质粒名称: | pFUGW ORANGE Tubb3-GFP KI _ mCherry-KASH |
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出品公司: | addgene |
目录编号: | 131508 |
存储实验室: | Harold MacGillavry |
载体骨架基本信息 |
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载体名称: | pFUGW |
空载体大小: | 11480bp |
完整质粒大小: | 11472bp |
载体类型: | 哺乳细胞表达 |
载体抗性: | 氨苄霉素 |
生长温度: | 37 ℃ |
宿主菌: | NEB Stable |
拷贝数: | 高拷贝 |
插入基因信息 |
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插入基因名称: | gRNA and GFP donor |
物种来源: | M. musculus (mouse), R. norvegicus (rat) |
基因突变: | Tubb3 (a.k.a. 3200002H15Rik, M(beta)3, M(beta)6) |
启动子: | hUBC |
克隆方法: | 限制性内切酶 |
5’克隆位点: | 未知(未破坏) |
3’克隆位点: | 未知 (未破坏) |
5’测序引物 | GGCGAGTGTGTTTTGTGAAG |
3’测序引物 | GCAGCGTATCCACATAGCG |
文献引用方法 |
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材料和方法部分: | pFUGW ORANGE Tubb3-GFP KI _ mCherry-KASH was a gift from Harold MacGillavry (Addgene plasmid # 131508 ; http://n2t.net/addgene:131508 ; RRID:Addgene_131508) |
参考文献部分: | ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons. Willems J, de Jong APH, Scheefhals N, Mertens E, Catsburg LAE, Poorthuis RB, de Winter F, Verhaagen J, Meye FJ, MacGillavry HD. PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 10.1371/journal.pbio.3000665. 10.1371/journal.pbio.3000665 PubM |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pFUGW ORANGE Tubb3-GFP KI _ mCherry-KASH 11472 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Endogenous tagging of beta-3 Tubulin: C-terminal (amino acid position: STOP codon). ACCESSION . VERSION . KEYWORDS pFUGW ORANGE Tubb3-GFP KI _ mCherry-KASH SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11472) AUTHORS Willems J, de Jong APH, Scheefhals N, Mertens E, Catsburg LAE, Poorthuis RB, de Winter F, Verhaagen J, Meye FJ, MacGillavry HD TITLE ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons. JOURNAL PLoS Biol. 2020 Apr 10;18(4):e3000665. doi: 10.1371/journal.pbio.3000665. PUBMED 32275651 REFERENCE 2 (bases 1 to 11472) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..11472 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 277..394 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 436..676 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 436..456 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 607..626 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 704..779 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" CDS 906..1622 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 906..1619 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(948..969) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1209..1228) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1556..1577 /label=EGFP-C /note="EGFP, forward primer" promoter 1741..2952 /label=UbC promoter /note="human ubiquitin C promoter" primer_bind 2789..2808 /label=hUBCpro-F /note="Human Ubiquitin C (UbC) promoter, forward primer" CDS 2999..3709 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MTVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMN FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGAL KGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYER AEGRHSTGGMDELYK" primer_bind complement(3147..3165) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3415..3434 /label=mCherry-F /note="mCherry, forward primer" misc_feature 4001..4589 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(4054..4074) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(4472..4483) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(4592..4608) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4593..4609) /label=pBluescriptKS /note="For pBluescript vector" LTR 5114..5294 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" primer_bind complement(5320..5337) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5326..5550 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 5596..6024 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5683..5702) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5893..5914 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6033..6053) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 6038..6367 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 6218..6353 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6280..6299 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 6415..6462 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 6481..6855 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 6985..7106 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7022..7041) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7076..7095 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(7155..7171) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7155..7171) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(7168..7190) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 7179..7195 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(7203..7233) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 7248..7269 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(7386..7403) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(7557..8145) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7637..7656) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8316..9176) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 8939..8958 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9177..9281) /gene="bla" /label=AmpR promoter primer_bind complement(9356..9375) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 9547..9926 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 9928..10126 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 10081..10101 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 10144..10324 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 10371..10496 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 10989..11222 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 11407..11451 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" ORIGIN 1 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 61 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 121 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa 181 ggtggagaga gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg 241 ccaattctgc agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg 301 ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga 361 attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga 421 tccagtttgg ttagtgaggg cctatttccc atgattcctt catatttgca tatacgatac 481 aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa 541 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 601 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 661 atcttgtgga aaggacgaaa caccgctgcg agcaacttca cttgttttag agctagaaat 721 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 781 tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg 841 cgccaattct gcagacaaat ggctctagaa gcttgctgcg agcaacttca cttgggggat 901 caggcgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 961 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 1021 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 1081 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 1141 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 1201 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 1261 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 1321 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 1381 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 1441 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 1501 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 1561 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 1621 aatgtgctgc gagcaacttc acttgggctc gaggatccta ggacgcgtcc taggacgcgt 1681 ccagaggtac ccgttacata acttacggta aatggcctaa cccgtgtcgg ctccagatct 1741 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 1801 ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag 1861 cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag 1921 gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 1981 aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat 2041 gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt 2101 cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct 2161 ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc 2221 caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg 2281 cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga 2341 ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt 2401 cgctaatgcg ggaaagctct tattcgggtg agatgggctg gggcaccatc tggggaccct 2461 gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 2521 tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 2581 gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 2641 cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 2701 cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 2761 gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 2821 ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 2881 atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 2941 tttttgttag acgaagcttg ggctgcaggt cgactctaga ggatccccgg gtaccggtat 3001 gacggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa 3061 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg 3121 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc 3181 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca 3241 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg 3301 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga 3361 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt 3421 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg 3481 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc 3541 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt 3601 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga 3661 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt ccggactcag 3721 atctcgagag gaggaggagg agacagacag caggatgccc cacctcgaca gccccggcag 3781 ctcccagccg agacgctcct tcctctcaag ggtgatcagg gcagcgctac cgttgcagct 3841 gcttctgctg ctgctgctgc tcctggcctg cctgctacct gcctctgaag atgactacag 3901 ctgcacccag gccaacaact ttgcccgatc cttctacccc atgctgcggt acaccaacgg 3961 gccacctccc acctaggaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa 4021 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata 4081 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc 4141 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg 4201 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac 4261 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat 4321 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt 4381 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat 4441 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc 4501 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag 4561 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgag acctagaaaa 4621 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga 4681 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat 4741 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 4801 gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc acacacaagg 4861 ctacttccct gattggcaga actacacacc agggccaggg atcagatatc cactgacctt 4921 tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag ccaatgaagg 4981 agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc cggagagaga 5041 agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc gagagctgca 5101 tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta 5161 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg 5221 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg 5281 gaaaatctct agcagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 5341 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 5401 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 5461 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 5521 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 5581 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 5641 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 5701 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 5761 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 5821 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 5881 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 5941 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 6001 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa 6061 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 6121 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 6181 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 6241 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 6301 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 6361 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac 6421 aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc 6481 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 6541 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 6601 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 6661 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 6721 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 6781 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 6841 gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg 6901 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc 6961 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa 7021 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 7081 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc 7141 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 7201 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 7261 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 7321 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 7381 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 7441 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 7501 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 7561 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 7621 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 7681 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 7741 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 7801 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 7861 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 7921 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7981 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 8041 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 8101 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 8161 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 8221 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 8281 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 8341 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 8401 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 8461 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 8521 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 8581 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 8641 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 8701 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 8761 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 8821 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 8881 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 8941 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 9001 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 9061 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 9121 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 9181 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 9241 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 9301 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa 9361 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 9421 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca 9481 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata 9541 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 9601 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 9661 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 9721 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 9781 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 9841 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 9901 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 9961 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 10021 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 10081 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt 10141 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 10201 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 10261 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 10321 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac 10381 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta 10441 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta 10501 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga 10561 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta 10621 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat 10681 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt 10741 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag 10801 agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag acctggagga 10861 ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt aaaaattgaa 10921 ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga aaaaagagca 10981 gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac tatgggcgca 11041 gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt gcagcagcag 11101 aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac agtctggggc 11161 atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga tcaacagctc 11221 ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc ttggaatgct 11281 agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat ggagtgggac 11341 agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc gcaaaaccag 11401 caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt gtggaattgg 11461 tttaacataa ca //