagcgagaaccagtacttccagagcg 
               
               
                   
               
               
                 ccatctggagcggcttcatcaaggtgaagaaaagcgatgagtacaccttc 
               
               
                   
               
               
                 gctaccagcgctgacaaccacgtgaccatgtgggtggacgatcaggaagt 
               
               
                   
               
               
                 gatcaacaaggccagcaacagcaacaaaatccggctggagaagggccggc 
               
               
                   
               
               
                 tgtaccagatcaagatccagtaccagcgggaaaaccccaccgagaagggc 
               
               
                   
               
               
                 ctggacttcaagctgtactggaccgacagccagaacaagaaagaagtgat 
               
               
                   
               
               
                 cagcagcgataacctgcagctgcccgagctgaagcagaagagcagcaaca 
               
               
                   
               
               
                 gccggaaaaaacggagcaccagcgctggccccaccgtgcccgatcgggat 
               
               
                   
               
               
                 aacgatggcatccccgatagcctggaagtggagggctacaccgtggacgt 
               
               
                   
               
               
                 gaagaacaaacggaccttcctgagcccctggatcagcaacatccacgaga 
               
               
                   
               
               
                 agaaaggcctgaccaagtacaaaagcagccccgaaaaatggagcaccgct 
               
               
                   
               
               
                 agcgacccctacagcgatttcgagaaggtgaccggccggatcgacaaaaa 
               
               
                   
               
               
                 cgtgagccccgaagctcggcaccccctggtggctgcttaccccatcgtgc 
               
               
                   
               
               
                 acgtggacatggagaacatcatcctgagcaagaacgaagatcagagcacc 
               
               
                   
               
               
                 cagaacaccgacagcgagacccggaccatcagcaaaaacaccagcaccag 
               
               
                   
               
               
                 ccggacccacaccagcgaagtgcacggcaacgccgaggtgcacgctagct 
               
               
                   
               
               
                 tcttcgatatcggcggcagcgtgagcgctggcttcagcaacagcaacagc 
               
               
                   
               
               
                 agcaccgtggctatcgatcacagcctgagcctggctggcgaacggacctg 
               
               
                   
               
               
                 ggctgaaaccatgggcctgaacaccgctgataccgctcggctgaacgcta 
               
               
                   
               
               
                 acatccggtacgtgaacaccggcaccgctcccatctacaacgtgctgccc 
               
               
                   
               
               
                 accaccagcctggtgctgggcaagaaccagaccctggccaccatcaaggc 
               
               
                   
               
               
                 caaggaaaaccagctgagccagatcctggcccccaacaactactacccca 
               
               
                   
               
               
                 gcaaaaacctggctcccatcgctctgaacgctcaggacgatttcagcagc 
               
               
                   
               
               
                 acccccatcaccatgaactacaaccagttcctggagctggagaagaccaa 
               
               
                   
               
               
                 acagctgcggctggataccgaccaggtgtacggcaacatcgccacctaca 
               
               
                   
               
               
                 acttcgaaaacggccgggtgcgggtggataccggcagcaactggagcgaa 
               
               
                   
               
               
                 gtgctgccccagatccaggaaaccaccgcccggatcatcttcaacggcaa 
               
               
                   
               
               
                 ggatctgaacctggtggaacggcggatcgctgctgtgaaccccagcgatc 
               
               
                   
               
               
                 ccctggagaccaccaaacccgacatgaccctgaaggaggccctgaagatc 
               
               
                   
               
               
                 gccttcggcttcaacgaacccaacggcaacctgcagtaccagggcaagga 
               
               
                   
               
               
                 catcaccgagttcgacttcaacttcgaccagcagaccagccagaacatca 
               
               
                   
               
               
                 aaaaccagctggccgagctgaacgccaccaacatctacaccgtgctggac 
               
               
                   
               
               
                 aagatcaagctgaacgccaagatgaacatcctgatccgggacaagcgttc 
               
               
                   
               
               
                 cactacgatcggaacaacatcgctgtgggcgctgatgaaagcgtggtgaa 
               
               
                   
               
               
                 ggaagctcaccgggaagtgatcaacagcagcaccgagggcctgctcctga 
               
               
                   
               
               
                 acatcgacaaggacatccggaagatcctgagcggctacatcgtggagatc 
               
               
                   
               
               
                 gaggacaccgagggcctgaaggaagtgatcaacgaccggtacgacatgct 
               
               
                   
               
               
                 gaacatcagcagcctgcggcaggacggcaagaccttcatcgacttcaaga 
               
               
                   
               
               
                 aatacaacgacaagctgcccctgtacatcagcaaccccaactacaaggtg 
               
               
                   
               
               
                 aacgtgtacgccgtgaccaaggagaacaccatcatcaaccccagcgaaaa 
               
               
                   
               
               
                 cggcgacaccagcaccaacggcatcaagaaaatcctgatcttcagcaaga 
               
               
                   
               
               
                 aaggctacgagatcggcggcccggtcgccaccatggtgagcaagggcgag 
               
               
                   
               
               
                 gagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgt 
               
               
                   
               
               
                 aaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacct 
               
               
                   
               
               
                 acggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtg 
               
               
                   
               
               
                 ccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcag 
               
               
                   
               
               
                 ccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgc 
               
               
                   
               
               
                 ccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaac 
               
               
                   
               
               
                 tacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccg 
               
               
                   
               
               
                 catcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggc 
               
               
                   
               
               
                 acaagctggagtacaactacaacagccacaacgtctatatcatggccgac 
               
               
                   
               
               
                 aagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcga 
               
               
                   
               
               
                 ggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcg 
               
               
                   
               
               
                 gcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtcc 
               
               
                   
               
               
                 gccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctgga 
               
               
                   
               
               
                 gttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagt 
               
               
                   
               
               
                 aactcgagccgcagatctttttccctctgccaaaaattatggggacatca 
               
               
                   
               
               
                 tgaagccccttgagcatctgacttctggctaataaaggaaatttatttca 
               
               
                   
               
               
                 ttgcaatagtgtgttggaattttttgtgtctctcactcggaaggacataa 
               
               
                   
               
               
                 gggcggccgcaacgacgagaacgaacgaagaacgctcgagagtcgattta 
               
               
                   
               
               
                 aatcccccctgtatcgatgcactgcctcgatgctgcatcgatgcacaaaa 
               
               
                   
               
               
                 tgctagcccgcctaatgag 
               
               
                   
               
               
                 SEQ ID NO: 2: SV40 enhancer (212 bp) 
               
               
                 ctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagc 
               
               
                   
               
               
                 aggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtg 
               
               
                   
               
               
                 gaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctc 
               
               
                   
               
               
                 aattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccct 
               
               
                   
