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  • pCMV-VSV-G

    價格:1250元

    聯系方式:I47-825O-882O

    買家導航

    載體基本信息

    出品公司: Addgene
    試劑名稱: pCMV-VSV-G
    質粒類型: 哺乳動物載體;逆病毒包裝;雙質粒包裝系統;信封載體
    高拷貝/低拷貝: 高拷貝
    克隆方法: 限制性內切酶,多克隆位點
    啟動子: CMV
    載體大小: 6507 bp
    5' 測序引物及序列: CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’
    3' 測序引物及序列: --
    載體標簽:
    載體抗性: Ampicillin
    篩選標記:
    克隆菌株: DH5α等
    宿主細胞(系): 包裝細胞系如293T
    備注: 逆病毒包裝載體pCMV-VSV-G需要與pUMVC共同使用,使用方法見下文。
    產品目錄號: #8454
    穩定性: 瞬表達
    組成型/誘導型: 組成型
    病毒/非病毒: 非病毒

    載體質粒圖譜和多克隆位點信息

    pCMV-VSV-G載體圖譜



    載體簡介

    雙質粒系統逆病毒包裝質粒pCMV-VSV-G使用方法
    
    Day 1
    
    1.      Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if desired)
    
    
    Day 2
    
    1.      Set up (use polypropylene tubes for this; polystyrene tubes DO NOT work!):
    
               1 µg retroviral DNA encoding gene X
               1 µg packaging plasmid Mix
                    the packaging plasmid (pUMVC3) at a 8:1 ratio with the envelope plasmid (pCVM-VSV-G)-a total of 1µg
               DME without serum to 94μL total
               6 μL Fugene
    
           Mix and wait 15 to 30 minutes at room temperature
    
           Add to 293T cells without touching the sides of the dish (DO NOT CHANGE MEDIA)
    
           If you are using amphotropic virus then move immediately to BL2+ in a secondary container, which has an absorbent material. 
    
           (This does not mean a couple of hours; it means Immediately!).  The rest of this protocol is the same for all viruses---the BL2+ safety practices are in place if you are using amphotropic viruses 
    
    
    Day 3
    
    1.      Change the media to whatever media you wish to use when infecting target cells. 293T cells are easily detached so remember not to put the media directly onto to cells, but rather “run” it down the side of the dish. Remember that you will get the highest titer virus when your cells are “happy.”
    
    2.      Plate out your target cells
    
    
    Day 4
    
    1.      Remove the medium from the 293T cells and use a 0.45 u syringe filter to remove any 293T cells. DO NOT use the 0.2 u filter, as it is likely to shear the envelope from your virus
    making it noninfectious
    
            Note: After filtering, the filter should be removed and placed in the biohazard bag in the hood and the syringe rinsed with bleach and decontaminated for a minimum of 20 min. It is useful to place a plastic beaker with bleach in the hood in advance
    
    2.      Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target cells
    
    3.      Carry out infection for 1 to 4 hours. Remove virus and replace with fresh media
    
            Note: If you wish to do a second infection the following day, it is important to put fresh media on the cells and not let the virus remain on the cells overnight. The media contains huge amounts of envelope, both associated and unassociated with viral particles, which will bind all the cell surface receptors required for virus adsorption, resulting in their down-regulation. Hence, if you don’t change the media after the initial infection, very few receptors will be available for the next round of infection. In addition, very few cells tolerate the presence of high levels of the VSV-G envelope for extended periods of time (i.e. a lot of your cells may die)
    
    
    Day 5 +
    
    1.      Allow the cells to recover and begin to express the virus-encoded genes. The cells usually require 48 hours for this to occur
    
    2.      Add drug if you are scoring for the presence of a vector that carries a drug resistance marker. Prior to this step it is advisable that you titrate the drug to be used for selection in order to know precisely how much to add. In addition, it is necessary to bring an extra plate of uninfected cells (often referred to as “canaries”) which will function as a positive control in the kill assay.  Add drug to both plates. When your canaries are dead, you can remove the drug.
    
