LOCUS Exported 10386 bp ds-DNA circular SYN 13-SEP-2019 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10386) AUTHORS waldripzachary TITLE Direct Submission JOURNAL Exported Friday, Sep 13, 2019 from SnapGene Viewer 4.3.11 https://www.snapgene.com FEATURES Location/Qualifiers source 1..10386 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 106..293 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" CDS complement(346..1020) /codon_start=1 /gene="S. cerevisiae TRP1" /product="phosphoribosylanthranilate isomerase, required for tryptophan biosynthesis" /label=TRP1 /note="yeast auxotrophic marker" /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA KK" promoter complement(1021..1122) /gene="S. cerevisiae TRP1" /label=TRP1 promoter primer_bind complement(1137..1153) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" rep_origin complement(1539..2127) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(2296..3111) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin 3470..4634 /label=2u ori /note="yeast 2u plasmid origin of replication" promoter 4728..4746 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 4764..5032 /label=SNR52 promoter /note="promoter for the S. cerevisiae small nucleolar RNA gene SNR52" misc_RNA 5051..5126 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" terminator 5130..5149 /label=SUP4 terminator /note="transcription terminator for the S. cerevisiae SUP4 tRNA gene" promoter 5261..5702 /gene="S. cerevisiae GAL1" /label=GAL1 promoter /note="inducible promoter, regulated by Gal4" protein_bind 5261..5378 /label=UAS /bound_moiety="Gal4" /note="upstream activating sequence mediating Gal4-dependent induction" misc_feature 5723..6172 /label=Protein A tag gene 6191..10294 /label=deSPCas91.1 CDS 10307..10327 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 10331..10351 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 10355..10375 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" ORIGIN 1 caattgtcgt agatcttcgt cagcagagct tcaccattga agggctggcg gttggggtta 61 ttcgcaacgg cgactggctg tgaggatccg ccaagctaat tccgggcgaa tttcttatga 121 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt 181 gactcttagg ttttaaaacg aaaattcttg ttcttgagta actctttcct gtaggtcagg 241 ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg 301 gcaagtgcac aaacaatact taaataaata ctactcagta ataacctatt tcttagcatt 361 tttgacgaaa tttgctattt tgttagagtc ttttacacca tttgtctcca cacctccgct 421 tacatcaaca ccaataacgc catttaatct aagcgcatca ccaacatttt ctggcgtcag 481 tccaccagct aacataaaat gtaagctttc ggggctctct tgccttccaa cccagtcaga 541 aatcgagttc caatccaaaa gttcacctgt cccacctgct tctgaatcaa acaagggaat 601 aaacgaatga ggtttctgtg aagctgcact gagtagtatg ttgcagtctt ttggaaatac 661 gagtctttta ataactggca aaccgaggaa ctcttggtat tcttgccacg actcatctcc 721 atgcagttgg acgatatcaa tgccgtaatc attgaccaga gccaaaacat cctccttagg 781 ttgattacga aacacgccaa ccaagtattt cggagtgcct gaactatttt tatatgcttt 841 tacaagactt gaaattttcc ttgcaataac cgggtcaatt gttctctttc tattgggcac 901 acatataata cccagcaagt cagcatcgga atctagagca cattctgcgg cctctgtgct 961 ctgcaagccg caaactttca ccaatggacc agaactacct gtgaaattaa taacagacat 1021 actccaagct gcctttgtgt gcttaatcac gtatactcac gtgctcaata gtcaccaatg 1081 ccctccctct tggccctctc cttttctttt ttcgaccgaa ttaattcgta atcatggtca 1141 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 1201 agcataaagt gtaaagcctg gggtgcctaa tgagtgaggt aactcacatt aattgcgttg 1261 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctggatta atgaatcggc 1321 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 1381 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 1441 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 1501 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 1561 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 1621 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 1681 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 1741 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 1801 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 1861 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 1921 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 1981 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 2041 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 2101 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 2161 gctcagtgga acgaaaactc acgttaaggg attttggtca tgtggatgat ccagttcgat 2221 ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt gttacaacca 2281 attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat 2341 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc 2401 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc 2461 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc 2521 accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac 2581 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt 2641 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt 2701 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc 2761 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttgcctggga tcgcagtggt 2821 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa 2881 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt 2941 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc 3001 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt 3061 ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca taacacccct 3121 tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg 3181 tgcaatgtaa catcagagat tttgagacac aacgtggctt actaggatcg atccgacatg 3241 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 3301 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 3361 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 3421 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa cgcatttaag 3481 cataaacacg cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata 3541 taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc 3601 tcgttttcgg aaacgctttg aagttcctat tccgaagttc ctattctcta gctagaaagt 3661 ataggaactt cagagcgctt ttgaaaacca aaagcgctct gaagacgcac tttcaaaaaa 3721 ccaaaaacgc accggactgt aacgagctac taaaatattg cgaataccgc ttccacaaac 3781 attgctcaaa agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc 3841 atccaccttt cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat 3901 ctctagtatt actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga 3961 aaacaatacg aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa 4021 ccgttcataa ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag 4081 tatgcgcaat ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc 4141 acccgcagct tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga 4201 agagaaaata gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag 4261 ttatcaagag actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt 4321 gttagaaaaa tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct 4381 ttgttggtaa aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt 4441 ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag 4501 attcttcgtt ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc 4561 agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc 4621 acagatgctt cgttgcttgc atgcaacttc ttttcttttt ttttcttttc tctctccccc 4681 gttgttacta gtggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac 4741 taaagggaac aaaagctgga gcttctttga aaagataatg tatgattatg ctttcactca 4801 tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta catgtacgtt 4861 tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt 4921 ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt 4981 ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga tcccagtttc 5041 atgtactgtc gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac 5101 ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt tttatgtctt cgagtcatgt 5161 aattagttat gtcacgctta cgttcacgcc ctccccccac atccgctcta accgaaaaac 5221 tagtctcacc atatccgcaa tgacaaaaaa aatgatggaa cggattagaa gccgccgagc 5281 gggcgacagc cctccgacgg aagactctcc tccgtgcgtc ctcgtcttca ccggtcgcgt 5341 tcctgaaacg cagatgtgcc tcgcgccgca ctgctccgaa caataaagat tctacaatac 5401 tagcttttat ggttatgaag aggaaaaatt ggcagtaacc tggccccaca aaccttcaaa 5461 ttaacgaatc aaattaacaa ccataggatg ataatgcgat tagtttttta gccttatttc 5521 tggggtaatt aatcagcgaa gcgatgattt ttgatctatt aacagatata taaatggaaa 5581 agctgcataa ccactttaac taatactttc aacattttca gtttgtatta cttcttattc 5641 aaatgtcata aaagtatcaa caaaaaattg ttaatatacc tctatacttt aacgtcaagg 5701 agaaaaaaca gaattcgagc tcatgcgtac gctgcaggtc gacggatccc ctcaacaaaa 5761 caaaaccgcg gctcttgcgc aacacgatga agccgtggac aacaaattca acaaagaaca 5821 acaaaacgcg ttctatgaga tcttacattt acctaactta aacgaagaac aacgaaacgc 5881 cttcatccaa agtttaaaag atgacccaag ccaaagcgct aaccttttag cagaagctaa 5941 aaagctaaat gatgctcagg cgccgaaagt agacaacaaa ttcaacaaag aacaacaaaa 6001 cgcgttctat gagatcttac atttacctaa cttaaacgaa gaacaacgaa acgccttcat 6061 ccaaagttta aaagatgacc caagccaaag cgctaacctt ttagcagaag ctaaaaagct 6121 aaatgatgct caggcgccga aagtagacgc gaattccgcg gctcttgcta gcgccccggg 6181 ttcaggttca atggacaaga agtattctat cggactggcc atcgggacta atagcgtcgg 6241 gtgggccgtg atcactgacg agtacaaggt gccctctaag aagttcaagg tgctcgggaa 6301 caccgaccgg cattccatca agaaaaatct gatcggagct ctcctctttg attcagggga 6361 gaccgctgaa gcaacccgcc tcaagcggac tgctagacgg cggtacacca ggaggaagaa 6421 ccggatttgt taccttcaag agatattctc caacgaaatg gcaaaggtcg acgacagctt 6481 cttccatagg ctggaagaat cattcctcgt ggaagaggat aagaagcatg aacggcatcc 6541 catcttcggt aatatcgtcg acgaggtggc ctatcacgag aaatacccaa ccatctacca 6601 tcttcgcaaa aagctggtgg actcaaccga caaggcagac ctccggctta tctacctggc 6661 cctggcccac atgatcaagt tcagaggcca cttcctgatc gagggcgacc tcaatcctga 6721 caatagcgat gtggataaac tgttcatcca gctggtgcag acttacaacc agctctttga 6781 agagaacccc atcaatgcaa gcggagtcga tgccaaggcc attctgtcag cccggctgtc 6841 aaagagccgc agacttgaga atcttatcgc tcagctgccg ggtgaaaaga aaaatggact 6901 gttcgggaac ctgattgctc tttcacttgg gctgactccc aatttcaagt ctaatttcga 6961 cctggcagag gatgccaagc tgcaactgtc caaggacacc tatgatgacg atctcgacaa 7021 cctcctggcc cagatcggtg accaatacgc cgaccttttc cttgctgcta agaatctttc 7081 tgacgccatc ctgctgtctg acattctccg cgtgaacact gaaatcacca aggcccctct 7141 ttcagcttca atgattaagc ggtatgatga gcaccaccag gacctgaccc tgcttaaggc 7201 actcgtccgg cagcagcttc cggagaagta caaggaaatc ttctttgacc agtcaaagaa 7261 tggatacgcc ggctacatcg acggaggtgc ctcccaagag gaattttata agtttatcaa 7321 acctatcctt gagaagatgg acggcaccga agagctcctc gtgaaactga atcgggagga 7381 tctgctgcgg aagcagcgca ctttcgacaa tgggagcatt ccccaccaga tccatcttgg 7441 ggagcttcac gccatccttc ggcgccaaga