               
               
                 aactccgcccag 
               
               
                   
               
               
                 SEQ ID NO: 3: PAS-BH (48 bp) 
               
               
                 cagctcccggagacggtcacagcttgtctgtaagcggatgccgggagc 
               
               
                   
               
               
                 SEQ ID NO: 4: pBR322 origin region between ROP and 
               
               
                 PAS-BL (pBR322 2067-2351) (285 bp) 
               
               
                 ctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagc 
               
               
                   
               
               
                 tcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaa 
               
               
                   
               
               
                 gcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccat 
               
               
                   
               
               
                 gacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggc 
               
               
                   
               
               
                 atcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgc 
               
               
                   
               
               
                 acagatgcgtaaggagaanataccgcatcaggcgc 
               
               
                   
               
               
                 SEQ ID NO: 5: ApaI-KpnI dual terminator (101 bp) 
               
               
                 GGGCCCACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTGGAC 
               
               
                   
               
               
                 TGCAAACAAATAGTCAAAAGCCTCCGGTCGGAGGCTTTTGACTTTGGTAC 
               
               
                   
               
               
                 C 
               
               
                   
               
               
                 SEQ ID NO: 6: NTC8685 (3095 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttagggtgcaaaggagagcctgta 
               
               
                   
               
               
                 agcgggcactcttccgtggtctggtggataaattcgcaagggtatcatgg 
               
               
                   
               
               
                 cggacgaccggggttcgagccccgtatccggccgtccgccgtgatccatg 
               
               
                   
               
               
                 cggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggg 
               
               
                   
               
               
                 gagtgctccttttggcttccttcccctaccggtctgcctcgcgcgtttcg 
               
               
                   
               
               
                 gtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcaca 
               
               
                   
               
               
                 gcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtc 
               
               
                   
               
               
                 agcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgagcga 
               
               
                   
               
               
                 tagcggagtgtatactggcttaactatgcggcatcagagcagattgtact 
               
               
                   
               
               
                 gagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaa 
               
               
                   
               
               
                 aataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgc 
               
               
                   
               
               
                 tcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaat 
               
               
                   
               
               
                 acggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaa 
               
               
                   
               
               
                 aaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttt 
               
               
                   
               
               
                 ttccataggctccgcccccctgacgagcatcacaaaaaatcgacgctcaa 
               
               
                   
               
               
                 gtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccc 
               
               
                   
               
               
                 cctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg 
               
               
                   
               
               
                 atacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagct 
               
               
                   
               
               
                 cacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc 
               
               
                   
               
               
                 tgtgtgcacgaaccccccgttcagcccgaccgctgcgcctatccggtaac 
               
               
                   
               
               
                 tatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagc 
               
               
                   
               
               
                 agccactggtaacaggattagcagagcgaggtatgtaggcggtgctacag 
               
               
                   
               
               
                 agttcttgaagtggtggcctaactacggctacactagaagaacagtattt 
               
               
                   
               
               
                 ggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtag 
               
               
                   
               
               
                 ctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt 
               
               
                   
               
               
                 gcaagcagcagattacgcgcagaaaaaaggatctcaagaagatcctttga 
               
               
                   
               
               
                 tcttttctacggggtctgacgctcagtggaacgaaaactcacgttaaggg 
               
               
                   
               
               
                 attttggtcatgagattatcaaaaaggatcttcacctagatccttaaatt 
               
               
                   
               
               
                 aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtct 
               
               
                   
               
               
                 gacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct 
               
               
                   
               
               
                 atttcgttcatccatagttgcctgactcctgcaaaccacgttgtggtaga 
               
               
                   
               
               
                 attggtaaagagagtcgtgtaaaatatcgagttcgcacatcttgttgtct 
               
               
                   
               
               
                 gattattgatttttggcgaaaccatttgatcatatgacaagatgtgtatc 
               
               
                   
               
               
                 taccttaacttaatgattttgataaaaatcattaggtacccctgatcact 
               
               
                   
               
               
                 gtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcag 
               
               
                   
               
               
                 gcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtgga 
               
               
                   
               
               
                 aagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaa 
               
               
                   
               
               
                 ttagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaa 
               
               
                   
               
               
                 ctccgcccagttacggggtcattagttcatagcccatatatggagttccg 
               
               
                   
               
               
                 cgttacaaacttacggtaaatggcccgcctggctgaccgcccaacgaccc 
               
               
                   
               
               
                 ccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatag 
               
               
                   
               
               
                 ggactttccattgacgtcaatgggtggagtatttacggtaaactgcccac 
               
               
                   
               
               
                 ttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgt 
               
               
                   
               
               
                 caatgacggtaaatggcccgcctggcattatgcccagtacatgaccttat 
               
               
                   
               
               
                 gggactttcctacttggcagtacatctacgtattagtcatcgctattacc 
               
               
                   
               
               
                 atggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttga 
               
               
                   
               
               
                 ctcacggggatttccaagtctccaccccattgacgtcaatgggagtttgt 
               
               
                   
               
               
                 tttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgcc 
               
               
                   
               
               
                 ccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataag 
               
               
                   
               
               
                 cagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgct 
               
               
                   
               
               
                 gttttgacctccatagaagacaccgggaccgatccagcctccgcggctcg 
               
               
                   
               
               
                 catctctccttcacgcgcccgccgccctacctgaggccgccatccacgcc 
               
               
                   
               
               
                 ggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcg 
               
               
                   
               
               
                 tccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtcc 
               
               
                   
               
               
                 ggcgctcccttggagcctacctagactcagccggctctccacgctttgcc 
               
               
                   
               
               
                 tgaccctgcttgctcaactctagttctctcgttaacttaatgagacagat 
               
               
                   
               
               
                 agaaactggtcttgtagaaacagagtagtcgcctgcttttctgccaggtg 
               
               
                   
               
               
                 ctgacttctctcccctgggcttttttctttttctcaggttgaaaagaaga 
               
               
                   
               
               
                 agacgaagaagacgaagaagacaaaccgtcgtcgacagatctttttccct 
               
               
                   
               
               
                 ctgccaaaaattatggggacatcatgaagccccttgagcatctgacttct 
               
               
                   
               
               
                 ggctaataaaggaaatttattttcattgcaatagtgtgttggaatttttt 
               
               
                   
               
               
                 gtgtctctcactcggaaggacataagggcggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 7: NTC8485 (3016 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttaggctgcctcgcgcgtttcggt 
               
               
                   
               
               
                 gatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagc 
               
               
                   
               
               
                 ttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcag 
               
               
                   
               
               
                 cgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgat 
               
               
                   
               
               
                 agcggagtgtatactggcttaactatgcggcatcagagcagattgtactg 
               
               
                   
