| 出品公司: | SBI |
|---|---|
| 载体名称: | pCDH-CMV-MCS-EF1-copGFP-T2A-Puro |
| 质粒类型: | 慢病毒表达载体；cDNA表达载体；双启动子载体 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 启动子: | CMV |
| 载体大小: | -- |
| 5' 测序引物及序列: | CMV-F :CGCAAATGGGCGGTAGGCGTG |
| 3' 测序引物及序列: | -- |
| 载体标签: | 无 |
| 载体抗性: | 氨苄青霉素（Ampicillin） |
| 筛选标记: | GFP、Puromycin |
| 克隆菌株: | E.coli cells(RecA-)推荐: Stbl2 ,OmniMAX 2 T1R |
| 宿主细胞（系）: | 常用细胞系，如HeLa, HEK293, HT1080, H1299 |
| 备注: |
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro慢病毒表达载体是基于HIV的慢病毒载体； 用于cDNA表达和克隆；高效转染细胞，建立稳定细胞系； CMV启动子驱动目的基因的高水平表达，EF1a启动子驱动报告基因的中 等水平的表达。 |
| 产品目录号: | CD513B-1 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 慢病毒（HIV) |
买家导航
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro载体质粒基本信息
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro质粒图谱载体图谱和pCDH-CMV-MCS-EF1-copGFP-T2A-Puro载体序列质粒序列多克隆位点信息
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro质粒载体简介
背景简介：
This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.
基于HIV-1的pCDH 慢病毒载体特征：
Multiple Cloning Site (MCS)—for cloning the gene of interest in the MCS located downstream of the CMV promoter.
WPRE element—enhances stability and translation of the CMV-driven transcripts.
SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.
Hybrid RSV/5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.
Genetic elements (cPPT, gag, env, LTRs)—necessary for packaging, transducing, and stably integrating the vira expression construct into genomic DNA.
SV40 origin—for stable propagation of the pCDH plasmid in mammalian cells.
pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.
Ampicillin resistance gene—for selection in E.coli cells.
pCDH 慢病毒表达载体的优势：
Lentiviral expression vectors are the most effective vehicles for the delivery and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.
pCDH 慢病毒载体的包装载体及细胞系
The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently co-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system,please refer to the Lentivector Expression System user manual.
启动子的选择：
SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.
The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.
The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).
The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.
MSCV promoter is the 5’-LTR promoter of murine stem cell virus. When a portion of the U3 region of the 3’ HIV LTR was replaced with the U3 region of MSCV LTR, the resulted hybrid HIV/MSCV LTR has dramatically increased the transgene expression level in human CD34+ hematopoietic cells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acceptor sites derived from the lentiviral vector. Further studies found that additional CpG mutations in the MSCV LTR reduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV LTR. After integration into genomic DNA, 3’MSCV/LTR will replace the 5’LTR and provide a high level of expression of the target gene and reporter gene downstream.
SBI第三代慢病毒载体
SBI offers a third generation of the most popular HIV-1 based lentivector expression system which consists of three maincomponents:
(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)
(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)
(3) A pseudoviral particle producer cell line (e.g., 293TN cells)
2A Peptide-enabled dual expression system
Coexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgenes with an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines— however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).
The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006). The 2A-like sequences exist in several viruses and are used to mediate protein cleavage from a single open reading frame. Through a ribosomal skip mechanism, the 2A peptide prevents normal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reporter gene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.
pCDH-CMV-MCS-EF1-copGFP-T2A-Puro质粒序列载体序列
此序列来自于测序结果，仅供参考。
LOCUS Exported 8201 bp ds-DNA circular SYN 25-JUL-2016
DEFINITION pCDH-CMV-MCS-EF1-CoGFP-T2A-puro.
ACCESSION .
VERSION .
KEYWORDS pCDH-CMV-MCS-EF1-CoGFP-T2A-puro
SOURCE http://www.biofeng.com
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8201)
AUTHORS Biofeng Lab.
