质粒名称: | pDD162 (Peft-3::Cas9 + Empty sgRNA) |
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出品公司: | Addgene |
目录编号: | 47549 |
存储实验室: | Bob Goldstein |
载体骨架基本信息 |
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载体名称: | pCFJ601 |
载体抗性: | 氨苄青霉素 |
空载体大小: | 2300 bp |
完整质粒大小: | 8113 bp |
载体修饰: | 由pCFJ601产生,用Cas9替换Mos1转座酶,然后插入U6启动子::sgRNA基因。克隆方法为Gibson组装(选择“结扎独立克隆”作为通用描述)。 |
载体类型: | 蠕虫表达,CRISPR |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | Cas9 |
物种来源: | 合成 |
插入大小: | 4323 bp |
基因突变: | 线虫密码子优化及内含子合成 |
启动子 | eef-1A.1 (eft-3) |
标签/融合蛋白: |
NLS(插入的C端子)
HA(插入的C端子)
|
克隆方法: | 非连接克隆 |
5' 测序引物 | TCAGTTGGGAAACACTTTGCT |
3' 测序引物 | gcttgaaaggattttgcatttatc |
文献引用方法 |
|
材料和方法部分: | pDD162 (Peft-3::Cas9 + Empty sgRNA) was a gift from Bob Goldstein (Addgene plasmid # 47549 ; http://n2t.net/addgene:47549 ; RRID:Addgene_47549) |
参考文献部分: | Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. Nat Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641. 10.1038/nmeth.2641 PubMed 23995389 |
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LOCUS pDD162 (Peft-3::Cas9 + Empty sgRNA) 8113 bp ds-DNA circular SYN 13-NOV-2018 DEFINITION Cas9 + sgRNA plasmid that can be modified to cleave any Cas9 target site in the C. elegans genome.. ACCESSION . VERSION . KEYWORDS pDD162 (Peft-3::Cas9 + Empty sgRNA) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8113) AUTHORS Dickinson DJ, Ward JD, Reiner DJ, Goldstein B TITLE Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. JOURNAL Nat Methods. 2013 Sep 1. doi: 10.1038/nmeth.2641. PUBMED 23995389 REFERENCE 2 (bases 1 to 8113) AUTHORS . TITLE Direct Submission JOURNAL Exported Nov 13, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8113 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 1..589 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 490..509 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 716..738 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 735..751 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 735..751 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" protein_bind 771..791 /gene="mutant version of attB" /label=attB4 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" promoter 792..1391 /gene="eft-3" /label=eft-3 promoter /note="promoter for the C. elegans elongation factor 1-alpha gene (Mitrovich and Anderson, 2000)" CDS join(1408..2451,2503..3573,3625..4650,4702..5664) /codon_start=1 /product="S. pyogenes Cas9, codon optimized for C. elegans with synthetic introns" /label=Cas9 /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5680..5700 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5701..5727 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" terminator 5742..6065 /gene="tbb-2" /label=tbb-2 terminator /note="transcription terminator for the C. elegans tubulin beta-2 gene" protein_bind complement(6069..6089) /gene="mutant version of attB" /label=attB3 /bound_moiety="BP Clonase(TM)" /note="core recombination site for the Gateway(R) BP reaction" primer_bind complement(6097..6114) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6097..6113) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6106..6128) /label=M13/pUC Forward /note="In lacZ gene" promoter 6134..6483 /label=CeU6 promoter /note="promoter for the C. elegans R07E5.16 U6 snRNA gene (Dickinson et al., 2013)" misc_RNA 6485..6560 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind 6832..6854 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6892..6910) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6978..7082 /gene="bla" /label=AmpR promoter CDS 7083..7943 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGSDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7301..7320) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 601 cgcggccttt ttacggttcc tggtcttttg ctggcctttt gctcacatgt tctttcctgc 661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccagcgg 721 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat caactttgta 781 tagaaaagtt ggcacctttg gtcttttatt gtcaacttcc attggttctt ccattgtttc 841 tgttaaatta atgaattttt cataaaataa agacattata caatataaaa atgaagaatt 901 tattgaaaat aaactgccag