               
               
                 agagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaa 
               
               
                   
               
               
                 ataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgct 
               
               
                   
               
               
                 cggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaata 
               
               
                   
               
               
                 cggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa 
               
               
                   
               
               
                 aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttt 
               
               
                   
               
               
                 tccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagt 
               
               
                   
               
               
                 cagaggtggcgaaacccgacaggactataaagataccaggcgtttccccc 
               
               
                   
               
               
                 tggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat 
               
               
                   
               
               
                 acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctca 
               
               
                   
               
               
                 cgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctg 
               
               
                   
               
               
                 tgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaaca 
               
               
                   
               
               
                 tcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcag 
               
               
                   
               
               
                 ccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagag 
               
               
                   
               
               
                 ttcttgaagtggtggcctaactacggctacactagaagaacagtatttgg 
               
               
                   
               
               
                 atctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctc 
               
               
                   
               
               
                 ttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca 
               
               
                   
               
               
                 agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatc 
               
               
                   
               
               
                 ttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat 
               
               
                   
               
               
                 tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt 
               
               
                   
               
               
                 aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtct 
               
               
                   
               
               
                 gacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct 
               
               
                   
               
               
                 atttcgttcatccatagttgcctgactcctgcaaaccacgttgtggtaga 
               
               
                   
               
               
                 attggtaaagagagtcgtgtaaaatatcgagttcgcacatcttgttgtct 
               
               
                   
               
               
                 gattattgatttttggcgaaaccatttgatcatatgacaagatgtgtatc 
               
               
                   
               
               
                 taccttaacttaatgattttgataaaaatcattaggtacccctgatcact 
               
               
                   
               
               
                 gtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcag 
               
               
                   
               
               
                 gcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtgga 
               
               
                   
               
               
                 aagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaa 
               
               
                   
               
               
                 ttagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaa 
               
               
                   
               
               
                 ctccgcccaggatccgctctagatggccattgcatacgttgtatccatat 
               
               
                   
               
               
                 cataatatgtacatttatattggctcatgtccaacattaccgccatgttg 
               
               
                   
               
               
                 acattgattattgactagttattaatagtaatcaattacggggtcattag 
               
               
                   
               
               
                 ttcatagcccatatatggagttccgcgttacataacttacggtaaatggc 
               
               
                   
               
               
                 ccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac 
               
               
                   
               
               
                 gtatgttcccatagtaacgccaatagggactttccattgacgtcaatggg 
               
               
                   
               
               
                 tggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcat 
               
               
                   
               
               
                 atgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctg 
               
               
                   
               
               
                 gcattatgcccagtacatgaccttatgggactttcctacttggcagtaca 
               
               
                   
               
               
                 tctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac 
               
               
                   
               
               
                 atcaatgggcgtggatagcggtttgactcacggggatttccaagtctcca 
               
               
                   
               
               
                 ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggact 
               
               
                   
               
               
                 ttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtagg 
               
               
                   
               
               
                 cgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtca 
               
               
                   
               
               
                 gatcgcctggagacgccatccacgctgttttgacctccatagaagacacc 
               
               
                   
               
               
                 gggaccgatccagcctccgcggctcgcatctctccttcacgcgcccgccg 
               
               
                   
               
               
                 ccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctc 
               
               
                   
               
               
                 ccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaag 
               
               
                   
               
               
                 ctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctag 
               
               
                   
               
               
                 actcagccggctctccacgctttgcctgaccctgcttgctcaactctagt 
               
               
                   
               
               
                 tctctcgttaacttaatgagacagatagaaactggtcttgtagaaacaga 
               
               
                   
               
               
                 gtagtcgcctgcttttctgccaggtgctgacttctctcccctgggctttt 
               
               
                   
               
               
                 ttctttttctcaggttgaaaagaagaagacgaagaagacgaagaagacaa 
               
               
                   
               
               
                 accgtcgtcgacagatctttttccctctgccaaaattatggggacatcat 
               
               
                   
               
               
                 gaagccccttgagcatctgacttctggctaataaaggaaatttattttca 
               
               
                   
               
               
                 ttgcaatagtgtgttggaattttttgtgtctctcactcggaaggacataa 
               
               
                   
               
               
                 ggcggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 8: NTC8682 (3186 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttagggtgcaaaaggagagcctga 
               
               
                   
               
               
                 agcgggcactcttccgtggtctggtggataaattcgcaagggtatcatgg 
               
               
                   
               
               
                 cggacgaccggggttcgagccccgtatccggccgtccgccgtgatccatg 
               
               
                   
               
               
                 cggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggg 
               
               
                   
               
               
                 gagtgctccttttggcttccttcccctaccggtctgcctcgcgcgtttcg 
               
               
                   
               
               
                 gtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcaca 
               
               
                   
               
               
                 gcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtc 
               
               
                   
               
               
                 agcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcg 
               
               
                   
               
               
                 atagcggagtgtatactggcttaactatgcggcatcagagcagattgtac 
               
               
                   
               
               
                 tgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggaga 
               
               
                   
               
               
                 aaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcg 
               
               
                   
               
               
                 ctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaa 
               
               
                   
               
               
                 tacggttatccacagaatcaggggataacgcaggaaagaacatgtgagca 
               
               
                   
               
               
                 aaaggccagcaaaaggccaggaaccgtaaaaggccgcgttgctggcgttt 
               
               
                   
               
               
                 ttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag 
               
               
                   
               
               
                 tcagaggtggcgaaacccgacaggactataaagataccaggcgtttcccc 
               
               
                   
               
               
                 ctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgga 
               
               
                   
               
               
                 tacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctc 
               
               
                   
               
               
                 acgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct 
               
               
                   
               
               
                 gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaac 
               
               
                   
               
               
                 tatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagc 
               
               
                   
               
               
                 agccactggtacaggattagcagagcgaggtatgtaggcggtgctacaga 
               
               
                   
               
               
                 gttcttgaagtggtggcctaactacggctacactagaagaacagtatttg 
               
               
                   
               
               
                 gtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagc 
               
               
                   
               
               
                 tcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttg 
               
               
                   
               
               
                 caagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttga 
               
               
                   
               
               
                 tcttttctacggggtctgacgctcagtggaacgaaaactcacgttaaggg 
               
               
                   
               
               
                 attttggtcatgagattatcaaaaaggatcttcacctagatccttttaaa 
               
               
                   
               
               
                 ttaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt 
               
               
                   
               
               
                 ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgt 
               
               
                   
               
               
                 ctatttcgttcatccatagttgcctgactcctgcaaaccacgttgtggta 
               
               
                   
