LOCUS Exported 6147 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION BB1 encoding a FLAG tagged dCAS9 with a SV40 NLS. ACCESSION . VERSION . KEYWORDS BB1_23_dCAS9(3xFLAG_SV40NLS) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6147) AUTHORS Baumschabl M, Prielhofer R, Mattanovich D, Steiger MG TITLE Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds. JOURNAL ACS Synth Biol. 2020 Dec 18;9(12):3202-3209. doi: 10.1021/acssynbio.0c00214. Epub 2020 Nov 12. PUBMED 33180466 REFERENCE 2 (bases 1 to 6147) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6147 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 610..629 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(703..805) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" terminator 806..837 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" primer_bind 885..901 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 885..901 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 988..1053 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1057..5160 /codon_start=1 /product="catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=dCas9 /note="RNA-guided DNA-binding protein that lacks endonuclease activity due to the D10A mutation in the RuvC catalytic domain and the H840A mutation in the HNH catalytic domain" /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 5173..5193 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" primer_bind complement(5327..5344) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5327..5343) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 5384..5971 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5872..5891 /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 5983..6010 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(6034..702) /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="MSHIQRETSCLKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" ORIGIN 1 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat 61 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt 121 gacgactgaa tccggtgaga atggcaagag tttatgcatt tctttccaga cttgttcaac 181 aggccagcca ttacgctcgt catcaaaatc actcgcacca accaaaccgt tattcattcg 241 tgattgcgcc tgagcgagac gaaatacgcg atcgccgtta aaaggacaat tacaaacagg 301 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc 361 aggatattct tctaatacct ggaatgctgt tttccctggg atcgcagtgg tgagtaacca 421 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag 481 ccagtttagc ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt 541 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg 601 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa 661 tcgcggcttc aagcaagacg tttcccgttg aatatggctc attttagctt ccttagctcc 721 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa 781 gttggaacct cttacgtgcc gatcaagtca aaagcctccg gtcggaggct tttgactttc 841 tgctatggag gtcaggtatg atttaaatgg tcagtattga gcctcaggaa acagctatga 901 catcaagctg actagataat ctagctgatc gtggaccgat catacgtata atgccgtaag 961 atcacgcgcg aattcgaaga cgccatggac tacaaagacc atgacggtga ttataaagat 1021 catgacatcg attacaagga tgacgatgac aagctcatgg acaagaagta ctccattggg 1081 ctcgctatcg gcacaaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg 1141 agcaaaaaat tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt 1201 ggcgccctcc tgttcgactc cggggagacg gccgaagcca cgcggctcaa aagaacagca 1261 cggcgcagat atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat 1321 gagatggcta aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag 1381 gaggataaaa agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac 1441 catgaaaagt acccaaccat atatcatctg aggaagaagc ttgtagacag tactgataag 1501 gctgacttgc ggttgatcta tctcgcgctg gcgcatatga tcaaatttcg gggacacttc 1561 ctcatcgagg gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg 1621 gttcagactt acaatcagct tttcgaagag aacccgatca acgcatccgg agttgacgcc 1681 aaagcaatcc tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag 1741 ctccctgggg agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg 1801 acccccaact ttaaatctaa cttcgacctg gccgaagatg ccaagcttca actgagcaaa 1861 gacacctacg atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac 1921 ctttttttgg cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg 1981 aacacggaga tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac 2041 caccaagact tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag 2101 gaaattttct tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc 2161 caggaggaat tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag 2221 ctgctggtaa agcttaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga 2281 