LOCUS Exported 8935 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION All-in-one vector for CRISPR/Cas9-mediated homology-independent knock-in system. The plasmid contains eSpCas9(1.1) and two sgRNA expression cassettes. The first gRNA cloning site is empty.. ACCESSION . VERSION . KEYWORDS peSpCas9(1.1)-2×sgRNA (empty, donor) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8935) AUTHORS Katoh Y, Michisaka S, Nozaki S, Funabashi T, Hirano T, Takei R, Nakayama K TITLE Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated homology-independent knock-in system. JOURNAL Mol Biol Cell. 2017 Feb 8. pii: mbc.E17-01-0051. doi: 10.1091/mbc.E17-01-0051. PUBMED 28179459 REFERENCE 2 (bases 1 to 8935) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8935 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 268..343 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 434..674 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 703..778 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 869..1154 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 1156..1433 /label=chicken beta-actin promoter intron 1434..1662 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" regulatory 1674..1683 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1683..1748 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1755..1775 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1800..5900 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system, mutated to improve targeting specificity (Slaymaker et al., 2016)" /label=eSpCas9(1.1) /note="carries the mutations K848A, K1003A, and R1060A" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5901..5948 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" primer_bind complement(5976..5993) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 5982..6189 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 6198..6338 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 6198..6327 /label=AAV2 ITR rep_origin 6413..6868 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6500..6519) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6710..6731 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6885..6904) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7004..7026 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7064..7082) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7150..7254 /gene="bla" /label=AmpR promoter CDS 7255..8115 /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(7473..7492) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8286..8874 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 8775..8794 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 421 aaatggctct agagagggcc tatttcccat gattccttca tatttgcata tacgatacaa 481 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa 541 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa 601 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat 661 cttgtggaaa ggacgaaaca ccgcatcgta cgcgtacgtg ttgttttaga gctagaaata 721 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt 781 ttttgtttta gagctagaaa tagcaagtta aaataaggct agtccgtttt tagcgcgtgc 841 gccaattctg cagacaaatg gcggtacccg ttacataact tacggtaaat ggcccgcctg 901 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc 961 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 1021 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 1081 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 1141 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc 1201 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg 1261 cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg 1321 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta 1381 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg 1441 ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg 1501 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc 1561 tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg 1621 tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca 1681 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg 1741 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg 1801 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca 1861 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca 1921 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca 1981 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc 2041 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg 2101 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca 2161 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac 2221 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga 2281 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg 2341 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca 2401 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc 2461 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga 2521 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg 2581 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga 2641 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc 2701 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga 2761 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc 2821 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct 2881 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa 2941 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc 3001 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca 3061 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga 3121 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat 3181 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg 3241 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc 3301 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata 3361 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg 3421 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga 3481 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg 3541 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca 3601 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga 3661 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc 3721 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc 3781 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt 3841 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc 3901 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg 3961 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga 4021 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg 4081 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga 4141 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg 4201 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata 4261 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg 4321 tgcctcagag ctttctggcg gacgactcca tcgacaacaa ggtgctgacc agaagcgaca 4381 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact 4441 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca 4501 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg 4561 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta 4621 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc 4681 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc 4741 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc 4801 ctgcgctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga 4861 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca 4921 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aaggcgcctc 4981 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca 5041 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga 5101 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca 5161 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt 5221 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag 5281 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc 5341 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact 5401 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga 5461 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact 5521 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc 5581 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc 5641 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca 5701 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg 5761 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg 5821 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc 5881 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa 5941 agaaaaagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 6001 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 6061 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 6121 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 6181 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc 6241 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 6301 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct 6361 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc 6421 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 6481 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 6541 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6601 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc 6661 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 6721 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt 6781 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 6841 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat 6901 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 6961 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 7021 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 7081 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 7141 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 7201 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 7261 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 7321 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 7381 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 7441 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 7501 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 7561 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 7621 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 7681 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 7741 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 7801 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 7861 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 7921 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt 7981 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 8041 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 8101 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 8161 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 8221 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 8281 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 8341 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 8401 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 8461 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 8521 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 8581 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 8641 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 8701 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 8761 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 8821 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 8881 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt //