质粒名称: | pDestTol2pA2-U6:gRNA |
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出品公司: | 爱迪基因 |
目录编号: | 63157 |
存储实验室: | Leonard Zon |
载体骨架基本信息 |
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载体名称: | pDestTol2pA2 (Tol2Kit#394) |
载体出品公司: | -- |
载体抗性: | 氯霉素和氨苄青霉素 |
空载体大小: | 6354 bp |
完整质粒大小: | -- |
载体修饰: | 添加一个斑马鱼U6启动子,驱动一个含有BseRI位点的gRNA支架来克隆种子 |
载体类型: | CRISPR;网关的Tol2目标向量 |
筛选标记: | -- |
高拷贝/低拷贝: | -- |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | -- |
物种来源: | -- |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | GAGGATCATAATCAGCCATA |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pDestTol2pA2-U6:gRNA was a gift from Leonard Zon (Addgene plasmid # 63157 ; http://n2t.net/addgene:63157 ; RRID:Addgene_63157) |
参考文献部分: | A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish. Ablain J, Durand EM, Yang S, Zhou Y, Zon LI. Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 10.1016/j.devcel.2015.01.032. 10.1016/j.devcel.2015.01.032 PubMed 25752963 |
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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 pDestTol2pA2-U6:gRNA 6352 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Destination vector for Gateway based on vector #394 from the Tol2Kit, containing a gRNA scaffold under the control of a zebrafish U6 promoter.. ACCESSION . VERSION . KEYWORDS pDestTol2pA2-U6:gRNA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6352) AUTHORS Ablain J, Durand EM, Yang S, Zhou Y, Zon LI TITLE A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish. JOURNAL Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 10.1016/j.devcel.2015.01.032. PUBMED 25752963 REFERENCE 2 (bases 1 to 6352) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6352 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 20..124 /gene="bla" /label=AmpR promoter CDS 125..985 /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 complement(343..362) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1156..1744 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1645..1664 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1898..1915 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2032..2053 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2068..2098 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2106..2122 /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)." primer_bind 2111..2133 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2130..2146 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2165..2185 /label=T3 /note="T3 promoter, forward primer" promoter 2167..2185 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 2222..2238 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 2222..2238 /label=pBluescriptSK /note="For pBluescript vector" polyA_signal 2910..3044 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2947..2966) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3001..3020 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" misc_RNA 3443..3518 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind 3531..3547 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 3555..3678 /gene="mutant version of attR" /label=attR3 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 3703..3733 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 3787..4467 /codon_start=1 /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE DPAY" primer_bind complement(3853..3872) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 4787..5092 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 4971..4990 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5133..5257) /gene="mutant version of attR" /label=attR4 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(5276..5292) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6188..6209 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 61 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 121 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 181 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 241 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 301 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 361 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 421 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 481 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 541 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 601 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 661 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 721 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 781 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 841 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 901 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 961 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 1021 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 1081 catgaccaaa atgccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 1141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 1201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 1261 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 1321 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 1381 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 1441 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 1501 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 1561 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 1621 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 1681 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 1741 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 1801 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 1861 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 1921 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 1981 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 2041 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 2101 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 2161 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg 2221 ctctagaact agtggatctg ctgggcttgc tgaaggtagg gggtcaagaa ccagaggtgt 2281 aaagtacttg agtaatttta cttgattact gtacttaagt attatttttg gggattttta 2341 ctttacttga gtacaattaa aaatcaatac ttttactttt acttaattac atttttttag 2401 aaaaaaaagt actttttact ccttacaatt ttatttacag tcaaaaagta cttatttttt 2461 ggagatcact tcattctatt ttcccttgct attaccaaac caattgaatt gcgctgatgc 2521 ccagtttaat ttaaatgtta tttattctgc ctatgaaaat cgttttcaca ttatatgaaa 2581 ttggtcagac atgttcattg gtcctttgga agtgacgtca tgtcacatct attaccacaa 2641 tgcacagcac cttgacctgg aaattaggga aattataaca gtcaatcagt ggaagaaaat 2701 ggaggaagta tgtgattcat cagcagctgc gagcagcaca gtccaaaatc agccacagga 2761 tcaagagcac ccgtggccgt atcttcgcag atctgatcta gaggatcata atcagccata 2821 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 2881 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 2941 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt 3001 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcatcatc gatgcgtctt 3061 ttgttctggt catcaaggag gggggaatgt tctgcgcatg cctgtggggg agggagaagg 3121 acacgtcact gaaaacgtcc ctgcatcaca ccgagacacc caatcactca agccgagacc 3181 agataatttt gcatatgctt taaagtttga aaaataccac ggtaaaccct cacacaaact 3241 ctggatttga gatctttcag gttttatcag tttgcaggtt tatgtcacca tgatataggg 3301 tcagacttga tctaagggag ctgaataagt ggtttagtca ctcaccacct cccaaaaaca 3361 tacccagaag tccctggtat atatagctct ccctccagct cttggttcgg ctagcactcc 3421 tcagtgacga ggagtcagta gcgttttaga gctagaaata gcaagttaaa ataaggctag 3481 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggtacc gtaaaacgac 3541 ggccagtgaa ttatcaacta tgtataataa agttgaacga gaaacgtaaa atgatataaa 3601 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca 3661 acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact ttatgcttcc 3721 ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg agctaaggaa 3781 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt 3841 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag 3901 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc 3961 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa 4021 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa 4081 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac 4141 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt 4201 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta 4261 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg 4321 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc 4381 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcaggcgggg 4441 cgtaatctag aggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg 4501 atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta 4561 tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc 4621 atatatgatg tcaatatctc cggttcggta agcacaacca tgcagaatga agcccgtcgt 4681 ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt 4741 attgaaatga acggctcctt tgccgacgag aacaggggct ggtgaaatgc agtttaaggt 4801 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat 4861 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa 4921 agtcccccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac 4981 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca 5041 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg 5101 ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac 5161 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat 5221 tttacgtttc tcgttcaact tttctataca aagttgatag cttggcgtaa tcatggtcat 5281 agctgtttcc tgctcgaggg cccatctggc ctgtgtttca gacaccaggg agtctctgct 5341 cacgtttcct gctatttgca gcctctctat caagactaat acacctcttc ccgcatcggc 5401 tgcctgtgag aggcttttca gcactgcagg attgcttttc agccccaaaa gagctaggct 5461 tgacactaac aattttgaga atcagcttct actgaagtta aatctgaggt tttacaactt 5521 tgagtagcgt gtactggcat tagattgtct gtcttatagt ttgataatta aatacaaaca 5581 gttctaaagc aggataaaac cttgtatgca tttcatttaa tgttttttga gattaaaagc 5641 ttaaacaaga atctctagtt ttctttcttg cttttacttt tacttcctta atactcaagt 5701 acaattttaa tggagtactt ttttactttt actcaagtaa gattctagcc agatactttt 5761 acttttaatt gagtaaaatt ttccctaagt acttgtactt tcacttgagt aaaatttttg 5821 agtacttttt acacctctgt caagaactcc tggacaaacc tctgacctgt gtggaacaga 5881 gtggatatgg gtgtctgaac agatattcac gtcttttgca gatcagaggg catttctggt 5941 gttgtgcaat gacctgggtc caacaggtag gcgtactcgt atccaagagc attgttggac 6001 aggaggtagt ctccattcct ataaagagga aggaatggca ccatgtagta gccgtcattg 6061 tggccaattg gtgcattggt acgtgggtag atggaccggg gaggctgatg agttcttagc 6121 cagcgtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 6181 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 6241 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 6301 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt gg //
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