TITLE Direct Submission
JOURNAL Exported Monday, July 25, 2016 from SnapGene Viewer 3.1.4
http://www.snapgene.com
COMMENT ORIGDB|GenBank
FEATURES Location/Qualifiers
source 1..8201
/organism="pCDH-CMV-MCS-EF1-Puro"
/mol_type="other DNA"
/note="color: #ffffff"
misc_feature 7..234
/note="RSV"
/note="color: #993366"
misc_feature 234..414
/note="3'LTR"
/note="truncHIV-1 3'LTR"
/note="color: #66cc99; direction: RIGHT"
misc_feature 235..414
/note="5'LTR"
/note="HIV-1 5'LTR"
/note="color: #ffcc99; direction: RIGHT"
misc_feature 522..566
/note="psi pack"
/note="HIV-1 psi pack"
/note="color: #3366cc"
gene 567..919
/product="gag"
/note="gag"
/note="color: #666699; direction: RIGHT"
CDS 954..1454
/codon_start=1
/note="ORF frame 3"
/note="color: #ff9966"
/translation="MRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALF
LGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQA
RILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIGITRPGWSGTEKLTI
TQA"
misc_feature 1076..1308
/note="RRE"
/note="color: #66cc99; direction: RIGHT"
CDS 1309..1797
/codon_start=1
/note="Env"
/note="color: #333399"
/translation="WGFGVALENSFAPLLCLGMLVGVINLWNRLESHDLDGVGQRN*QL
HKLNTLLN*RIAKPARKE*TRIIGIR*MGKFVELV*HNKLAVVYKIIHNDSRRLGRFKN
SFCCTFYSE*S*AGIFTIIVSDPPPNPEGTRQARRNRRRRWRERQRQIHSISERISTVS
"
CDS 1415..2056
/codon_start=1
/note="ORF frame 2"
/note="color: #ff9966"
/translation="MEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF
NITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEG
IEEEGGERDRDRSIRLVNGSRRYRLTFKRKGGIGGYSAGERIVDIIATDIQTKELQKQI
TKFKILSILVLCPVHDLMGLSYLAVHLRISHRYYHGDAVLAVHQWAWIAV"
misc_feature 1798..1916
/note="cPPT"
/note="color: #ffcc00"
promoter 1922..2271
/note="CMV promoter"
/note="color: #ffffff; direction: RIGHT"
misc_feature 2164..2184
/note="CMV_fwd_primer"
/note="color: #c0c0c0"
misc_feature 2208..2227
/note="pCEP_fwd_primer"
/note="color: #c0c0c0"
misc_feature 2210..2234
/note="LNCX_primer"
/note="color: #c0c0c0"
misc_feature 2278..2320
/note="MCS"
/note="color: #00ffff"
promoter 2315..2860
/note="EF1-alpha promoter"
/note="EF1-alpha"
/note="color: #ffffff; direction: RIGHT"
misc_feature 2873..3631
/note="CoGFP"
/note="color: #00ff00; direction: RIGHT"
misc_feature 3630..3683
/note="T2A"
/note="color: #ff0000"
misc_feature 3690..4286
/note="Puro R"
/note="color: #99ccff; direction: RIGHT"
misc_feature 4298..4873
/note="WPRE"
/note="color: #66cc99; direction: RIGHT"
CDS 4386..4931
/codon_start=1
/note="ORF frame 2"
/note="color: #ff9966"
/translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVFA
DATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGKSS
SFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRISLWA
ASPPGTFKTNDLQGSCRS"
CDS 4399..4989
/codon_start=1
/note="ORF frame 3"
/note="color: #ff9966"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQPP
LVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPFLG
CSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPPPRL
VPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRK"
misc_feature 4941..4962
/note="U3PPT"
/note="color: #ffcc00"
misc_feature 4941..4956
/note="cPPT"
/note="color: #ffcc00"
misc_feature 4958..5010
/note="Delta-U3"
/note="delta U3 "
/note="color: #3366cc"
misc_feature 5011..5191
/note="HIV-1_5_LTR"
/note="color: #66cc99; direction: RIGHT"
misc_feature 5011..5191
/note="Delta-3'LTR"
/note="truncHIV-1 Delta-3'LTR"
/note="color: #ffcc99; direction: RIGHT"
terminator 5266..5385
/note="SV40 PA terminator"
/note="SV40_PA_terminator"
/note="color: #c0c0c0"
misc_feature 5354..5373
/note="EBV_rev_primer"
/note="color: #3366cc"
rep_origin 5403..5480
/direction=RIGHT
/note="SV40 ori"
/note="color: #ffff00"
misc_feature 5465..5484
/note="SV40pro_F_primer"
/note="color: #3366cc"
promoter complement(5572..5590)
/note="M13_reverse_primer"
/note="color: #800000; direction: LEFT"
misc_feature 5589..5611
/note="M13_pUC_rev_primer"
/note="color: #3366cc"
promoter complement(5625..5654)
/note="lac promoter"
/note="color: #000000; direction: LEFT"
repeat_region complement(5912..6584)
/note="pUC Ori"
/note="pUC origin"
/note="color: #ffff00; direction: LEFT"
gene complement(6737..7597)
/gene="Ampicillin"
/note="Ampicillin"
/note="color: #ccffcc; direction: LEFT"
CDS complement(6737..7597)
/codon_start=1
/note="ORF frame 2"
/note="color: #ff9966"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7639..7667)
/note="AmpR promoter"
/note="color: #000000; direction: LEFT"
misc_feature 7826..7848
/note="pGEX_3_primer"
/note="color: #3366cc"
misc_feature complement(8047..8190)
/note="lacZ a"
/note="color: #808080; direction: LEFT"
misc_feature 8161..8183
/note="M13_pUC_fwd_primer"
/note="color: #3366cc"
promoter 8176..8192
/note="M13_forward20_primer"
/note="color: #800000; direction: RIGHT"
ORIGIN
1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt
1921 atcgatacta gtattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
1981 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
2041 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
2101 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
2161 tgacgcaaat gggcggtagg cgtgtacggt gggaggttta tataagcaga gctcgtttag
2221 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagattct
2281 agagctagcg aattcgaatt taaatcggat ccgcggccgc gaaggatctg cgatcgctcc
2341 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
2401 gtcggcaatt gaacgggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
2461 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
2521 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacagctgaa gcttcgaggg
2581 gctcgcatct ctccttcacg cgcccgccgc cctacctgag gccgccatcc acgccggttg
2641 agtcgcgttc tgccgcctcc cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta
