pCDF1-MCS2-EF1-Puro

价格： ¥800 - 1200

品牌：Ybscience

货号：YB-0983

产品详情

载体基本信息

出品公司:	Ybscience
载体名称:	pCDF1-MCS2-EF1-Puro
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:	CMV
载体大小:	6606 bp
5' 测序引物及序列:	LNCX-F AGCTCGTTTAGTGAACCGTCAGATC
3' 测序引物及序列:	EF1a-R
载体标签:	无
载体抗性:	氨苄青霉素（Ampicillin）
筛选标记:	嘌呤霉素
克隆菌株:	stbl3或者 E. coli(RecA-)：OmniMAX 2
宿主细胞（系）:	常用细胞系(e.g.HeLa, HEK293, HT1080, H1299)
备注:	pCDF1-MCS2-EF1-Puro慢病毒表达载体是基于FIV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；
稳定性:	稳表达
组成型/诱导型:	组成型
病毒/非病毒:	慢病毒（FIV)

载体质粒图谱和多克隆位点信息

pCDF1-MCS2-EF1-Puro载体图谱
pCDF1-MCS2-EF1-Puro多克隆位点

pCDF1-MCS2-EF1-Puro 载体特征

载体简介

背景简介：A. Purpose of this ManualThis manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDF lentivectors. Specifically, it provides critical instructions on amplification and cloning the cDNA into the pCDF Vectors, and verifying final expression constructs. This manual does not include information on packaging the pCDF expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. B. Advantages of the Lentivector Expression System Lentiviral expression vectors are the most effective vehicles for delivering 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-tomedium 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.The lentiviral cDNA expression system consists of three main components:(1) The lentiviral expression vector (e.g., pCDF1-MCS2-EF1-Puro)(2) The lentiviral packaging plasmids (e.g., pPACKF1 Packaging Plasmid mix)(3) A pseudoviral particle producer cell line (e.g., 293TN cells) 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 cotransfected 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 Systems user manual.SBI’s novel pCDF Vectors are derived from feline immunodeficiency virus (FIV; Poeschla, 2003; for Safety Guidelines when working with these vectors, see section G). These pCDF Vectors, developed at SBI, are self-inactivating as a result of a deletion in the U3 region of 3’ ΔLTR (see Appendix for Vector Features). Upon integration into the genome, the 5’ LTR promoter is inactivated, which prevents formation of replication-competent viral particles.When expressed, the hybrid CMV/FIV 5’ LTR drives high level transcription of the viral construct and produces a transcript that contains all the necessary functional elements (i.e., Psi, RRE, and cPPT) for efficient packaging. When this construct is expressed in HEK 293 cells that also express viral coat proteins (i.e., a packaging cell line), the pCDF transcripts are efficiently packaged into pseudoviral particles. After isolation, these pseudoviral particles containing the RNA version of the pCDF expression cassette can be efficiently transduced into any mammalian target cells. Following transduction into the target cells, this expression cassette is reverse transcribed and integrated into the genome of the target cell. The pCDF Vectors also contain a bacterial origin of replication and ampicillin resistance (AmpR) gene for propagation and selection in E.coli. The pCDF1-MCS2-EF1-Puro Vector (Cat. # CD110B-1) contains a puromycin resistance gene, under the control of a constitutive EF1 promoter and a WPRE regulatory element, to enable selection of target cells stably expressing the cDNA template. The pCDF1-MCS2-EF1-copGFP Vector (Cat. # CD111B-1) contains a copGFP gene under the control of a EF1 promoter and WPRE element. CopGFP is a novel fluorescent protein ,derived from copepod plankton (Panalina sp.), which is similar to EGFP but has a brighter color This gene serves as a reporter for the transfected or transduced cells. pCDF Cloning and Expression LentivectorsThe FIV derived pCDF vectors contain the following features: CMV promoter—promotes a high level of expression of your gene of interest in a wide variety of cell lines. Multiple Cloning Site (MCS)—for cloning the gene of interest in MCS located downstream of CMV promoter. WPRE element—enhances stability and translation of the CMVdriven transcripts. SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts. Optional second expression cassette—provides expression of puromycin resistance gene or copGFP reporter under control of constitutive elongation factor 1 (EF1) promoter for selection or FACS analysis of transduced cells. Hybrid CMV-5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells. Genetic elements (cPPT, GAG, LTRs)—necessary for packaging, transducing, and stably integrating the viral expression construct into genomic DNA. SV40 origin—for stable propagation of the pCDF 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.

