LOCUS Exported 6389 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level0 Golden Gate part: CDS, SpCas9-h (no stop codon). ACCESSION . VERSION . KEYWORDS pICSL90005 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6389) AUTHORS Raitskin O, Schudoma C, West A, Patron NJ TITLE Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering. JOURNAL PLoS One. 2019 Feb 27;14(2):e0211598. doi: 10.1371/journal.pone.0211598. eCollection 2019. PUBMED 30811422 REFERENCE 2 (bases 1 to 6389) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6389 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(40..58) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 126..230 /gene="bla" /label=AmpR promoter CDS 452..1243 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 1414..2002 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1903..1922 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2156..2173 /label=L4440 /note="L4440 vector, forward primer" CDS 2251..6354 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /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 6367..6387 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" ORIGIN 1 ttcgtgagac cacgaagtgg ctcttcagtg gacgaaaggg cctcgtgata cgcctatttt 61 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 121 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 181 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgcgctcac 241 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca 301 tggcttgtta tgactgtttt tttttggggt acagtctatg cctcgggcat ccaagcagca 361 agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg ttacgcagca 421 gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg ccgaagtatc 481 gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc 541 cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagcg atattgattt 601 gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct 661 tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat 721 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg 781 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga 841 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc 901 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt 961 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt 1021 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga 1081 ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc 1141 ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt 1201 ccattacgta aaaggcgaga tcaccaaggt agtcggcaaa taactgtcag accaagttta 1261 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 1321 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 1381 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 1441 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 1501 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 1561 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 1621 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 1681 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 1741 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 1801 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 1861 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 1921 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 1981 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 2041 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 2101 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 2161 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 2221 gccgattcat taatcactct gtggtctcaa atggacaaga agtactccat tgggctcgat 2281 atcggcacaa acagcgtcgg ctgggccgtc attacggacg agtacaaggt gccgagcaaa 2341 aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc 2401 ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc 2461 agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg 2521 gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat 2581 aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa 2641 aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac 2701 ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc 2761 gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag 2821 acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca 2881 atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct 2941 ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc 3001 aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc 3061 tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt 3121 ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg 3181 gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa 3241 gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt 3301 ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag 3361 gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg 3421 gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc 3481 ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac 3541 ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac 3601 tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa 3661 gagactatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc 3721 ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa 3781 cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc 3841 acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac 3901 ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ttatttcaaa 3961 aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc 4021 ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag 4081 gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag 4141 atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag 4201 ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc 4261 cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac 4321 cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa 4381 gcacaagttt ctggccaggg ggacagtctc cacgagcaca tcgctaatct tgcaggtagc 4441 ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta 4501 atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc 4561 cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa 4621 ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag 4681 ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc 4741 aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct caaagatgat 4801 tctattgata ataaagtgtt gacaagatcc gataaaaata gagggaagag tgataacgtc 4861 ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa 4921 ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag 4981 ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac 5041 gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt 5101 cgagaggtga aagttattac tctgaagtct aagctggttt cagatttcag aaaggacttt 5161 cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat 5221 gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac 5281 ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc 5341 aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt 5401 acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga 5461 gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg 5521 caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc 5581 ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa 5641 tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag 5701 aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag 5761 cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc 5821 aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg 5881 aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct 5941 aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg atctcccgaa 6001 gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc 6061 gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg 6121 ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc 6181 cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc 6241 atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag 6301 tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg 6361 gctgacccca agaagaagag gaaggtggg //