    

    載體序列

    Please note that depositor's sequence is not 100% complete. The flanking sequence of VSV-G actually contains XhoI (at both the 5' and 3'end).
    
    LOCUS       pCMV-VSV-G Sequencing Result	6507 bp 	DNA	SYN
    DEFINITION  pCMV-VSV-G Sequencing Result
    ACCESSION   
    KEYWORDS    
    SOURCE      
      ORGANISM  other sequences; artificial sequences; vectors.
    FEATURES             Location/Qualifiers
         source          1..6507
                         /organism="pCMV-VSV-G Sequencing Result"
                         /mol_type="other DNA"
         promoter        84..660
                         /label="CMV_immearly_promoter"
         misc_feature    148..974
                         /label="5_LTR"
         misc_feature    163..450
                         /label="CAG_enhancer"
         misc_feature    617..637
                         /label="CMV_fwd_primer"
         promoter        618..687
                         /label="CMV_promoter"
         promoter        630..749
                         /label="CMV2_promoter"
         misc_feature    661..680
                         /label="pCEP_fwd_primer"
         misc_feature    663..687
                         /label="LNCX_primer"
         misc_feature    778..1350
                         /label="bGlob_int"
         CDS             1322..2971
                         /label="ORF frame 2"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         gene            1436..2959
                         /label="vsv-G"
                         /gene="vsv-G"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         misc_feature    2936..2968
                         /label="VSVG Tag"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         terminator      3118..3644
                         /label="rb_glob_PA_terminator"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         misc_feature    complement(3689..3711)
                         /label="M13_pUC_rev_primer"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         promoter        complement(3725..3754)
                         /label="lac_promoter"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         gene            complement(4837..5697)
                         /label="Ampicillin"
                         /gene="Ampicillin"
                         /translation="MFMPSSLSYSSWATCWLLCCLIILAKNSSTDPSRNSDTMKCLLY
                         LAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKS
                         HKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLN
                         PGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNS
                         TTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQ
                         YCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILD
                         YSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAP
                         ILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDL
                         HLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFF
                         IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK*"
         CDS             complement(4837..5697)
                         /label="ORF frame 1"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                         IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                         YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                         DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                         LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                         EIGASLIKHW*"
         promoter        complement(5739..5767)
                         /label="AmpR_promoter"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                         IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                         YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                         DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                         LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                         EIGASLIKHW*"
         misc_feature    complement(6284..6439)
                         /label="lacZ_a"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                         IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                         YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                         DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                         LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                         EIGASLIKHW*"
         misc_feature    6410..6432
                         /label="M13_pUC_fwd_primer"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                         IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                         YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                         DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                         LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                         EIGASLIKHW*"
         promoter        6425..6441
                         /label="M13_forward20_primer"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                         IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                         YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                         DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                         LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                         EIGASLIKHW*"
         promoter        6451..6469
                         /label="T7_promoter"
                         /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                         IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                         YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                         DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                         LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                         EIGASLIKHW*"
    ORIGIN
        1 gagcttggcc cattgcatac gttgtatcca tatcataata tgtacattta tattggctca
       61 tgtccaacat taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt
      121 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      181 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      241 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      301 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      361 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      421 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      481 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      541 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      601 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      661 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
