LOCUS Exported 8327 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION hCas9 under control of pPFK300 for direct cloning of HH-sgRNA-HDV PCR products for episomal expression in P. pastoris and selection on G418. ACCESSION . VERSION . KEYWORDS BB3cK_pGAP_23*_pPFK300_Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8327) AUTHORS Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R. TITLE CRISPR/Cas9-mediated homology directed genome editing in Pichia pastoris JOURNAL (2018) In Press REFERENCE 2 (bases 1 to 8327) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8327 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(70..658) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(150..169) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 745..1221 /label=GAP promoter /note="promoter for Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase" CDS 1988..6091 /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 6104..6124 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" terminator 6196..6443 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(6212..6230) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" gene 6463..7819 /label=kanMX /note="yeast selectable marker conferring kanamycin resistance (Wach et al., 1994)" promoter 6463..6806 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 6807..7616 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin" /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(6874..6893) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" terminator 7622..7819 /label=TEF terminator /note="Ashbya gossypii TEF terminator" misc_feature 7831..7 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" ORIGIN 1 agcgatcgcg ctgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 61 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 661 agatcctttg atcttttcta cggggtggag cgcaaaaaac cccgcccctg acagggcggg 721 gttttttcgc gatcggagga tccttttttg tagaaatgtc ttggtgtcct cgtccaatca 781 ggtagccatc tctgaaatat ctggctccgt tgcaactccg aacgacctgc tggcaacgta 841 aaattctccg gggtaaaact taaatgtgga gtaatggaac cagaaacgtc tcttcccttc 901 tctctccttc caccgcccgt taccgtccct aggaaatttt actctgctgg agagcttctt 961 ctacggcccc cttgcagcaa tgctcttccc agcattacgt tgcgggtaaa acggaggtcg 1021 tgtacccgac ctagcagccc agggatggaa aagtcccggc cgtcgctggc aataatagcg 1081 ggcggacgca tgtcatgaga ttattggaaa ccaccagaat cgaatataaa aggcgaacac 1141 ctttcccaat tttggtttct cctgacccaa agactttaaa tttaatttat ttgtccctat 1201 ttcaatcaat tgaacaacta tcaaaacacc atggcgtctt cggatccctc gaggaagacg 1261 cgcttttagt gtacatctga taatatagta ctaccacgta tgataatgta gagaataggc 1321 ttccttgtcg agtgtgtttg cagttttctt gagtttcaag gtttaaatgc tggtatatta 1381 gttcatcgaa ggtttcagcc aatagcacct taaatcaatc aaactaattc gactcttacg 1441 aaagagccta ctgtgtttag tatcgaagtc gtttaccttt catgttgaat agcttcctct 1501 ctgaccctaa catttcaaga tcctcctaaa gttacccgga ttgtgaaatt ctaatgatcc 1561 acctgcccaa tgcatttttt ctttattcag tttacctttt ttacctaata tacgagcttg 1621 ttaaagtaag tggcactgca atactaggct tattgttgat attatgatga atcgttttca 1681 caaacttgat ttcctgtgaa ctcaccatgt actaaggaaa aaaacatgca tcaccatctg 1741 aatatttgac cgctccgggg agcctgccat taggcaaaca ccaactagga acctacacca 1801 agttatgaag gagcttggaa aggattataa aaaatgcgat tacctaatac acacacgatg 1861 cctgagcggt taatgtgaaa tttcccggcc atcgatcgcc ccacaatcag actattttcg 1921 ctcatacaat aaaaagtctc ctgtttccac ccgagtatca ccagaacaaa gtaacaaata 1981 caaaaccatg gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggttg 2041 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac 2101 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac 2161 ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg 2221 gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt 2281 ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat 2341 ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct 2401 gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct 2461 ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa 2521 cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga 2581 gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa 2641 atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt 2701 tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct 2761 ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct 2821 gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga 2881 cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag 2941 cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct 3001 tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg 3061 ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc 3121 catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct 3181 gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga 3241 actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga 3301 aaagattgag aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg 3361 aaattccaga ttcgcgtgga tgactcgcaa atcagaagag actatcactc cctggaactt 3421 cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt 3481 tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt 3541 cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc 3601 attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa 3661 agttaccgtg aaacagctca aagaggacta tttcaaaaag attgaatgtt tcgactctgt 3721 tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct 3781 gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga 3841 cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac 3901 ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg 3961 atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac 4021 aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca 4081 tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga 4141 cagccttcac gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact 4201 gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa 4261 tatcgttatc gagatggccc gagagaacca aactacccag aagggacaga agaacagtag 4321 ggaaaggatg aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga 4381 acacccagtt gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa 4441 cggcagggac atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt 4501 ggatcatatc gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac 4561 aagatccgat aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa 4621 aatgaaaaat tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga 4681 taatctgact aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa 4741 aaggcagctt gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg 4801 catgaacacc aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct 4861 gaagtctaag ctcgtctcag atttcagaaa ggactttcag ttttataagg tgagagagat 4921 caacaattac caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat 4981 caaaaaatat cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt 5041 taggaaaatg atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt 5101 ttacagcaat attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg 5161 gaagcgacca cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag 5221 ggatttcgcg acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac 5281 cgaagtacag accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa 5341 gctgatcgca cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac 5401 agtcgcttac agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa 5461 aagcgtcaag gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc 5521 catcgacttt ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct 5581 tcccaagtac tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg 5641 cgagctgcag aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct 5701 ggccagccac tatgaaaagc tcaaagggtc ccccgaagat aatgagcaga agcagctgtt 5761 cgtggaacaa cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa 5821 aagagtgatc ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag 5881 ggataagccc atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt 5941 gggcgcgcct gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc 6001 tacaaaggag gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac 6061 aagaatcgac ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa 6121 ggtgtgagct tcgtccgacg gcggcccgac gggtccgagg cctcggagat ccgtccccct 6181 tttcctttgt cgatatcatg taattagtta tgtcacgctt acattcacgc cctcccccca 6241 catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc cctatttatt 6301 tttttatagt tatgttagta ttaagaacgt tatttatatt tcaaattttt cttttttttc 6361 tgtacagacg cgtgtacgca tgtaacatta tactgaaaac cttgcttgag aaggttttgg 6421 gacgctcgaa ggctttaatt tgcaagctgg cgctaattca tggacatgga ggcccagaat 6481 accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac 6541 atttagccca tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg 6601 gcgcgaagca aaaattacgg ctcctcgctg cagacgtgcg agcagggaaa cgctcccctc 6661 acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa aggttaggat 6721 ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg atacagttct 6781 cacatcacat ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt tcgaggccgc 6841 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg 6901 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc 6961 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact 7021 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg 7081 catggttact caccactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc 7141 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga 7201 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat 7261 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc 7321 ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg 7381 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt 7441 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga 7501 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg 7561 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag 7621 tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat 7681 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat 7741 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg 7801 aatgctggtc gctatactgg cttccttttc atcacgtgct ataaaaataa ttataattta 7861 aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa 7921 aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc 7981 ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt 8041 agaacaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat 8101 atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga 8161 aattttttaa acctttgttt attttttttt cttcattccg taactcttct accttcttta 8221 tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca 8281 aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggca //