LOCUS Exported 8923 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Mammalian expression vector encoding WT ScCas9. ACCESSION . VERSION . KEYWORDS pScCas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8923) AUTHORS Chatterjee P, Jakimo N, Jacobson JM TITLE Minimal PAM specificity of a highly similar SpCas9 ortholog. JOURNAL Sci Adv. 2018 Oct 24;4(10):eaau0766. doi: 10.1126/sciadv.aau0766. eCollection 2018 Oct. PUBMED 30397647 REFERENCE 2 (bases 1 to 8923) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8923 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 1..72 /label=5' beta-globin insulator /note="insulator upstream of the human beta-globin locus (Farrell et al., 2002)" terminator 77..123 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" polyA_signal 141..189 /note="synthetic polyadenylation signal" promoter 226..1404 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 457..1395 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1352..1372 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1517..5641 /codon_start=1 /product="Cas9 endonuclease from the Streptococcus canis Type II CRISPR/Cas system (Chatterjee et al., 2018)" /label=ScCas9 /note="recognizes minimal 5'-NNG-3' PAM sequences" /translation="MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKK NLMGALLFDSGETAEATRLKRTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEES FLVEEDKKNERHPIFGNLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAHIIK FRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESPLDEIEVDAKGILSARLSKSKRL EKLIAVFPNEKKNGLFGNIIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLGQ IGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDEHHQDLALLKTLVR QQFPEKYAEIFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEEL LAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRI PYYVGPLARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKV LPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEIIGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTL FEDREMIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLK SDGFSNRNFMQLIHDDSLTFKEEIEKAQVSGQGDSLHEQIADLAGSPAIKKGILQTVKI VDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKELESQILKENPVEN TQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSVE NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEADKAGFIKRQL VETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDINN YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFY SNIMNFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKT EVQTGGFSKESILSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEKGKAKKL KSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIKKELIFKLPKYSLFELENGRRRMLAS ATELQKANELVLPQHLVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKY ILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKYTSFGASGGFTFLDLDVKQGRLRY QTVTEVLDATLIYQSITGLYETRTDLSQLGGD" CDS 5654..5674 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" polyA_signal 5712..5833 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5723..5742) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5777..5796 /label=SV40pA-R /note="SV40 polyA, reverse primer" terminator 5948..5994 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" terminator 6024..6160 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" misc_feature 6205..6276 /label=3' beta-globin insulator /note="insulator downstream of the human beta-globin locus (Farrell et al., 2002)" rep_origin complement(6502..7090) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6582..6601) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 