      721 catagaagac accgggaccg atccagcctc cggtcgaccg atcctgagaa cttcagggtg
      781 agtttgggga cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga
      841 gggggcaaag ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga
      901 ccctcatgat aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt
      961 attttctttt cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt
     1021 tttaaattca cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt
     1081 caaggcaatc agggtatatt atattgtact tcagcacagt tttagagaac aattgttata
     1141 attaaatgat aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt
     1201 attggtagaa acaactacac cctggtcatc atcctgcctt tctctttatg gttacaatga
     1261 tatacactgt ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac
     1321 catgttcatg ccttcttctc tttcctacag ctcctgggca acgtgctggt tgttgtgctg
     1381 tctcatcatt ttggcaaaga attcctcgac ggatccctcg aggaattctg acactatgaa
     1441 gtgccttttg tacttagcct ttttattcat tggggtgaat tgcaagttca ccatagtttt
     1501 tccacacaac caaaaaggaa actggaaaaa tgttccttct aattaccatt attgcccgtc
     1561 aagctcagat ttaaattggc ataatgactt aataggcaca gccttacaag tcaaaatgcc
     1621 caagagtcac aaggctattc aagcagacgg ttggatgtgt catgcttcca aatgggtcac
     1681 tacttgtgat ttccgctggt atggaccgaa gtatataaca cattccatcc gatccttcac
     1741 tccatctgta gaacaatgca aggaaagcat tgaacaaacg aaacaaggaa cttggctgaa
     1801 tccaggcttc cctcctcaaa gttgtggata tgcaactgtg acggatgccg aagcagtgat
     1861 tgtccaggtg actcctcacc atgtgctggt tgatgaatac acaggagaat gggttgattc
     1921 acagttcatc aacggaaaat gcagcaatta catatgcccc actgtccata actctacaac
     1981 ctggcattct gactataagg tcaaagggct atgtgattct aacctcattt ccatggacat
     2041 caccttcttc tcagaggacg gagagctatc atccctggga aaggagggca cagggttcag
     2101 aagtaactac tttgcttatg aaactggagg caaggcctgc aaaatgcaat actgcaagca
     2161 ttggggagtc agactcccat caggtgtctg gttcgagatg gctgataagg atctctttgc
     2221 tgcagccaga ttccctgaat gcccagaagg gtcaagtatc tctgctccat ctcagacctc
     2281 agtggatgta agtctaattc aggacgttga gaggatcttg gattattccc tctgccaaga
     2341 aacctggagc aaaatcagag cgggtcttcc aatctctcca gtggatctca gctatcttgc
     2401 tcctaaaaac ccaggaaccg gtcctgcttt caccataatc aatggtaccc taaaatactt
     2461 tgagaccaga tacatcagag tcgatattgc tgctccaatc ctctcaagaa tggtcggaat
     2521 gatcagtgga actaccacag aaagggaact gtgggatgac tgggcaccat atgaagacgt
     2581 ggaaattgga cccaatggag ttctgaggac cagttcagga tataagtttc ctttatacat
     2641 gattggacat ggtatgttgg actccgatct tcatcttagc tcaaaggctc aggtgttcga
     2701 acatcctcac attcaagacg ctgcttcgca acttcctgat gatgagagtt tattttttgg
     2761 tgatactggg ctatccaaaa atccaatcga gcttgtagaa ggttggttca gtagttggaa
     2821 aagctctatt gcctcttttt tctttatcat agggttaatc attggactat tcttggttct
     2881 ccgagttggt atccatcttt gcattaaatt aaagcacacc aagaaaagac agatttatac
     2941 agacatagag atgaaccgac ttggaaagta actcaaatcc tgcacaacag attcttcatg
     3001 tttggaccaa atcaacttgt gataccatgc tcaaagaggc ctcaattata tttgagtttt
     3061 taatttttat gaaaaaaaaa aaaaaaaacg gaattcctcg agggatccgt cgaggaattc
     3121 actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct
     3181 cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc
     3241 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     3301 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
     3361 aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa
     3421 ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
     3481 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
     3541 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
     3601 tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cggatcggcc
     3661 gcaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3721 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     3781 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     3841 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     3901 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3961 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4021 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4081 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4141 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4201 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4261 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4321 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4381 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4441 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4501 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4561 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4621 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4681 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4741 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4801 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4861 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4921 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4981 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5041 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5101 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5161 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5221 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5281 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5341 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5401 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5461 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5521 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5581 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5641 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5701 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5761 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5821 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5881 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5941 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     6001 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     6061 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     6121 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     6181 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     6241 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     6301 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     6361 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     6421 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattggag
     6481 ctccaccgcg gtggcggccg ctctaga
    //
    
    

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