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。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 1 (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. LOCUS Exported 8201 bp ds-DNA circular SYN 25-JUL-2016DEFINITION pCDH-CMV-MCS-EF1-CoGFP-T2A-puro.ACCESSION .VERSION .KEYWORDS pCDH-CMV-MCS-EF1-CoGFP-T2A-puroSOURCE http://www.biofeng.com ORGANISM synthetic DNA constructREFERENCE 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.comCOMMENT ORIGDB|GenBankFEATURES 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