7836..8630 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(7890..7909) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 8500..8519 /label=Neo-F /note="Neomycin resistance gene, forward primer" terminator 8643..8779 /label=rrnG terminator /note="transcription terminator from the E. coli ribosomal RNA rrnG operon (Albrechtsen et al., 1991)" ORIGIN 1 catcttggac cattagctcc acaggtatct tcttccctct agtggtcata acagcagctt 61 cagctacctc tcaattcaaa aaacccctca agacccgttt agaggcccca aggggttatg 121 ctatcaatcg ttgcgttaca cacacaaaaa accaacacac atccatcttc gatggatagc 181 gattttatta tctaactgct gatcgagtgt agccagatct cgtgaggctc cggtgcccgt 241 cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat 301 tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg 361 ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcactagt cgccgtgaac 421 gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc 481 gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg 541 cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgtggcct 601 tgcgcttaag gagccccttc gcctcgtgct tgagttgtgg cctggcctgg gcgctggggc 661 cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta 721 gccatttaaa atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta 781 aatgcgggcc aagatcagca cactggtatt tcggtttttg gggccgcggg cggcgacggg 841 gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga 901 atcggacggg ggtagtctca agctgcccgg cctgctctgg tgcctggcct cgcgccgccg 961 tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa 1021 agatggccgc ttcccggccc tgctgcaggg agcacaaaat ggaggacgcg gcgctcggga 1081 gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct 1141 tcatgtgact ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctccagcttt 1201 tggagtacgt cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact 1261 gagtgggtgg agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt 1321 gccctttttg agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt 1381 tttcttccat ttcaggtgtc gtgaaaacta cccctaaaag ccaaaagatc tttgtcgatc 1441 ctaccatcca ctcgacacac ccgccagcgg ccgctgccaa gcttccgagc tctcgaattc 1501 aaaggaggta cccaccatgg aaaaaaaata ctcaatcgga ttggatatcg gcactaactc 1561 agttggatgg gctgtaatta cagacgatta caaggtccct agcaaaaagt ttaaggtgct 1621 gggtaatacg aatcgcaaga gtattaagaa gaatctgatg ggtgcattac tgtttgattc 1681 tggcgaaaca gccgaagcta ctcgtttaaa gcgcaccgcc cgccgtcgtt atacacgccg 1741 caagaatcgc attcgctatc ttcaagaaat cttcgctaat gaaatggcga agctggacga 1801 ctctttcttc cagcgtttgg aggaaagttt tcttgtggag gaggacaaaa aaaatgaacg 1861 tcaccccatt ttcgggaact tagcggacga ggtcgcctat caccgtaact atcccactat 1921 ttaccacctg cgcaaaaagc ttgcggattc tccagaaaag gctgaccttc gcctgattta 1981 tctggcattg gcacatatca tcaaatttcg tgggcacttc cttattgaag gaaaattgaa 2041 cgcagaaaat tctgatgtcg caaaactgtt ctaccagttg atccagacat ataatcaatt 2101 attcgaggag agtcccctgg atgaaatcga agtggatgca aagggtatcc tgtccgcccg 2161 tctgtcaaag agtaaacgtc ttgaaaagct gattgcggtg tttcccaatg aaaaaaagaa 2221 cggtttgttc gggaacatta tcgctctggc ccttggttta acccctaact ttaagtcaaa 2281 ctttgacctg actgaggacg cgaaattaca gctttctaaa gatacttatg acgatgactt 2341 agatgagctt ctggggcaaa ttggggacca gtacgccgat cttttcagtg cagccaagaa 2401 tcttagtgac gcgattttgc tttcagacat cttacgttcc aacagcgaag taacaaaggc 2461 gccactgagc gcctctatgg ttaagcgtta tgacgagcac catcaggacc ttgcacttct 2521 taagactctg gtgcgtcaac agtttccaga aaagtacgca gagatcttca aggacgatac 2581 aaaaaacgga tacgccgggt atgtcggaat tggaattaaa catcgcaagc gcacaactaa 2641 actggcgaca caagaagagt tctacaaatt cattaaacct attcttgaga aaatggacgg 2701 ggccgaggag ctgttggcca aactgaaccg tgacgacctt ttacgtaaac