agagaaaaag tatgcaacac tcccgccgag agtgtttgaa 961 atggtgtacg gtacattttc gtgctaggag ttagatgtgc aggcagcaac gagaggggga 1021 gagatttttt tgggccttgt gaaattaacg tgagttttct ggtcatctga ctaatcatgt 1081 tggttttttg ttggtttatt ttgtttttat ctttgttttt atccagatta ggaaatttaa 1141 attttatgaa tttataatga ggtcaaacat tcagtcccag cgtttttcct gttctcactg 1201 tttagtcgaa tttttatttt aggctttcaa caaatgttct aactgtctta tttgtgacct 1261 cactttttat atttttttaa tttttaaaaa tattagaagt ttctaggata attttttcga 1321 cttttattct ctctaccgtc cgcactcttc ttacttttaa attaaattgt ttttttttca 1381 gttgggaaac actttgctct agaaaaaatg gacaaaaaat acagcatcgg ccttgacatc 1441 ggaacgaact ccgtcggatg ggccgtcatc accgacgagt acaaggtccc atccaagaag 1501 ttcaaggtcc tcggaaacac cgaccgtcac tccatcaaga agaacctcat cggagccctc 1561 ctcttcgact ccggagagac cgccgaggcc acccgtctca agcgtaccgc ccgtcgtcgt 1621 tacacccgtc gtaagaaccg tatctgctac ctccaagaga tcttctccaa cgagatggcc 1681 aaggtcgacg actccttctt ccaccgtctc gaggagtcct tcctcgtcga ggaggacaag 1741 aagcacgagc gtcacccaat cttcggaaac atcgtcgacg aggtcgccta ccacgagaag 1801 tacccaacca tctaccacct ccgtaagaag ctcgtcgact ccaccgacaa ggccgacctc 1861 cgtctcatct acctcgccct cgcccacatg atcaagttcc gtggacactt cctcatcgag 1921 ggagacctca acccagacaa ctccgacgtc gacaagctct tcatccaact cgtccaaacc 1981 tacaaccaac tcttcgagga gaacccaatc aacgcctccg gagtcgacgc caaggccatc 2041 ctctccgccc gtctctccaa gtcccgtcgt ctcgagaacc tcatcgccca actcccagga 2101 gagaagaaga acggactctt cggaaacctc atcgccctct ccctcggact caccccaaac 2161 ttcaagtcca acttcgacct cgccgaggac gccaagctcc aactctccaa ggacacctac 2221 gacgacgacc tcgacaacct cctcgcccaa atcggagacc aatacgccga cctcttcctc 2281 gccgccaaga acctctccga cgccatcctc ctctccgaca tcctccgtgt caacaccgag 2341 atcaccaagg ccccactctc cgcctccatg atcaagcgtt acgacgagca ccaccaagac 2401 ctcaccctcc tcaaggccct cgtccgtcaa caactcccag agaagtacaa ggtaagttta 2461 aacatatata tactaactaa ccctgattat ttaaattttc aggagatctt cttcgaccaa 2521 tccaagaacg gatacgccgg atacatcgac ggaggagcct cccaagagga gttctacaag 2581 ttcatcaagc caatcctcga gaagatggac ggaaccgagg agctcctcgt caagctcaac 2641 cgtgaggacc tcctccgtaa gcaacgtacc ttcgacaacg gatccatccc acaccaaatc 2701 cacctcggag agctccacgc catcctccgt cgtcaagagg acttctaccc attcctcaag 2761 gacaaccgtg agaagatcga gaagatcctc accttccgta tcccatacta cgtcggacca 2821 ctcgcccgtg gaaactcccg tttcgcctgg atgacccgta agtccgagga gaccatcacc 2881 ccatggaact tcgaggaggt cgtcgacaag ggagcctccg cccaatcctt catcgagcgt 2941 atgaccaact tcgacaagaa cctcccaaac gagaaggtcc tcccaaagca ctccctcctc 3001 tacgagtact tcaccgtcta caacgagctc accaaggtca agtacgtcac cgagggaatg 3061 cgtaagccag ccttcctctc cggagagcaa aagaaggcca tcgtcgacct cctcttcaag 3121 accaaccgta aggtcaccgt caagcaactc aaggaggact acttcaagaa gatcgagtgc 3181 ttcgactccg tcgagatctc cggagtcgag gaccgtttca acgcctccct cggaacctac 3241 cacgacctcc tcaagatcat caaggacaag gacttcctcg acaacgagga gaacgaggac 3301 atcctcgagg acatcgtcct caccctcacc ctcttcgagg accgtgagat gatcgaggag 3361 cgtctcaaga cctacgccca cctcttcgac gacaaggtca tgaagcagct taagcgtcgt 3421 cgttacaccg gatggggacg tctctcccgt aagctcatca acggaatccg tgacaagcaa 3481 tccggaaaga ccatcctcga cttcctcaag tccgacggat tcgccaaccg taacttcatg 3541 caactcatcc acgacgactc cctcaccttc aaggtaagtt taaacagttc ggtactaact 3601 aaccatacat atttaaattt tcaggaggac atccaaaagg cccaagtctc cggacaagga 3661 gactccctcc acgagcacat cgccaacctc gccggatccc cagccatcaa gaagggaatc 3721 ctccaaaccg tcaaggtcgt cgacgagctc gtcaaggtca tgggacgtca caagccagag 3781 aacatcgtca tcgagatggc ccgtgagaac caaaccaccc aaaagggaca aaagaactcc 3841 cgtgagcgta tgaagcgtat cgaggaggga atcaaggagc tcggatccca aatcctcaag 3901 gagcacccag tcgagaacac ccaactccaa aacgagaagc tctacctcta ctacctccaa 3961 aacggacgtg acatgtacgt cgaccaagag ctcgacatca accgtctctc cgactacgac 4021 gtcgaccaca tcgtcccaca atccttcctc aaggacgact ccatcgacaa caaggtcctc 4081 acccgttccg acaagaaccg tggaaagtcc gacaacgtcc catccgagga ggtcgtcaag 4141 aagatgaaga actactggcg tcaactcctc aacgccaagc tcatcaccca acgtaagttc 4201 gacaacctca ccaaggctga gcgtggagga ctctccgagc tcgacaaggc cggattcatc 4261 aagcgtcaac tcgtcgagac ccgtcaaatc accaagcacg tcgcccaaat cctcgactcc 4321 cgtatgaaca ccaagtacga cgagaacgac aagctcatcc gtgaggtcaa ggtcatcacc 4381 ctcaagtcca agctcgtctc cgacttccgt aaggacttcc