               
               
                 gaattggtaaagagagtcgtgtaaaatatcgagttcgcacatcttgttgt 
               
               
                   
               
               
                 ctgattattgatttttggcgaaaccatttgatcatatgacaagatgtgta 
               
               
                   
               
               
                 tctaccttaacttaatgattttgataaaaatcattaggtacccctgatca 
               
               
                   
               
               
                 ctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagc 
               
               
                   
               
               
                 aggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtg 
               
               
                   
               
               
                 gaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctc 
               
               
                   
               
               
                 aattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccct 
               
               
                   
               
               
                 aactccgcccagttacggggtcattagttcatagcccatatatggagttc 
               
               
                   
               
               
                 cgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacga 
               
               
                   
               
               
                 cccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa 
               
               
                   
               
               
                 tagggactttccattgacgtcaatgggtggagtatttacggtaactgccc 
               
               
                   
               
               
                 acttggcagacatcaatgtatcatatgccaagtacgccccctattgacgt 
               
               
                   
               
               
                 caatgacggtaaatggcccgcctggcattatgcccagacatgaccttatg 
               
               
                   
               
               
                 ggactttcctacttggcaggtacatctacgtattagtcatcgctattacc 
               
               
                   
               
               
                 atggtgatgcggttttggcagtacatcatgggcgtggatagcggtttgac 
               
               
                   
               
               
                 tcacggggatttccaagtctccaccccattgacgtcaatgggagtttgtt 
               
               
                   
               
               
                 ttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccc 
               
               
                   
               
               
                 cattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc 
               
               
                   
               
               
                 agagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctg 
               
               
                   
               
               
                 ttttgacctccatagaagacaccgggaccgatccagcctccgcggctcgc 
               
               
                   
               
               
                 atctctccttcacgcgcccgccgccctacctgaggccgccatccacgccg 
               
               
                   
               
               
                 gttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgt 
               
               
                   
               
               
                 ccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccg 
               
               
                   
               
               
                 gcgctcccttggagcctacctagactcagccggctctccacgctttgcct 
               
               
                   
               
               
                 gaccctgcttgctcaactctagttctctcgttaactaatgagacagatag 
               
               
                   
               
               
                 aaactggtcttgtagaaacagagtagtcgcctgcttttctgccaggtgct 
               
               
                   
               
               
                 gacttctctcccctgggcttttttctttttctcaggttgaaaagaagaag 
               
               
                   
               
               
                 acgaagaagacgaagaagacaaagccgccaccatggatgcaatgaagaga 
               
               
                   
               
               
                 gggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccag 
               
               
                   
               
               
                 cggtaccggatccgtcgacggggggagatctttttccctctgccaaaaat 
               
               
                   
               
               
                 tatggggacatcatgaagccccttgagcatctgacttctggctaataaag 
               
               
                   
               
               
                 gaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcac 
               
               
                   
               
               
                 tcggaaggacataagggcggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 9: NTC8482 (3107 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttaggctgcctcgcgcgtttcggt 
               
               
                   
               
               
                 gatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagc 
               
               
                   
               
               
                 ttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcag 
               
               
                   
               
               
                 cgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgat 
               
               
                   
               
               
                 agcggagtgtatactggcttaactatgcggcatcagagcagattgtactg 
               
               
                   
               
               
                 agagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaa 
               
               
                   
               
               
                 ataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgct 
               
               
                   
               
               
                 cggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaata 
               
               
                   
               
               
                 cggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa 
               
               
                   
               
               
                 aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttt 
               
               
                   
               
               
                 tccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagt 
               
               
                   
               
               
                 cagaggtggcgaaacccgacaggactataaagataccaggcgtttccccc 
               
               
                   
               
               
                 tggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat 
               
               
                   
               
               
                 acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctca 
               
               
                   
               
               
                 cgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctg 
               
               
                   
               
               
                 tgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaact 
               
               
                   
               
               
                 atcgtcttgagtccaacccggtaagacacgacttatcgccactggcagca 
               
               
                   
               
               
                 gccactggtaacaggattagcagagcgaggtatgtaggcggtgctacaga 
               
               
                   
               
               
                 gttcttgaagtggtggcctaactacggctacactagaagaacagtatttg 
               
               
                   
               
               
                 gtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagc 
               
               
                   
               
               
                 tcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttg 
               
               
                   
               
               
                 caagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttga 
               
               
                   
               
               
                 tcttttctacggggtctgacgctcagtggaacgaaaactcacgttaaggg 
               
               
                   
               
               
                 attttggtcatgagattatcaaaaaggatcttcacctagatccttttaaa 
               
               
                   
               
               
                 ttaaaaatgattttaaatcaatctaaagtatatatgagtaaacttggtct 
               
               
                   
               
               
                 gacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct 
               
               
                   
               
               
                 atttcgttcatccaagttgcctgactcctgcaaaccacgtttgtggtaga 
               
               
                   
               
               
                 attggtaaagagagtcgtgtaaaatatcgagttcgcacatcttgttgtct 
               
               
                   
               
               
                 gattattgatttttggcgaaaccatttgatcatatgacaagatgtgtatc 
               
               
                   
               
               
                 taccttaacttaatgattttgataaaaatcattaggtacccctgatcact 
               
               
                   
               
               
                 gtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcag 
               
               
                   
               
               
                 gcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtgga 
               
               
                   
               
               
                 aagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaa 
               
               
                   
               
               
                 ttagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaa 
               
               
                   
               
               
                 ctccgcccaggatccgctctagatggccattgcatacgttgtatccatat 
               
               
                   
               
               
                 cataatatgtacatttatattggctcatgtccaacattaccgccatgttg 
               
               
                   
               
               
                 acattgattattgactagttattaatagtaatcaattacggggtcattag 
               
               
                   
               
               
                 ttcatagcccatatatggagttccgcgttacataacttacggtaaatggc 
               
               
                   
               
               
                 ccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgac 
               
               
                   
               
               
                 gtatgttcccatagtaacgccaatagggactttccattgacgtcaatggg 
               
               
                   
               
               
                 tggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcat 
               
               
                   
               
               
                 atgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctg 
               
               
                   
               
               
                 gcattatgcccagtacatgaccttatgggactttcctacttggcagtaca 
               
               
                   
               
               
                 tctacgtattagtcatcgctattaccatggtgatgcggttttggcagtac 
               
               
                   
               
               
                 atcaatgggcgtggatagcggtttgactcacggggatttccaagtctcca 
               
               
                   
               
               
                 ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggact 
               
               
                   
               
               
                 ttccaaaatgtcgtaacaactccgccccattgacgcaatgggcggtaggc 
               
               
                   
               
               
                 gtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcag 
               
               
                   