2701 agtttaaagc tcaggtcgag accgggcctt tgtccggcgc tcccttggag cctacctaga
2761 ctcagccggc tctccacgct ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg
2821 ttttctgttc tgcgccgtta cagatccaag ctgtgaccgg cgcctacgct agatggagag
2881 cgacgagagc ggcctgcccg ccatggagat cgagtgccgc atcaccggca ccctgaacgg
2941 cgtggagttc gagctggtgg gcggcggaga gggcaccccc aagcagggcc gcatgaccaa
3001 caagatgaag agcaccaaag gcgccctgac cttcagcccc tacctgctga gccacgtgat
3061 gggctacggc ttctaccact tcggcaccta ccccagcggc tacgagaacc ccttcctgca
3121 cgccatcaac aacggcggct acaccaacac ccgcatcgag aagtacgagg acggcggcgt
3181 gctgcacgtg agcttcagct accgctacga ggccggccgc gtgatcggcg acttcaaggt
3241 ggtgggcacc ggcttccccg aggacagcgt gatcttcacc gacaagatca tccgcagcaa
3301 cgccaccgtg gagcacctgc accccatggg cgataacgtg ctggtgggca gcttcgcccg
3361 caccttcagc ctgcgcgacg gcggctacta cagcttcgtg gtggacagcc acatgcactt
3421 caagagcgcc atccacccca gcatcctgca gaacgggggc cccatgttcg ccttccgccg
3481 cgtggaggag ctgcacagca acaccgagct gggcatcgtg gagtaccagc acgccttcaa
3541 gacccccatc gccttcgcca gatcccgcgc tcagtcgtcc aattctgccg tggacggcac
3601 cgccggaccc ggctccaccg gatctcgctg agggcagagg aagtcttcta acatgcggtg
3661 acgtggagga gaatcccggc ccttccggga tgaccgagta caagcccacg gtgcgcctcg
3721 ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc
3781 ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag
3841 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
3901 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
3961 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
4021 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
4081 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
4141 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
4201 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
4261 ggtgcatgac ccgcaagccc ggtgcctgag tcgacaatca acctctggat tacaaaattt
4321 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
4381 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
4441 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
4501 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
4561 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
4621 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
4681 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
4741 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
4801 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
4861 tctccctttg ggccgcctcc ccgcctggta cctttaagac caatgactta caaggcagct
4921 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
4981 cgaaaataag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
5041 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
5101 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
5161 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
5221 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
5281 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
5341 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
5401 ctatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
5461 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
5521 gaggcttttt tggaggccta gacttttgca gagacggccc aaattcgtaa tcatggtcat
5581 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
5641 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
5701 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
5761 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
5821 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
5881 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
5941 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
6001 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
6061 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
6121 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
6181 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
6241 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
6301 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
6361 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
6421 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
6481 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
6541 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
6601 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
6661 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
6721 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
6781 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
6841 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
6901 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
6961 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
7021 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
7081 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
7141 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
7201 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
7261 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
7321 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
7381 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
7441 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
7501 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
7561 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
7621 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
7681 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
7741 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
7801 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
7861 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
7921 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
7981 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
8041 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
8101 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
8161 cccagtcacg acgttgtaaa acgacggcca gtgccaagct g
//
