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-20℃
6个月
pCMV-VSV-G
大量
钰博生物
1ug/5ug
出品公司: | Ybscience |
---|---|
试剂名称: | pCMV-VSV-G |
质粒类型: | 哺乳动物载体;逆病毒包装;双质粒包装系统;信封载体 |
高拷贝/低拷贝: | 高拷贝 |
克隆方法: | 限制性内切酶,多克隆位点 |
启动子: | CMV |
载体大小: | 6507 bp |
5' 测序引物及序列: | CMV fwd 5’CGCAAATGGGCGGTAGGCGTG 3’ |
3' 测序引物及序列: | -- |
载体标签: | 无 |
载体抗性: | Ampicillin |
筛选标记: | 无 |
克隆菌株: | DH5α等 |
宿主细胞(系): | 包装细胞系如293T |
备注: | 逆病毒包装载体pCMV-VSV-G需要与pUMVC共同使用,使用方法见下文。 |
稳定性: | 瞬表达 |
组成型/诱导型: | 组成型 |
病毒/非病毒: | 非病毒 |
双质粒系统逆病毒包装质粒pCMV-VSV-G使用方法Day 11. Plate 5 x 105 293T cells in 6 cm2 dishes containing 5 mL of media. (This can be scaled up if desired)Day 21. 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 31. 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 cellsDay 41. 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 virusmaking 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 advance2. Add 8 to 10 μg/ml of polybrene (Hexadimethrine bromide) or protamine sulfate to the target cells3. 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 occur2. 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.
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 SYNDEFINITION pCMV-VSV-G Sequencing ResultACCESSION 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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