               
               
                 atcgcctggagacgccatccacgctgttttgacctccatagaagacaccg 
               
               
                   
               
               
                 ggaccgatccagcctccgcggctcgcatctctccttcacgcgcccgccgc 
               
               
                   
               
               
                 cctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcc 
               
               
                   
               
               
                 cgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagc 
               
               
                   
               
               
                 tcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctaga 
               
               
                   
               
               
                 ctcagccggctctccacgctttgcctgaccctgcttgctcaactctagtt 
               
               
                   
               
               
                 ctctcgttaacttaatgagacagatagaaactggtcttgtagaaacagag 
               
               
                   
               
               
                 tagtcgcctgcttttctgccaggtgctgacttctctcccctgggcttttt 
               
               
                   
               
               
                 tctttttctcaggttgaaaagaagaagacgaagaagacgaagaagacaaa 
               
               
                   
               
               
                 gccgccaccatggatgcaatgaagagagggctctgctgtgtgctgctgct 
               
               
                   
               
               
                 gtgtggagcagtcttcgtttcgcccagcggtaccggatccgtcgacgggg 
               
               
                   
               
               
                 ggagatctttttccctctgccaaaaattatggggacatcatgaagcccct 
               
               
                   
               
               
                 tgagcatctgacttctggctaataaaggaaatttattttcattgcaatag 
               
               
                   
               
               
                 tgtgttggaattttttgtgtctctcactcggaaggacataaggcggccgc 
               
               
                   
               
               
                 tagc 
               
               
                   
               
               
                 SEQ ID NO: 10: NTC7685 (3863 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttagggtgcaaaaggagagcctgt 
               
               
                   
               
               
                 aagcgggcactcttccgtggtctggtggataaattcgcaagggtatcatg 
               
               
                   
               
               
                 gcggacgaccggggttcgagccccgtatccggccgtccgccgtgatccat 
               
               
                   
               
               
                 gcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacggg 
               
               
                   
               
               
                 ggagtgctccttttggcttccttcccctaccggtctgcctcgcgcgtttc 
               
               
                   
               
               
                 ggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcac 
               
               
                   
               
               
                 agcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgt 
               
               
                   
               
               
                 cagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagc 
               
               
                   
               
               
                 gatagcggagtgtatactggcttaactatgcggcatcagagcagattgta 
               
               
                   
               
               
                 ctgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggag 
               
               
                   
               
               
                 aaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgc 
               
               
                   
               
               
                 gctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggta 
               
               
                   
               
               
                 atacggttatccacagaatcaggggataacgcaggaaagaacatgtgagc 
               
               
                   
               
               
                 aaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgt 
               
               
                   
               
               
                 ttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca 
               
               
                   
               
               
                 agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc 
               
               
                   
               
               
                 ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg 
               
               
                   
               
               
                 gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagc 
               
               
                   
               
               
                 tcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctggg 
               
               
                   
               
               
                 ctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggta 
               
               
                   
               
               
                 actatcgtcttgagtccaacccggtaagacacgacttatcgccactggca 
               
               
                   
               
               
                 gcagccactggtaacaggattagcagagcgaggtatgaggcggtgctaca 
               
               
                   
               
               
                 gagttcttgaagtggtggcctaactacggctacactagaagaacagtatt 
               
               
                   
               
               
                 tggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggta 
               
               
                   
               
               
                 gctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtt 
               
               
                   
               
               
                 tgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatccttt 
               
               
                   
               
               
                 gatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaag 
               
               
                   
               
               
                 ggattttggtcatgagattatcaaaaaggatcttcacctagatcctttta 
               
               
                   
               
               
                 aattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttg 
               
               
                   
               
               
                 gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct 
               
               
                   
               
               
                 gtctatttcgttcatccatagttgcctgactcctgcaaaccacgttgtgt 
               
               
                   
               
               
                 ctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatga 
               
               
                   
               
               
                 acaataaaactgtctgcttacataaacagtaatacaaggggtgttatgag 
               
               
                   
               
               
                 ccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaaca 
               
               
                   
               
               
                 tggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaa 
               
               
                   
               
               
                 tcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagtt 
               
               
                   
               
               
                 gtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgaga 
               
               
                   
               
               
                 tggtcagactaaactggctgacggaatttatgcctcttccgaccatcaag 
               
               
                   
               
               
                 cattttatccgtactcctgatgatgcatggttactcaccactgcgatccc 
               
               
                   
               
               
                 cgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaa 
               
               
                   
               
               
                 atattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcct 
               
               
                   
               
               
                 gtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggc 
               
               
                   
               
               
                 gcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacg 
               
               
                   
               
               
                 agcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagctt 
               
               
                   
               
               
                 ttgccattctcaccggattcagtcgtcactcatggtgatttctcacttga 
               
               
                   
               
               
                 taaccttatttttgacgaggggaaattaataggttgtattgatgttggac 
               
               
                   
               
               
                 gagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgc 
               
               
                   
               
               
                 ctcggtgagttttctccttcattacagaaacggctttttcaaaaatatgg 
               
               
                   
               
               
                 tattgataatcctgatatgaataaattgcagtttcatttgatgctcgatg 
               
               
                   
               
               
                 agtttttctaaccctgatcactgtggaatgtgtgtcagttagggtgtgga 
               
               
                   
               
               
                 aagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaa 
               
               
                   
               
               
                 ttagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaa 
               
               
                   
               
               
                 gtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgccccta 
               
               
                   
               
               
                 actccgcccatcccgcccctaactccgcccagttacggggtcattagttc 
               
               
                   
               
               
                 atagcccataatggagttccgcgttacataacttacggtaaatggcccgc 
               
               
                   
               
               
                 ctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtat 
               
               
                   
               
               
                 gttcccatagtaacgccaatagggactttccattgacgtcaatgggtgga 
               
               
                   
               
               
                 gtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgc 
               
               
                   
               
               
                 caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcat 
               
               
                   
               
               
                 tatgcccagtacatgaccttatgggactttcctacttggcagtacatcta 
               
               
                   
               
               
                 cgtattagtcatcgctattaccatggtgatgcggttttggcagtacatca 
               
               
                   
               
               
                 atgggcgtggatagcggtttgactcacggggatttccaagtctccacccc 
               
               
                   
               
               
                 attgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttcc 
               
               
                   
               
               
                 aaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtg 
               
               
                   
               
               
                 tacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatc 
               
               
                   
               
               
                 gcctggagacgccatccacgctgttttgacctccatagaagacaccggga 
               
               
                   
               
               
                 ccgatccagcctccgcggctcgcatctctccttcacgcgcccgccgccct 
               
               
                   
               
               
                 acctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgc 
               
               
                   