agcatccccc accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat 2341 ttctacccct ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata 2401 ccctactatg taggccccct cgcccgggga aattccagat tcgcgtggat gactcgcaaa 2461 tcagaagaga ctatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc 2521 cagtccttca tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt 2581 cctaaacact ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa 2641 tacgtcacag aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc 2701 gtggacctcc tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaggactat 2761 ttcaaaaaga ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac 2821 gcatccctgg gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac 2881 aatgaggaga acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat 2941 agggagatga ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg 3001 aaacagctca agaggcgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat 3061 gggatccgag acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt 3121 gccaacagga acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc 3181 cagaaagcac aagtttctgg ccagggggac agtctgcacg agcacatcgc taatcttgca 3241 ggtagcccag ctatcaaaaa gggaatactg cagaccgtta aggtcgtgga tgaactcgtc 3301 aaagtaatgg gaaggcataa gcccgagaat atcgttatcg agatggcccg agagaaccaa 3361 actacccaga agggacagaa gaacagtagg gaaaggatga agaggattga agagggtata 3421 aaagaactgg ggtcccaaat ccttaaggaa cacccagttg aaaacaccca gcttcagaat 3481 gagaagctct acctgtacta cctgcagaac ggcagggaca tgtacgtgga tcaggaactg 3541 gacatcaatc ggctctccga ctacgacgtg gatgccatcg tgccccagtc ttttctcaaa 3601 gatgattcta ttgataataa agtgttgaca agatccgata aaaatagagg gaagagtgat 3661 aacgtcccct cagaagaagt tgtcaagaaa atgaaaaatt attggcggca gctgctgaac 3721 gccaaactga tcacacaacg gaagttcgat aatctgacta aggctgaacg aggtggcctg 3781 tctgagttgg ataaagccgg cttcatcaaa aggcagcttg ttgagacacg ccagatcacc 3841 aagcacgtgg cccaaattct cgattcacgc atgaacacca agtacgatga aaatgacaaa 3901 ctgattcgag aggtgaaagt tattactctg aagtctaagc tagtctcaga tttcagaaag 3961 gactttcagt tttataaggt gagagagatc aacaattacc accatgcgca tgatgcctac 4021 ctgaatgcag tggtaggcac tgcacttatc aaaaaatatc ccaagcttga atctgaattt 4081 gtttacggag actataaagt gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa 4141 ataggcaagg ccaccgctaa gtacttcttt tacagcaata ttatgaattt tttcaagacc 4201 gagattacac tggccaatgg agagattcgg aagcgaccac ttatcgaaac aaacggagaa 4261 acaggagaaa tcgtgtggga caagggtagg gatttcgcga cagtccggaa ggtcctgtcc 4321 atgccgcagg tgaacatcgt taaaaagacc gaagtacaga ccggaggctt ctccaaggaa 4381 agtatcctcc cgaaaaggaa cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc 4441 aagaaatacg gcggattcga ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa 4501 gtggagaaag ggaagtctaa aaaactcaaa agcgtcaagg aactgctggg catcacaatc 4561 atggagcgat caagcttcga aaaaaacccc atcgactttc tcgaggcgaa aggatataaa 4621 gaggtcaaaa aagacctcat cattaagctt cccaagtact ctctctttga gcttgaaaac 4681 ggccggaaac gaatgctcgc tagtgcgggc gagctgcaga aaggtaacga gctggcactg 4741 ccctctaaat acgttaattt cttgtatctg gccagccact atgaaaagct caaagggtcc 4801 cccgaagata atgagcagaa gcagctgttc gtggaacaac acaaacacta ccttgatgag 4861 atcatcgagc aaataagcga attctccaaa agagtgatcc tcgccgacgc taacctcgat 4921 aaggtgcttt ctgcttacaa taagcacagg gataagccca tcagggagca ggcagaaaac 4981 attatccact tgtttactct gaccaacttg ggcgctcctg cagccttcaa gtacttcgac 5041 accaccatag acagaaagcg gtacacctct acaaaggagg tcctggacgc cacactgatt 5101 catcagtcaa ttacggggct ctatgaaaca agaatcgacc tctctcagct cggtggagac 5161 agcagggctg accccaagaa gaagaggaag gtgtgagctt gcgtcttcga attcgaggcc 5221 gcatatcatt caggacgagc ctcagactcc agcgtaactg gactgcaatc aactcactgg 5281 ctcaccttcc ggtccacgat cagctagaat caagctgact agataaactg gccgtcgttt 5341 tacacgggtg ggcctttctt cggtagaaaa tcaaaggatc ttcttgagat cctttttttc 5401 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 5461 cggatcaaga gctaccaact ctttttccga ggtaactggc ttcagcagag cgcagatacc 5521 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 5581 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 5641 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 5701 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 5761 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 5821 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 5881 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcatc gatttttgtg 5941 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcagaaa ggcccacccg 6001 aaggtgagcc aggtgattac atttaggtcc tcattagaaa aactcatcga gcatcaagtg 6061 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg 6121 taatgaagga gaaaactcac cgaggca //