LOCUS Exported 7177 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION regulated dCas9 generator. ACCESSION . VERSION . KEYWORDS pdCas9_CL SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7177) AUTHORS Huang HH, Bellato M, Qian Y, Cardenas P, Pasotti L, Magni P, Del Vecchio D TITLE dCas9 regulator to neutralize competition in CRISPRi circuits. JOURNAL Nat Commun. 2021 Mar 16;12(1):1692. doi: 10.1038/s41467-021-21772-6. PUBMED 33727557 REFERENCE 2 (bases 1 to 7177) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7177 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 65..83 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" misc_RNA 159..234 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 289..310 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" promoter 329..357 /label=trc promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" RBS 395..403 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 409..4515 /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" terminator 4539..4610 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4626..4653 /label=T7Te terminator /note="phage T7 early transcription terminator" misc_feature 4660..4680 /label=BioBrick suffix /note="universal suffix for all parts" CDS complement(4820..5635) /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" primer_bind 5543..5562 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5627..5646 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin complement(6230..6774) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(6309..6328) /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 agtctattcc atggtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 61 taaaaatagg cgtatcacga ggcagggcgc gccaaaaaga gtattgactt cgcatctttt 121 tgtacctata atagattcag tattgactat taatcatcgt tttagagcta gaaatagcaa 181 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 241 tccaattatt gaacacccta acgggtgttt tttttttttt ggtctcccga attcgcggcc 301 gcttctagag ggcgcgccaa aaagagtatt gactattaat catccggctc gtataataga 361 ttcactctag acgagaggaa acgcggtttt aatataagga ggttattaat ggataagaaa 421 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa 481 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa 541 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc 601 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag 661 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct 721 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat 781 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat 841 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt 901 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta 961 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt 1021 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat 1081 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg 1141 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta 1201 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat 1261 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat 1321 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc 1381 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca 1441 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat 1501 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat 1561 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc 1621 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga 1681 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg 1741 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg 1801 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa 1861 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat 1921 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg 1981 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag 2041 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta 2101 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa 2161 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa 2221 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc 2281 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat 2341 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga 2401 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa 2461 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt 2521 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt 2581 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt 2641 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca 2701 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc 2761 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact 2821 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg 2881 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa 2941 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt 3001 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga 3061 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa 3121 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact 3181 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat 3241 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct 3301 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc 3361 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt 3421 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag 3481 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac 3541 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa 3601 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc 3661 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga 3721 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa 3781 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta 3841 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta 3901 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct 3961 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt 4021 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat 4081 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag 4141 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat 4201 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat 4261 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa 4321 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt 4381 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat 4441 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag 4501 ctaggaggtg actaactcga gtaaggatct ccaggcatca aataaaacga aaggctcagt 4561 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc 4621 acactggctc accttcgggt gggcctttct gcgtttatat actagtagcg gccgctgcag 4681 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 4741 cactcaaagg cggtaatctc gagtcccgtc aagtcagcgt aatgctctgc cagtgttaca 4801 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat 4861 tcatatcagg attatcgata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 4921 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 4981 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 5041 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc 5101 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 5161 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 5221 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 5281 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 5341 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 5401 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 5461 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 5521 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 5581 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac 5641 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat 5701 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa 5761 cttttgctga gttgaaggat cagatcacgc atcttcccga caacgcagac cgttccgtgg 5821 caaagcaaaa gttcaaaatc accaactggt ccacctacaa caaagctctc atcaaccgtg 5881 gctccctcac tttctggctg gatgatgggg cgattcaggc ctggtatgag tcagcaacac 5941 cttcttcacg aggcagacct cagcgctagc ggagtgtata ctggcttact atgttggcac 6001 tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt 6061 cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg 6121 gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat 6181 gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc 6241 tccgccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac 6301 aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc 6361 tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc 6421 tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg 6481 ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac 6541 atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag 6601 tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc 6661 cagttacctc ggttcaaaga gttggtagct cagagaacct tcaaaaaacc gccctgcaag 6721 gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat 6781 catcttatta aggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 6841 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 6901 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 6961 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 7021 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 7081 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 7141 cgcagaagtg gtcctgcaac tttatccgcc tccatcc //