               
               
                 ctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctca 
               
               
                   
               
               
                 ggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactc 
               
               
                   
               
               
                 agccggctctccacgctttgcctgaccctgcttgctcaactctagttctc 
               
               
                   
               
               
                 tcgttaacttaatgagacagatagaaactggtcttgtagaaacagagtag 
               
               
                   
               
               
                 tcgcctgcttttctgccaggtgctgacttctctcccctgggcttttttct 
               
               
                   
               
               
                 ttttctcaggttgaaaagaagaagacgaagaagacgaagaagacaaaccg 
               
               
                   
               
               
                 tcgtcgacagatctttttccctctgccaaaaattatggggacatcatgaa 
               
               
                   
               
               
                 gccccttgagcatctgacttctggctaataaaggaaatttattttcattg 
               
               
                   
               
               
                 caatagtgtgttggaattttttgtgtctctcactcggaaggacataaggg 
               
               
                   
               
               
                 cggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 11: NTC7485 (3784 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttaggctgcctcgcgcgtttcggt 
               
               
                   
               
               
                 gatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagc 
               
               
                   
               
               
                 ttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcag 
               
               
                   
               
               
                 cgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgat 
               
               
                   
               
               
                 agcggagtgtatactggcttaactatgcggcatcagagcagattgtactg 
               
               
                   
               
               
                 agagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaa 
               
               
                   
               
               
                 ataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgct 
               
               
                   
               
               
                 cggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaata 
               
               
                   
               
               
                 cggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa 
               
               
                   
               
               
                 aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttt 
               
               
                   
               
               
                 tccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagt 
               
               
                   
               
               
                 cagaggtggcgaaacccgacaggactataaagataccaggcgtttccccc 
               
               
                   
               
               
                 tggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat 
               
               
                   
               
               
                 acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctca 
               
               
                   
               
               
                 cgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctg 
               
               
                   
               
               
                 tgtgcacgaaccccccgttcagcccgaccgctgcgcctatccggtaacta 
               
               
                   
               
               
                 tcgtcttgagtccaacccggaagacacgacttatcgccactggcagcagc 
               
               
                   
               
               
                 cactggtaacaggattagcagagcgaggatgtaggcggtgctacagagtt 
               
               
                   
               
               
                 cttgatggtggcctaactacggctacactagaagaacagtatttggtatc 
               
               
                   
               
               
                 tgcgctctgctgaagccagtaccttcggaaaaagagttggtagctcttga 
               
               
                   
               
               
                 tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagca 
               
               
                   
               
               
                 gcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt 
               
               
                   
               
               
                 ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttg 
               
               
                   
               
               
                 gtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaa 
               
               
                   
               
               
                 atgaagtttaaatcaatctaaagtatatatgagtaaacttggtctgacag 
               
               
                   
               
               
                 ttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttc 
               
               
                   
               
               
                 gttcatccatagttgcctgactcctgcaaaccacgttgtgtctcaaaatc 
               
               
                   
               
               
                 tctgatgttacattgcacaagataaaaatatatcatcatgaacaataaaa 
               
               
                   
               
               
                 ctgtctgcttacataaacagtaatacaaggggtgttatgagccatattca 
               
               
                   
               
               
                 acgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctg 
               
               
                   
               
               
                 atttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcg 
               
               
                   
               
               
                 acaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaa 
               
               
                   
               
               
                 acatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagac 
               
               
                   
               
               
                 taaactggctgacggaatttatgcctcttccgaccatcaagcattttatc 
               
               
                   
               
               
                 cgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaac 
               
               
                   
               
               
                 agcattccaggtattagagaatatcctgattcaggtgaaaatattgttga 
               
               
                   
               
               
                 tgcgctggcattcctgcgccggttgcattcgattcctgtttgtaattgtc 
               
               
                   
               
               
                 cttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatg 
               
               
                   
               
               
                 aataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctg 
               
               
                   
               
               
                 gcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcac 
               
               
                   
               
               
                 cggattcagtcgtcactcatggtgatttctcacttgataaccttattttt 
               
               
                   
               
               
                 gacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgc 
               
               
                   
               
               
                 agaccgataccaggatcttgccatcctatggaactgcctcggtgagtttt 
               
               
                   
               
               
                 ctccttcattacagaaacggctttttcaaaaatatggtattgataatcct 
               
               
                   
               
               
                 gatatgaataaattgcagtttcatttgatgctcgatgagtttttctaacc 
               
               
                   
               
               
                 ctgatcactgtggaatgtgtgtcagttagggtgtggaaagtccccaggct 
               
               
                   
               
               
                 ccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacc 
               
               
                   
               
               
                 aggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcat 
               
               
                   
               
               
                 gcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcc 
               
               
                   
               
               
                 cgcccctaactccgcccaggatccgctctagatggccattgcatacgttg 
               
               
                   
               
               
                 tatccatatcataatatgtacatttatattggctcatgtccaacattacc 
               
               
                   
               
               
                 gccatgttgacattgattattgactagttattaatagtaatcaattacgg 
               
               
                   
               
               
                 ggtcattagttcatagcccatatatggagttccgcgttacataacttacg 
               
               
                   
               
               
                 gtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtc 
               
               
                   
               
               
                 aataatgacgtatgttcccatagtaacgccaatagggactttccattgac 
               
               
                   
               
               
                 gtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaa 
               
               
                   
               
               
                 gtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatg 
               
               
                   
               
               
                 gcccgcctggcattatgcccagtacatgaccttatgggactttcctactt 
               
               
                   
               
               
                 ggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttt 
               
               
                   
               
               
                 tggcagtacatcaatgggcgtggatagcggtttgactcacggggatttcc 
               
               
                   
               
               
                 aagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatc 
               
               
                   
               
               
                 aacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatg 
               
               
                   
               
               
                 ggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagt 
               
               
                   
               
               
                 gaaccgtcagatcgcctggagacgccatccacgctgttttgacctccata 
               
               
                   
               
               
                 gaagacaccgggaccgatccagcctccgcggctcgcatctctccttcacg 
               
               
                   
               
               
                 cgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttc 
               
               
                   
               
               
                 tgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggta 
               
               
                   
               
               
                 agtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggag 
               
               
                   
               
               
                 cctacctagactcagccggctctccacgctttgcctgaccctgcttgctc 
               
               
                   
               
               
                 aactctagttctctcgttaacttaatgagacagatagaaactggtcttgt 
               
               
                   
               
               
                 agaaacagagtagtcgcctgcttttctgccaggtgctgacttctctcccc 
               
               
                   
               
               
                 tgggcttttttctttttctcaggttgaaaagaagaagacgaagaagacga 
               
               
                   