agcgcacctt 2761 tgacaacggt tccattcctc accagatcca tcttaaagaa ttgcacgcaa ttttgcgtcg 2821 tcaggaggag ttttatccat ttctgaaaga gaatcgtgag aaaattgaga aaatccttac 2881 gttccgcatc ccctactatg ttggtcctct ggctcgcggc aactcccgct tcgcgtggtt 2941 gacacgtaag tctgaggagg ctatcactcc ctggaacttc gaggaagtgg tggacaaagg 3001 agccagcgca cagagcttta tcgaacgtat gacaaatttc gacgaacagc ttccaaacaa 3061 aaaagtctta ccgaagcata gtcttttata tgagtatttc accgtctata atgagttaac 3121 gaaagtgaag tacgttactg agcgcatgcg taagcccgag tttttaagtg gcgagcaaaa 3181 aaaggctatt gtcgatcttc tgtttaagac taaccgcaaa gtcaccgtga aacaacttaa 3241 agaagattat tttaagaaga tcgaatgttt cgattcggta gaaattattg gcgtagagga 3301 tcgtttcaac gctagtttgg ggacatacca tgacttactt aagatcatca aggacaagga 3361 ttttttggat aatgaagaaa acgaggatat tctggaggac attgtattga ccctgacact 3421 gttcgaggac cgtgaaatga ttgaagagcg tttaaaaaca tatgcgcacc tgtttgacga 3481 taaagtgatg aagcaattaa agcgccgtca ttacactggg tggggtcgtc tttctcgtaa 3541 aatgattaac ggtatccgtg ataaacagtc tggtaagacg attctggact tccttaagag 3601 cgatggcttc tcgaaccgta actttatgca actgattcac gatgactccc tgacgtttaa 3661 agaagaaatt gagaaggctc aagtctctgg gcaaggggac agtctgcacg agcaaattgc 3721 tgaccttgcc ggaagtcccg cgattaagaa aggaatcttg caaacagtaa aaattgtgga 3781 tgaattggtt aaagtgatgg gacataaacc cgaaaatatt gtcatcgaga tggcgcgcga 3841 gaaccagacc actaccaagg ggcttcaaca atcgcgtgaa cgtaagaaac gcatcgaaga 3901 aggcattaag gagttggaga gccaaattct gaaagaaaat ccagtagaga atactcagtt 3961 acagaatgaa aaattgtatc tttattactt acagaacgga cgcgatatgt acgtggatca 4021 agagctggac atcaatcgcc tgagtgacta cgacgtagat catatcgttc ctcagtcgtt 4081 tatcaaggat gacagcatcg acaataaagt gctgacgcgt agtgtcgaaa atcgcggaaa 4141 atcggataat gtgccatcgg aggaagtcgt gaaaaaaatg aagaattatt ggcgccaact 4201 tttgaacgcc aagttgatta cccaacgtaa attcgataat cttaccaagg ctgaacgcgg 4261 gggcctgtcc gaagcagaca aagcagggtt tattaagcgt caacttgtag agacacgtca 4321 gattacaaag catgtcgccc gcattcttga ttcgcgcatg aatactaagc gtgataaaaa 4381 tgacaagccc attcgtgagg ttaaagtaat tacactgaag tctaagctgg ttagtgactt 4441 tcgtaaagac ttccagttat acaaggtgcg cgatatcaat aattatcatc acgcccatga 4501 cgcttatctg aatgcggttg taggtacggc cctgatcaag aagtatccaa aattagagtc 4561 cgagttcgtg tacggtgatt acaaggtcta cgatgtccgt aaaatgattg ccaaatccga 4621 acaagaaatc ggtaaagcga ctgcaaaacg cttcttctat tctaacatta tgaatttctt 4681 taagaccgag gtaaaattgg cgaacgggga aattcgtaaa cgtccgttga tcgagacaaa 4741 cggagaaacg ggcgaggttg tttggaacaa agaaaaagac ttcgccactg ttcgtaaagt 4801 tctggcgatg ccgcaggtaa acatcgtaaa gaaaaccgaa gttcaaactg gaggattttc 4861 taaagaatca atcctttcta aacgtgaatc ggcgaagttg attccgcgca aaaaagggtg 4921 ggacactcgt aaatacggcg gttttgggag tccgactgta gcctatagta tcctggtggt 4981 tgcaaaggtc gagaagggaa aagctaaaaa actgaaatcc gttaaggtct tggttgggat 5041 cacgattatg gagaaaggaa gctatgaaaa ggaccctatt ggttttctgg aagccaaagg 5101 ctacaaggat atcaaaaagg agttgatctt caaattacct aaatattcat tgttcgaact 5161 ggagaacgga cgccgtcgta tgttagcttc tgcaaccgag ttgcagaagg ctaacgaact 5221 ggtattgcca caacatctgg tgcgtttact ttattataca cagaacatct cggctacgac 5281 aggctctaac aatttggggt acatcgagca acatcgtgaa gaattcaagg agattttcga 5341 aaaaattatt gatttctcgg agaagtatat cttgaaaaat aaagtgaatt caaacttgaa 5401 atcgagtttt gacgagcaat tcgcggtatc ggattccatt ctgttgtcca acagctttgt 5461 tagcctgttg aaatatacct cttttggcgc gtcgggagga ttcaccttcc tggaccttga 5521 cgttaagcaa gggcgcttgc gctaccagac ggttacggag gttctggatg cgaccttgat 5581 ctatcaaagc atcaccggac tgtacgagac acgtacagat ctgagccaac tgggaggaga 5641 ctcgcgggct gacccgaaaa agaagagaaa ggtctagtga gatacagcgt accttcagct 5701 cacagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 