| 出品公司: | SBI |
|---|---|
| 载体名称: | pCDH1-MCS2-EF1-copGFP |
| 质粒类型: | 慢病毒表达载体；cDNA表达载体 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 启动子: | EF1a |
| 载体大小: | 6771 bp |
| 5' 测序引物及序列: | 5’-GGGGTACAGTGCAGGGGAAAGAAT-3’ |
| 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 |
| 克隆菌株: | stbl3 |
| 宿主细胞（系）: | 大部分细胞类型（分裂与非分裂细胞）；初级分化细胞 |
| 备注: |
pCDH1-MCS2-EF1-copGFP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，表达水平高； 单一启动子，由EF1a启动子驱动目 的基因和下游报告基因转录为双顺反子； |
| 产品目录号: | CD521A-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
载体基本信息
载体图谱质粒图谱和多克隆位点信息
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.
载体序列
LOCUS pCDH1-MCS2-EF1-copGFP 6771 bp DNA SYN
DEFINITION pCDH1-MCS2-EF1-copGFP
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..6771
/organism="pCDH1-MCS2-EF1-copGFP"
/mol_type="other DNA"
misc_feature 287..307
/label="CMV_fwd_primer"
misc_feature 1165..1180
/label="cPPT"
misc_feature 1633..1653
/label="CMV_fwd_primer"
promoter 1634..1703
/label="CMV_promoter"
misc_feature 1677..1696
/label="pCEP_fwd_primer"
misc_feature 1679..1703
/label="LNCX_primer"
CDS 2366..3124
/label="ORF frame 2"
/translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNK
MKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGG
VLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGS
FARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEY
QHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSR*"
misc_feature 3133..3708
/label="WPRE"
/translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNK
MKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGG
VLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGS
FARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEY
QHAFKTPIAFARSRAQSSNSAVDGTAGPGSTGSR*"
CDS 3221..3961
/label="ORF frame 2"
/translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVF
ADATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGK
SSSFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRIS
LWAASPPVPMTELEDRFRKLFGTTSTTGDSTVDSEDEPPKKEKRVDWDEYWNPEIDSF
QCFVKLRGVSLLRTFEFSLEAPTDTINI*"
CDS 3234..3731
/label="ORF frame 3"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
misc_feature 3818..3843
/label="U3PPT"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
misc_feature 3820..3843
/label="U3PPT"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
terminator 4058..4177
/label="SV40_PA_terminator"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
misc_feature 4146..4165
/label="EBV_rev_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
rep_origin 4195..4272
/label="SV40_origin"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
misc_feature 4257..4276
/label="SV40pro_F_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
promoter complement(4364..4382)
/label="M13_reverse_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
misc_feature complement(4381..4403)
/label="M13_pUC_rev_primer"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
promoter complement(4417..4446)
/label="lac_promoter"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
gene complement(5529..6389)
/label="Ampicillin"
/gene="Ampicillin"
/translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP
PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF
LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP
PRRYR*"
CDS complement(5529..6389)
/label="ORF frame 2"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
promoter complement(6431..6459)
/label="AmpR_promoter"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
misc_feature complement(6618..6640)
/label="pGEX_3_primer"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW*"
ORIGIN
1 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
61 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
121 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
181 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
241 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
301 aggcgtgtac ggtgggaggt ctatataagc agagcttgtg aaacttcgag gagtctcttt
361 gttgaggact tttgagttct cccttgaggc tcccacagat acaataaata tttgagattg
421 aaccctgtcg agtatctgtg taatcttttt tacctgtgag gtctcggaat ccgggccgag
481 aacttcgcag ttggcgcccg aacagggact tgattgagag tgattgagga agtgaagcta
541 gagcaataga aagctgttaa gcagaactcc tgctgaccta aatagggaag cagtagcaga
601 cgctgctaac agtgagtatc tctagtgaag cagactcgag ctcataatca agtcattgtt
661 taaaggccca gataaattac atctggtgac tcttcgcgga ccttcaagcc aggagattcg
721 ccgagggaca gtcaacaagg taggagagat tctacagcaa catggggaat ggacaggggc
781 gagattggaa aatggccatt aagagatgta gtaatgttgc tgtaggagta ggggggaaga
841 gtaaaaaatt tggagaaggg aatttcagat gggccattag aatggctaat gtatctacag
901 gacgagaacc tggtgatata ccagagactt tagatcaact aaggttggtt atttgcgatt
961 tacaagaaag aagagaaaaa tttggatcta gcaaagaaat tgatatggca attcctgcat
1021 tgaggagaaa tggtaggcaa tgtggcatgt ctgaaaaaga ggaggaatga tgaagtatct
1081 cagacttatt ttataaggga gatactgtgc tgagttcttc cctttgagga aggtatgtca
1141 tatcctagac atagtctcaa ttttaaaaga agaggtagga taggagggat ggccccttat
1201 gaattattag cacaacaaga atccttaaga atacaagatt atttttctgc aataccacaa
1261 aaattgcaag cacagtggat ttattataaa gatcaaaaag ataagaaatg gaaaggacca
1321 atgagagtag aatactgggg acagggatca gtattattaa aggatgaaga gaagggatat
1381 tttcttataa tcgatactag tattatgccc agtacatgac cttatgggac tttcctactt
1441 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
1501 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
1561 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
1621 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