ggacttctac ccctttctta aggacaacag 7501 ggagaagatt gagaaaattc tcactttccg catcccctac tacgtgggac ccctcgccag 7561 aggaaatagc cggtttgctt ggatgaccag aaagtcagaa gaaactatca ctccctggaa 7621 cttcgaagag gtggtggaca agggagccag cgctcagtca ttcatcgaac ggatgactaa 7681 cttcgataag aacctcccca atgagaaggt cctgccgaaa cattccctgc tctacgagta 7741 ctttaccgtg tacaacgagc tgaccaaggt gaaatatgtc accgaaggga tgaggaagcc 7801 cgcattcctg tcaggcgaac aaaagaaggc aattgtggac cttctgttca agaccaatag 7861 aaaggtgacc gtgaagcagc tgaaggagga ctatttcaag aaaattgaat gcttcgactc 7921 tgtggagatt agcggggtcg aagatcggtt caacgcaagc ctgggtacct accatgatct 7981 gcttaagatc atcaaggaca aggattttct ggacaatgag gagaacgagg acatccttga 8041 ggacattgtc ctgactctca ctctgttcga ggaccgggaa atgatcgagg agaggcttaa 8101 gacctacgcc catctgttcg acgataaagt gatgaagcaa cttaaacgga gaagatatac 8161 cggatgggga cgccttagcc gcaaactcat caacggaatc cgggacaaac agagcggaaa 8221 gaccattctt gatttcctta agagcgacgg attcgctaat cgcaacttca tgcaacttat 8281 ccatgatgat tccctgacct ttaaggagga catccagaag gcccaagtgt ctggacaagg 8341 tgactcactg cacgagcata tcgcaaatct ggctggttca cccgctatta agaagggtat 8401 tctccagacc gtgaaagtcg tggacgagct ggtcaaggtg atgggtcgcc ataaaccaga 8461 gaacattgtc atcgagatgg ccagggaaaa ccagactacc cagaagggac agaagaacag 8521 cagggagcgg atgaaaagaa ttgaggaagg gattaaggag ctcgggtcac agatccttaa 8581 agagcacccg gtggaaaaca cccagcttca gaatgagaag ctctatctgt actaccttca 8641 aaatggacgc gatatgtatg tggaccaaga gcttgatatc aacaggctct cagactacga 8701 cgtggacgcc atcgtccctc agagcttcct cgcagacgac tcaattgaca ataaggtgct 8761 gactcgctca gacaagaacc ggggaaagtc agataacgtg ccctcagagg aagtcgtgaa 8821 aaagatgaag aactattggc gccagcttct gaacgcaaag ctgatcactc agcggaagtt 8881 cgacaatctc actaaggctg agaggggcgg actgagcgaa ctggacaaag caggattcat 8941 taaacggcaa cttgtggaga ctcggcagat tactaaacat gtcgcccaaa tccttgactc 9001 acgcatgaat accaagtacg acgaaaacga caaacttatc cgcgaggtga aggtgattac 9061 cctgaagtcc aagctggtca gcgatttcag aaaggacttt caattctaca aagtgcggga 9121 gatcaataac tatcatcatg ctcatgacgc atatctgaat gccgtggtgg gaaccgccct 9181 gatcaagaag tacccagcgc tggaaagcga gttcgtgtac ggagactaca aggtctacga 9241 cgtgcgcaag atgattgcca aatctgagca ggagatcgga aaggccaccg caaagtactt 9301 cttctacagc aacatcatga atttcttcaa gaccgaaatc acccttgcaa acggtgagat 9361 ccggaaggcg ccgctcatcg agactaatgg ggagactggc gaaatcgtgt gggacaaggg 9421 cagagatttc gctaccgtgc gcaaagtgct ttctatgcct caagtgaaca tcgtgaagaa 9481 aaccgaggtg caaaccggag gcttttctaa ggaatcaatc ctccccaagc gcaactccga 9541 caagctcatt gcaaggaaga aggattggga ccctaagaag tacggcggat tcgattcacc 9601 aactgtggct tattctgtcc tggtcgtggc taaggtggaa aaaggaaagt ctaagaagct 9661 caagagcgtg aaggaactgc tgggtatcac cattatggag cgcagctcct tcgagaagaa 9721 cccaattgac tttctcgaag ccaaaggtta caaggaagtc aagaaggacc ttatcatcaa 9781 gctcccaaag tatagcctgt tcgaactgga gaatgggcgg aagcggatgc tcgcctccgc 9841 tggcgaactt cagaagggta atgagctggc tctcccctcc aagtacgtga atttcctcta 9901 ccttgcaagc cattacgaga agctgaaggg gagccccgag gacaacgagc aaaagcaact 9961 gtttgtggag cagcataagc attatctgga cgagatcatt gagcagattt ccgagttttc 10021 taaacgcgtc attctcgctg atgccaacct cgataaagtc cttagcgcat acaataagca 10081 cagagacaaa ccaattcggg agcaggctga gaatatcatc cacctgttca ccctcaccaa 10141 tcttggtgcc cctgccgcat tcaagtactt cgacaccacc atcgaccgga aacgctatac 10201 ctccaccaaa gaagtgctgg acgccaccct catccaccag agcatcaccg gactttacga 10261 aactcggatt gacctctcac agctcggagg ggatgaggga gctgatccaa aaaagaagag 10321 aaaggtagat ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa aggtataggt 10381 ttaaac //