载体序列

LOCUS       pCDF1-MCS2-EF1-Puro	6606 bp 	DNA	SYNDEFINITION  pCDF1-MCS2-EF1-PuroACCESSION   KEYWORDS    SOURCE        ORGANISM  other sequences; artificial sequences; vectors.FEATURES             Location/Qualifiers     source          1..6606                     /organism="pCDF1-MCS2-EF1-Puro"                     /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"     gene            2358..2957                     /label="puro (variant)"                     /gene="puro (variant)"     CDS             2358..2957                     /label="ORF frame 3"                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR                     LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"     misc_feature    2966..3541                     /label="WPRE"                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR                     LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFL                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"     CDS             3054..3566                     /label="ORF frame 3"                     /translation="MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVF                     ADATPTGWGIATTCQLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGK                     SSSFPWLLACVATWILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPQTSRIS                     LWAASPPGYR*"     CDS             3067..3639                     /label="ORF frame 1"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     misc_feature    3653..3678                     /label="U3PPT"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     misc_feature    3655..3678                     /label="U3PPT"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     terminator      3893..4012                     /label="SV40_PA_terminator"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     misc_feature    3981..4000                     /label="EBV_rev_primer"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     rep_origin      4030..4107                     /label="SV40_origin"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     misc_feature    4092..4111                     /label="SV40pro_F_primer"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     promoter        complement(4199..4217)                     /label="M13_reverse_primer"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     misc_feature    complement(4216..4238)                     /label="M13_pUC_rev_primer"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     promoter        complement(4252..4281)                     /label="lac_promoter"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     gene            complement(5364..6224)                     /label="Ampicillin"                     /gene="Ampicillin"                     /translation="MLLLPVWLSFSPPCINPGCCLFMRSCGPLSGNVAWCALCLLTQP                     PLVGALPPPVSSFPGLSLSPSLLPRRNSSPPALPAAGQGLGCWALTIPWCCRGNHRPF                     LGCSPVLPPGFCAGRPSATSLRPSIQRTFLPAACCRLCGLFRVFAFALRRVGSPFGPP                     PRLGTDDRVRRSLQEAIWHDFYNGRQHSRF*"     CDS             complement(5364..6224)                     /label="ORF frame 2"                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA                     EIGASLIKHW*"     promoter        complement(6266..6294)                     /label="AmpR_promoter"                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA                     EIGASLIKHW*"     misc_feature    complement(6453..6475)                     /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 acgctagatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg 2401 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc 2461 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca 2521 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg 2581 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca 2641 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc 2701 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc 2761 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg 2821 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg 2881 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc 2941 gcaagcccgg tgcctgagtc gacaatcaac ctctggatta caaaatttgt gaaagattga 3001 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 3061 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 3121 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 3181 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 3241 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 3301 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 3361 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 3421 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 3481 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 3541 ccgcctcccc gcctgggtac cgatgacaga gttagaagat cgcttcagga agctatttgg 3601 cacgacttct acaacgggag acagcacagt agattctgaa gatgaacctc ctaaaaaaga 3661 aaaaagggtg gactgggatg agtattggaa ccctgaaatc gatagcttcc agtgctttgt 3721 gaaacttcga ggagtctctt tgttgaggac ttttgagttc tcccttgagg ctcccacaga 3781 tacaataaat atttgagatt gaaccctgtc gagtatctgt gtaatctttt ttacctgtga 3841 ggtctcggaa tccgggccga gaacttcgca gcgagctcat tgtaccgcga acttgtttat 3901 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 3961 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 4021 gctctagcta tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 4081 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 4141 tagtgaggag gcttttttgg aggcctagac ttttgcagag acggcccaaa ttcgtaatca 4201 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 4261 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 4321 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 4381 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 4441 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 4501 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 4561 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 4621 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 4681 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4741 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4801 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4861 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4921 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4981 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 5041 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 5101 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 5161 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 5221 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 5281 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 5341 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 5401 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 5461 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 5521 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 5581 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 5641 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 5701 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 5761 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 5821 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 5881 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 5941 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 6001 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 6061 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 6121 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 6181 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 6241 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 6301 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 6361 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 6421 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 6481 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 6541 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga 6601 gtgcac