               
               
                 agaagacaaaccgtcgtcgacagatctttttccctctgccaaaaattatg 
               
               
                   
               
               
                 gggacatcatgaagccccttgagcatctgacttctggctaataaaggaaa 
               
               
                   
               
               
                 tttattttcattgcaatagtgtgttggaattttttgtgtctctcactcgg 
               
               
                   
               
               
                 aaggacataaggcggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 12: NTC7682 (3954 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttagggtgcaaaaggagagcctgt 
               
               
                   
               
               
                 aagcgggcactcttccgtggtctggtggataaattcgcaagggtatcatg 
               
               
                   
               
               
                 gcggacgaccggggttcgagccccgtatccggccgtccgccgtgatccat 
               
               
                   
               
               
                 gcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacggg 
               
               
                   
               
               
                 ggagtgctccttttggcttccttcccctaccggtctgcctcgcgcgtttc 
               
               
                   
               
               
                 ggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcac 
               
               
                   
               
               
                 agcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgt 
               
               
                   
               
               
                 cagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagc 
               
               
                   
               
               
                 gatagcggagtgtatactggcttaactatgcggcatcagagcagattgta 
               
               
                   
               
               
                 ctgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggag 
               
               
                   
               
               
                 aaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgc 
               
               
                   
               
               
                 gctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggta 
               
               
                   
               
               
                 atacggttatccacagaatcaggggataacgcaggaaagaacatgtgagc 
               
               
                   
               
               
                 aaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgt 
               
               
                   
               
               
                 ttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctca 
               
               
                   
               
               
                 agtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc 
               
               
                   
               
               
                 ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg 
               
               
                   
               
               
                 gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaagct 
               
               
                   
               
               
                 cacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggc 
               
               
                   
               
               
                 tgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaa 
               
               
                   
               
               
                 ctatcgtcttgagtccaacccggtaagacacgacttatcgccactggcag 
               
               
                   
               
               
                 cagccactggtaacaggattagcagagcgaggtatgtaggcggtgctaca 
               
               
                   
               
               
                 gagttcttgaagtggtggcctaactacggctacactagaagaacagtatt 
               
               
                   
               
               
                 tggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggta 
               
               
                   
               
               
                 gctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtt 
               
               
                   
               
               
                 tgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatccttt 
               
               
                   
               
               
                 gatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaag 
               
               
                   
               
               
                 ggattttggtcatgagattatcaaaaaggatcttcacctagatcctttta 
               
               
                   
               
               
                 aattaaaaatgaagtttaaatcaatctaaagtatatatgagtaaacttgg 
               
               
                   
               
               
                 tctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctg 
               
               
                   
               
               
                 tctatttcgttcatccatagttgcctgactcctgcaaaccacgttgtgtc 
               
               
                   
               
               
                 tcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaa 
               
               
                   
               
               
                 caataaaactgtctgcttacataaacagtaatacaaggggtgttatgagc 
               
               
                   
               
               
                 catattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacat 
               
               
                   
               
               
                 ggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaat 
               
               
                   
               
               
                 caggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttg 
               
               
                   
               
               
                 tttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagat 
               
               
                   
               
               
                 ggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagc 
               
               
                   
               
               
                 attttatccgtactcctgatgatgcatggttactcaccactgcgatcccc 
               
               
                   
               
               
                 gggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaa 
               
               
                   
               
               
                 tattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctg 
               
               
                   
               
               
                 tttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcg 
               
               
                   
               
               
                 caatcacgaatgaataacggtttggttgatgcgagtgattttgatgacga 
               
               
                   
               
               
                 gcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttt 
               
               
                   
               
               
                 tgccattctcaccggattcagtcgtcactcatggtgatttctcacttgat 
               
               
                   
               
               
                 aaccttatttttgacgaggggaaattaataggttgtattgatgttggacg 
               
               
                   
               
               
                 agtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcc 
               
               
                   
               
               
                 tcggtgagttttctccttcattacagaaacggctttttcaaaaatatggt 
               
               
                   
               
               
                 attgataatcctgatatgaataaattgcagtttcatttgatgctcgatga 
               
               
                   
               
               
                 gtttttctaaccctgatcactgtggaatgtgtgtcagttagggtgtggaa 
               
               
                   
               
               
                 agtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaat 
               
               
                   
               
               
                 tagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaag 
               
               
                   
               
               
                 tatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaa 
               
               
                   
               
               
                 ctccgcccatcccgcccctaactccgcccagttacggggtcattagttca 
               
               
                   
               
               
                 tagcccatatggagttccgcgttacataacttacggtaaatggcccgcct 
               
               
                   
               
               
                 ggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgt 
               
               
                   
               
               
                 tcccatagtaacgccaatagggactttccattgacgtcaatgggtggagt 
               
               
                   
               
               
                 atttacggtaaactgcccacttggcagtacatcaagtgtatcatatgcca 
               
               
                   
               
               
                 agtacgccccctattgacgtcaatgacggtaaatggcccgcctggcatta 
               
               
                   
               
               
                 tgcccagtacatgaccttatgggactttcctacttggcagtacatctacg 
               
               
                   
               
               
                 tattagtcatcgctattaccatggtgatgcggttttggcagtacatcaat 
               
               
                   
               
               
                 gggcgtggatagcggtttgactcacggggatttccaagtctccaccccat 
               
               
                   
               
               
                 tgacgtcatgggagtttgttttggcaccaaaatcaacgggactttccaaa 
               
               
                   
               
               
                 atgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtac 
               
               
                   
               
               
                 ggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcc 
               
               
                   
               
               
                 tggagacgccatccacgctgttttgacctccatagaagacaccgggaccg 
               
               
                   
               
               
                 atccagcctccgcggctcgcatctctccttcacgcgcccgccgccctacc 
               
               
                   
               
               
                 tgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctg 
               
               
                   
               
               
                 tggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggt 
               
               
                   
               
               
                 cgagaccgggcctttgtccggcgctcccttggagcctacctagactcagc 
               
               
                   
               
               
                 cggctctccacgctttgcctgaccctgcttgctcaactctagttctctcg 
               
               
                   
               
               
                 ttaacttaatgagacagatagaaactggtcttgtagaaacagagtagtcg 
               
               
                   
               
               
                 cctgcttttctgccaggtgctgacttctctcccctgggcttttttctttt 
               
               
                   
               
               
                 tctcaggttgaaaagaagaagacgaagaagacgaagaagacaaagccgcc 
               
               
                   
               
               
                 accatggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtgg 
               
               
                   
               
               
                 agcagtcttcgtttcgcccagcggtaccggatccgtcgacggggggagat 
               
               
                   
               
               
                 ctttttccctctgccaaaaattatggggacatcatgaagccccttgagca 
               
               
                   