1681 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
1741 gaagattcta gagcccgggc gcgccggatc cagatcttaa ttaatttaaa tgaattcgcg
1801 gccgcgaagg atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca catcgcccac
1861 agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg
1921 cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg
1981 agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc
2041 cagaacacag ctgaagcttc gaggggctcg catctctcct tcacgcgccc gccgccctac
2101 ctgaggccgc catccacgcc ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc
2161 ctgaactgcg tccgccgtct aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc
2221 ggcgctccct tggagcctac ctagactcag ccggctctcc acgctttgcc tgaccctgct
2281 tgctcaactc tacgtctttg tttcgttttc tgttctgcgc cgttacagat ccaagctgtg
2341 accggcgcct acgctagacg ccaccatgga gagcgacgag agcggcctgc ccgccatgga
2401 gatcgagtgc cgcatcaccg gcaccctgaa cggcgtggag ttcgagctgg tgggcggcgg
2461 agagggcacc cccaagcagg gccgcatgac caacaagatg aagagcacca aaggcgccct
2521 gaccttcagc ccctacctgc tgagccacgt gatgggctac ggcttctacc acttcggcac
2581 ctaccccagc ggctacgaga accccttcct gcacgccatc aacaacggcg gctacaccaa
2641 cacccgcatc gagaagtacg aggacggcgg cgtgctgcac gtgagcttca gctaccgcta
2701 cgaggccggc cgcgtgatcg gcgacttcaa ggtggtgggc accggcttcc ccgaggacag
2761 cgtgatcttc accgacaaga tcatccgcag caacgccacc gtggagcacc tgcaccccat
2821 gggcgataac gtgctggtgg gcagcttcgc ccgcaccttc agcctgcgcg acggcggcta
2881 ctacagcttc gtggtggaca gccacatgca cttcaagagc gccatccacc ccagcatcct
2941 gcagaacggg ggccccatgt tcgccttccg ccgcgtggag gagctgcaca gcaacaccga
3001 gctgggcatc gtggagtacc agcacgcctt caagaccccc atcgccttcg ccagatcccg
3061 cgctcagtcg tccaattctg ccgtggacgg caccgccgga cccggctcca ccggatctcg
3121 ctaagtcgac aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa
3181 ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat
3241 tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
3301 tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc
3361 aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt
3421 ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg
3481 ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc
3541 ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
3601 ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct
3661 tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc
3721 ggtaccgatg acagagttag aagatcgctt caggaagcta tttggcacga cttctacaac
3781 gggagacagc acagtagatt ctgaagatga acctcctaaa aaagaaaaaa gggtggactg
3841 ggatgagtat tggaaccctg aaatcgatag cttccagtgc tttgtgaaac ttcgaggagt
3901 ctctttgttg aggacttttg agttctccct tgaggctccc acagatacaa taaatatttg
3961 agattgaacc ctgtcgagta tctgtgtaat cttttttacc tgtgaggtct cggaatccgg
4021 gccgagaact tcgcagcgag ctcattgtac cgcgaacttg tttattgcag cttataatgg
4081 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
4141 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
4201 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
4261 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
4321 tttggaggcc tagacttttg cagagacggc ccaaattcgt aatcatggtc atagctgttt
4381 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
4441 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
4501 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
4561 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
4621 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
4681 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
4741 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
4801 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
4861 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
4921 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
4981 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
5041 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
5101 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
5161 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
5221 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
5281 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
5341 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
5401 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
5461 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
5521 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
5581 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
5641 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
5701 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
5761 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
5821 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
5881 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
5941 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
6001 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
6061 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
6121 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
6181 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
6241 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
6301 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
6361 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
6421 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
6481 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
6541 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
6601 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
6661 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
6721 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca c
//