aattctacaa ggtccgtgag 4441 atcaacaact accaccacgc ccacgacgcc tacctcaacg ccgtagtagg aaccgccctc 4501 atcaagaagt acccaaagct cgagtccgag ttcgtctacg gagactacaa ggtctacgac 4561 gtccgtaaga tgatcgccaa gtccgagcaa gagatcggaa aggccaccgc caagtacttc 4621 ttctactcca acatcatgaa cttcttcaag gtaagtttaa acatgatttt actaactaac 4681 taatctgatt taaattttca gaccgagatc accctcgcca acggagagat ccgtaagcgt 4741 ccactcatcg agaccaacgg agagaccgga gagatcgtct gggacaaggg acgtgacttc 4801 gccacagtcc gtaaggtcct ctccatgcca caagtcaaca tcgtcaagaa gaccgaggtc 4861 caaaccggag gattctccaa ggagtccatc ctcccaaagc gtaactccga caagctcatc 4921 gcccgtaaga aggactggga cccaaagaag tacggaggat tcgactcccc aaccgtcgcc 4981 tactccgtcc tcgtcgtcgc caaggtcgag aagggaaagt ccaagaagct caagtccgtc 5041 aaggagctcc tcggaatcac catcatggag cgttcctcct tcgagaagaa cccaatcgac 5101 ttcctcgagg ccaagggata caaggaggtc aagaaggacc tcatcatcaa gctcccaaag 5161 tactccctct tcgagctcga gaacggacgt aagcgtatgc tcgcctccgc cggagagctc 5221 caaaagggaa acgagctcgc cctcccatcc aagtacgtca acttcctcta cctcgcctcc 5281 cactacgaga agctcaaggg atccccagag gacaacgagc aaaagcaact cttcgtcgag 5341 caacacaagc actacctcga cgagatcatc gagcaaatct ccgagttctc caagcgtgtc 5401 atcctcgccg acgccaacct cgacaaggtc ctctccgcct acaacaagca ccgtgacaag 5461 ccaatccgtg agcaagccga gaacatcatc cacctcttca ccctcaccaa cctcggagcc 5521 ccagccgcct tcaagtactt cgacaccacc atcgaccgta agcgttacac ctccaccaag 5581 gaggtcctcg acgccaccct catccaccaa tccatcaccg gactctacga gacccgtatc 5641 gacctctccc aactcggagg agacggagga tccggaccac caaagaagaa gcgtaaggtc 5701 tacccatacg acgtcccaga ctacgcctaa actagtgata aatgcaaaat cctttcaagc 5761 attcccttct tctctatcac tcttctttct ttttgtcaaa aaattctctc gctaatttat 5821 ttgctttttt aatgttatta ttttatgact ttttatagtc actgaaaagt ttgcatctga 5881 gtgaagtgaa tgctatcaaa atgtgattct gtctgatgta ctttcacaat ctctcttcaa 5941 ttccattttg aagtgcttta aacccgaaag gttgagaaaa atgcgagcgc tcaaatattt 6001 gtattgtgtt cgttgagtga cccaacaaaa agaggaaact ttattgtgcc gccaagaaaa 6061 aagtctcaca actttattat acatagttga taattcactg gccgtcgttt tacaacgtcg 6121 tgactgggaa aaccaaaaaa aactagcaat aaaggaataa aaaactgtac accttaaagg 6181 cgcacactct gttttgcaaa ttttattttt agttgtgaat tttctgctga gacctgaaaa 6241 tagcaacttt agtactacta taatttgtca accttttcaa aaaaagcatg caatttttga 6301 gaaactctta taaaagctat tattaaaaaa acaccttttt tccaaaatta ttccacaaaa 6361 aatatgttat gaaatgccta caccctctca cacacactct ttatactact ctgtcaaact 6421 cacgagatgt ctgccgcctc ttgtgttgcc cctatataaa cacctcctat tgcgagatgt 6481 cttggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa 6541 aagtggcacc gagtcggtgc tttttgtgaa atttctggcg taatagcgaa gaggcccgca 6601 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt 6661 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtgcaatct 6721 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 6781 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 6841 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga 6901 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 6961 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 7021 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 7081 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 7141 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 7201 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 7261 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 7321 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 7381 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 7441 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 7501 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 7561 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 7621 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 7681 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 7741 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 7801 ctcgcggtat cattgcagca ctggggtcag atggtaagcc ctcccgtatc gtagttatct 7861 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 7921 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 7981 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 8041 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 8101 tcaaaggatc ttc //
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