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  • pCDF1-MCS2-EF1-Puro

    價格:1800元

    聯系方式:I47-825O-882O

    買家導航

    載體基本信息

    出品公司: SBI
    載體名稱: pCDF1-MCS2-EF1-Puro
    質粒類型: 慢病毒表達載體;cDNA表達載體
    克隆方法: 多克隆位點,限制性內切酶
    啟動子: CMV
    載體大小: 6606 bp
    5' 測序引物及序列: LNCX-F AGCTCGTTTAGTGAACCGTCAGATC
    3' 測序引物及序列: EF1a-R
    載體標簽:
    載體抗性: 氨芐青霉素(Ampicillin)
    篩選標記: 嘌呤霉素
    克隆菌株: stbl3或者 E. coli(RecA-):OmniMAX 2
    宿主細胞(系): 常用細胞系(e.g.HeLa, HEK293, HT1080, H1299)
    備注: pCDF1-MCS2-EF1-Puro慢病毒表達載體是基于FIV的慢病毒載體;
    用于cDNA表達和克;高效轉染細胞,建立穩定細胞系 ,表達水平高;
    產品目錄號: CD110B-1
    穩定性: 穩表達
    組成型/誘導型: 組成型
    病毒/非病毒: 慢病毒(FIV)

    載體質粒圖譜和多克隆位點信息

    pCDF1-MCS2-EF1-Puro載體圖譜
    pCDF1-MCS2-EF1-Puro多克隆位點

    pCDF1-MCS2-EF1-Puro 載體特征

    載體簡介

    背景簡介:
    A. Purpose of this Manual
    This 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 Lentivectors
    The 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. 
    

    載體序列

    LOCUS       pCDF1-MCS2-EF1-Puro	6606 bp 	DNA	SYN
    DEFINITION  pCDF1-MCS2-EF1-Puro
    ACCESSION   
    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
    //
    
    

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