pCDH1-MCS2-EF1-copGFP

载体基本信息

载体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 systemCoexpression 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	SYNDEFINITION pCDH1-MCS2-EF1-copGFPACCESSION 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