5761 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 5821 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 5881 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtattggcc catctctatc 5941 ggtatcgtag cataacccct tggggcctct aaacgggtct tgaggggttt tttgtgcccc 6001 tcgggccgga ttgctatcta ccggcattgg cgcagaaaaa aatgcctgat gcgacgctgc 6061 gcgtcttata ctcccacata tgccagattc agcaacggat acggcttccc caacttgccc 6121 acttccatac gtgtcctcct taccagaaat ttatccttaa ggtcgtcagc tatcctgcag 6181 gcgatctctc gatttcgatc aagacattcc tttaatggtc ttttctggac accactaggg 6241 gtcagaagta gttcatcaaa ctttcttccc tccctaatct cattggttac cttgggctat 6301 cgaaacttaa ttaaccagtc aagtcagcta cttggcgaga tcgacttgtc tgggtttcga 6361 ctacgctcag aattgcgtca gtcaagttcg atctggtcct tgctattgca cccgttctcc 6421 gattacgagt ttcatttaaa tcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 6481 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 6541 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 6601 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 6661 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 6721 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 6781 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 6841 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 6901 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 6961 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 7021 aaccaccgct ggtagcggtg gtttttttgt ttgtaagcag cagattacgc gcagaaaaaa 7081 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 7141 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 7201 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 7261 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 7321 agttgcattt aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg 7381 tccgatccat cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt 7441 gcgccttggc tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc 7501 gagatcggga tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat 7561 ccgaggaata tcgaaatcgg ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag 7621 tctcgacgat ccatatcgtt gcttggcagt cagccagtcg gaatccagct tgggacccag 7681 gaagtccaat cgtcagatat tgtactcaag cctggtcacg gcagcgtacc gatctgttta 7741 aacctagata ttgatagtct gatcggtcaa cgtataatcg agtcctagct tttgcaaaca 7801 tctatcaaga gacaggatca gcaggaggct ttcgcatgat tgaacaagat ggattgcacg 7861 caggttctcc ggcggcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 7921 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgtccg gttctttttg 7981 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 8041 ggctggcgac gacgggcgtt ccttgcgcgg ctgtgctcga cgttgtcact gaagcgggaa 8101 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 8161 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 8221 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 8281 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 8341 aactgttcgc caggctcaag gcgtctatgc ccgacggcga ggatctcgtc gtgacccacg 8401 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 8461 gtggccgtct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 8521 ctgaagagct tggcggcgaa tgggctgacc gcttccttgt gctttacggt atcgccgcgc 8581 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga ccgattctag 8641 gtgcattggc gcagaaaaaa atgcctgatg cgacgctgcg cgtcttatac tcccacatat 8701 gccagattca gcaacggata cggcttcccc aacttgccca cttccatacg tgtcctcctt 8761 accagaaatt tatccttaag gtcgtttaaa ctcgactctg gctctatcga atctccgtcg 8821 tttcgagctt acgcgaacag ccgtggcgct catttgctcg tcgggcatcg aatctcgtca 8881 gctatcgtca gcttaccttt ttggcagcga tcgcggctcc cga //