               
               
                 tctgacttctggctaataaaggaaatttattttcattgcaatagtgtgtt 
               
               
                   
               
               
                 ggaattttttgtgtctctcactcggaaggacataagggcggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 13: NTC7482 (3876 bp) 
               
               
                 ccgcctaatgagcgggcttttttttcttaggctgcctcgcgcgtttcggt 
               
               
                   
               
               
                 gatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagc 
               
               
                   
               
               
                 ttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcag 
               
               
                   
               
               
                 cgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgat 
               
               
                   
               
               
                 agcggagtgtaactggcttaactatgcggcatcagagcagattgtactga 
               
               
                   
               
               
                 gagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaa 
               
               
                   
               
               
                 taccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctc 
               
               
                   
               
               
                 ggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatac 
               
               
                   
               
               
                 ggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaa 
               
               
                   
               
               
                 ggccagcaaaaggccaggaaccgtaaaaggccgcgttgctggcgtttttc 
               
               
                   
               
               
                 cataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtca 
               
               
                   
               
               
                 gaggtggcgaaacccgacaggactataaagataccaggcgtttccccctg 
               
               
                   
               
               
                 gaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac 
               
               
                   
               
               
                 ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacg 
               
               
                   
               
               
                 ctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtg 
               
               
                   
               
               
                 tgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat 
               
               
                   
               
               
                 cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagc 
               
               
                   
               
               
                 cactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagt 
               
               
                   
               
               
                 tcttgaagtggtggcctaactacggctacactagaagaacagtatttggt 
               
               
                   
               
               
                 atctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctc 
               
               
                   
               
               
                 ttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca 
               
               
                   
               
               
                 agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatc 
               
               
                   
               
               
                 ttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat 
               
               
                   
               
               
                 tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt 
               
               
                   
               
               
                 aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtct 
               
               
                   
               
               
                 gacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtct 
               
               
                   
               
               
                 atttcgttcatccatagttgcctgactcctgcaaaccacgttgtctcaaa 
               
               
                   
               
               
                 atctctgatgttacattgcacaagataaaaatatatcatcatgaacaata 
               
               
                   
               
               
                 aaactgtctgcttacataaacagtaatacaaggggtgttatgagccatat 
               
               
                   
               
               
                 tcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatg 
               
               
                   
               
               
                 ctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggt 
               
               
                   
               
               
                 gcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttct 
               
               
                   
               
               
                 gaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtca 
               
               
                   
               
               
                 gactaaactggctgacggaatttatgcctcttccgaccatcaagcatttt 
               
               
                   
               
               
                 atccgtactcctgatgatgcatggttactcaccactgcgatccccgggaa 
               
               
                   
               
               
                 aacagcattccaggtattagaagaatatcctgattcaggtgaaaatattg 
               
               
                   
               
               
                 ttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgt 
               
               
                   
               
               
                 aattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatc 
               
               
                   
               
               
                 acgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgta 
               
               
                   
               
               
                 atggctggcctgttgaacaagtctggaaagaaatgcataagcttttgcca 
               
               
                   
               
               
                 ttctcaccggattcagtcgtcactcatggtgatttctcacttgataacct 
               
               
                   
               
               
                 tatttttgacgaggggaaattaataggttgtattgatgttggacgagtcg 
               
               
                   
               
               
                 gaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggt 
               
               
                   
               
               
                 gagttttctccttcattacagaaacggctttttcaaaaatatggtattga 
               
               
                   
               
               
                 taatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttt 
               
               
                   
               
               
                 tctaaccctgatcactgtggaatgtgtgtcagttagggtgtggaaagtcc 
               
               
                   
               
               
                 ccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtc 
               
               
                   
               
               
                 agcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgc 
               
               
                   
               
               
                 aaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccg 
               
               
                   
               
               
                 cccatcccgcccctaactccgcccaggatccgctctagatggccattgca 
               
               
                   
               
               
                 tacgttgtatccatatcataatatgtacatttatattggctcatgtccaa 
               
               
                   
               
               
                 cattaccgccatgttgacattgattattgactagttattaatagtaatca 
               
               
                   
               
               
                 attacggggtcattagttcatagcccatatatggagttccgcgttacata 
               
               
                   
               
               
                 acttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccat 
               
               
                   
               
               
                 tgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttc 
               
               
                   
               
               
                 cattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagt 
               
               
                   
               
               
                 acatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacg 
               
               
                   
               
               
                 gtaaatggcccgcctggcattatgcccagtacatgaccttatgggacttt 
               
               
                   
               
               
                 cctacttggcagtacatctacgtattagtcatcgctattaccatggtgat 
               
               
                   
               
               
                 gcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggg 
               
               
                   
               
               
                 gatttccaagtctccaccccattgacgtcaatgggagtttgttttggcac 
               
               
                   
               
               
                 caaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgac 
               
               
                   
               
               
                 gcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctc 
               
               
                   
               
               
                 gtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgac 
               
               
                   
               
               
                 ctccatagaagacaccgggaccgatccagcctccgcggctcgcatctctc 
               
               
                   
               
               
                 cttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagt 
               
               
                   
               
               
                 cgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgt 
               
               
                   
               
               
                 ctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcc 
               
               
                   
               
               
                 cttggagcctacctagactcagccggctctccacgctttgcctgaccctg 
               
               
                   
               
               
                 cttgctcaactctagttctctcgttaacttaatgagacagatagaaactg 
               
               
                   
               
               
                 gtcttgtagaaacagagtagtcgcctgcttttctgccaggtgctgacttc 
               
               
                   
               
               
                 tctcccctgggcttttttctttttctcaggttgaaaagaagaagacgaag 
               
               
                   
               
               
                 aagacgaagaagacaaagccgccaccatggatgcaatgaagagagggctc 
               
               
                   
               
               
                 tgctgtgtgctgctgctgtgtggagcagtcttcgtttcgcccagcggtac 
               
               
                   
               
               
                 cggatccgtcgacggggggagatctttttccctctgccaaaaattatggg 
               
               
                   
               
               
                 gacatcatgaagccccttgagcatctgacttctggctaataaaggaaatt 
               
               
                   
               
               
                 tattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaa 
               
               
                   
               
               
                 ggacataagggcggccgctagc 
               
               
                   
               
               
                 SEQ ID NO: 14: NTC8482, NTC8682, NTC7482, NTC7682 
               
               
                 optimized TPA-SalI secretion signal (87 bp) 
               
               
                 atggatgcaatgaagagagggctctgctgtgtgctgctgctgtgtggagc 
               
               
                   
               
               
                 agtcttcgtttcgcccagcggtaccggatccgtcgac