LOCUS Exported 6211 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION MoClo golden gate assembly CD part for dCas9 (Catalytically-inactive S. pyogenes Cas9, for gRNA-targeted repression). Please see Supplemental Documents for annotated Genbank file.. ACCESSION . VERSION . KEYWORDS C41m SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6211) TITLE CIDAR MoClo Extension REFERENCE 2 (bases 1 to 6211) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6211 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 2..4108 /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" misc_feature 4119..4139 /label=BioBrick suffix /note="universal suffix for all parts" primer_bind complement(4145..4162) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4316..4904) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4396..4415) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5075..5935) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5698..5717 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5936..6040) /gene="bla" /label=AmpR promoter primer_bind 6108..6126 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 6130..6173 /label=bacterial terminator /note="putative bacterial transcription terminator" misc_feature 6176..6197 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" ORIGIN 1 aatggataag aaatactcaa taggcttagc tatcggcaca aatagcgtcg gatgggcggt 61 gatcactgat gaatataagg ttccgtctaa aaagttcaag gttctgggaa atacagaccg 121 ccacagtatc aaaaaaaatc ttataggggc tcttttattt gacagtggag agacagcgga 181 agcgactcgt ctcaaacgga cagctcgtag aaggtataca cgtcggaaga atcgtatttg 241 ttatctacag gagatttttt caaatgagat ggcgaaagta gatgatagtt tctttcatcg 301 acttgaagag tcttttttgg tggaagaaga caagaagcat gaacgtcatc ctatttttgg 361 aaatatagta gatgaagttg cttatcatga gaaatatcca actatctatc atctgcgaaa 421 aaaattggta gattctactg ataaagcgga tttgcgctta atctatttgg ccttagcgca 481 tatgattaag tttcgtggtc attttttgat tgagggagat ttaaatcctg ataatagtga 541 tgtggacaaa ctatttatcc agttggtaca aacctacaat caattatttg aagaaaaccc 601 tattaacgca agtggagtag atgctaaagc gattctttct gcacgattga gtaaatcaag 661 acgattagaa aatctcattg ctcagctccc cggtgagaag aaaaatggct tatttgggaa 721 tctcattgct ttgtcattgg gtttgacccc taattttaaa tcaaattttg atttggcaga 781 agatgctaaa ttacagcttt caaaagatac ttacgatgat gatttagata atttattggc 841 gcaaattgga gatcaatatg ctgatttgtt tttggcagct aagaatttat cagatgctat 901 tttactttca gatatcctaa gagtaaatac tgaaataact aaggctcccc tatcagcttc 961 aatgattaaa cgctacgatg aacatcatca agacttgact cttttaaaag ctttagttcg 1021 acaacaactt ccagaaaagt ataaagaaat cttttttgat caatcaaaaa acggatatgc 1081 aggttatatt gatgggggag ctagccaaga agaattttat aaatttatca aaccaatttt 1141 agaaaaaatg gatggtactg aggaattatt ggtgaaacta aatcgtgaag atttgctgcg 1201 caagcaacgg acctttgaca acggctctat tccccatcaa attcacttgg gtgagctgca 1261 tgctattttg agaagacaag aagactttta tccattttta aaagacaatc gtgagaagat 1321 tgaaaaaatc ttgacttttc gaattcctta ttatgttggt ccattggcgc gtggcaatag 1381 tcgttttgca tggatgactc ggaagtctga agaaacaatt accccatgga attttgaaga 1441 agttgtcgat aaaggtgctt cagctcaatc atttattgaa cgcatgacaa actttgataa 1501 aaatcttcca aatgaaaaag tactaccaaa acatagtttg ctttatgagt attttacggt 1561 ttataacgaa ttgacaaagg tcaaatatgt tactgaagga atgcgaaaac cagcatttct 1621 ttcaggtgaa cagaagaaag ccattgttga tttactcttc aaaacaaatc gaaaagtaac 1681 cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa tgttttgata gtgttgaaat 1741 ttcaggagtt gaagatagat ttaatgcttc attaggtacc taccatgatt tgctaaaaat 1801 tattaaagat aaagattttt tggataatga agaaaatgaa gatatcttag aggatattgt 1861 tttaacattg accttatttg aagataggga gatgattgag gaaagactta aaacatatgc 1921 tcacctcttt gatgataagg tgatgaaaca gcttaaacgt cgccgttata ctggttgggg 1981 acgtttgtct cgaaaattga ttaatggtat tagggataag caatctggca aaacaatatt 2041 agattttttg aaatcagatg gttttgccaa tcgcaatttt atgcagctga tccatgatga 2101 tagtttgaca tttaaagaag acattcaaaa agcacaagtg tctggacaag gcgatagttt 2161 acatgaacat attgcaaatt tagctggtag ccctgctatt aaaaaaggta ttttacagac 2221 tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg cataagccag aaaatatcgt 2281 tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc cagaaaaatt cgcgagagcg 2341 tatgaaacga atcgaagaag gtatcaaaga attaggaagt cagattctta aagagcatcc 2401 tgttgaaaat actcaattgc aaaatgaaaa gctctatctc tattatctcc aaaatggaag 2461 agacatgtat gtggaccaag aattagatat taatcgttta agtgattatg atgtcgatgc 2521 cattgttcca caaagtttcc ttaaagacga ttcaatagac aataaggtct taacgcgttc 2581 tgataaaaat cgtggtaaat cggataacgt tccaagtgaa gaagtagtca aaaagatgaa 2641 aaactattgg agacaacttc taaacgccaa gttaatcact caacgtaagt ttgataattt 2701 aacgaaagct gaacgtggag gtttgagtga acttgataaa gctggtttta tcaaacgcca 2761 attggttgaa actcgccaaa tcactaagca tgtggcacaa attttggata gtcgcatgaa 2821 tactaaatac gatgaaaatg ataaacttat tcgagaggtt aaagtgatta ccttaaaatc 2881 taaattagtt tctgacttcc gaaaagattt ccaattctat aaagtacgtg agattaacaa 2941 ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt ggaactgctt tgattaagaa 3001 atatccaaaa cttgaatcgg agtttgtcta tggtgattat aaagtttatg atgttcgtaa 3061 aatgattgct aagtctgagc aagaaatagg caaagcaacc gcaaaatatt tcttttactc 3121 taatatcatg aacttcttca aaacagaaat tacacttgca aatggagaga ttcgcaaacg 3181 ccctctaatc gaaactaatg gggaaactgg agaaattgtc tgggataaag ggcgagattt 3241 tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat attgtcaaga aaacagaagt 3301 acagacaggc ggattctcca aggagtcaat tttaccaaaa agaaattcgg acaagcttat 3361 tgctcgtaaa aaagactggg atccaaaaaa atatggtggt tttgatagtc caacggtagc 3421 ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa tcgaagaagt taaaatccgt 3481 taaagagtta ctagggatca caattatgga aagaagttcc tttgaaaaaa atccgattga 3541 ctttttagaa gctaaaggat ataaggaagt taaaaaagac ttaatcatta aactacctaa 3601 atatagtctt tttgagttag aaaacggtcg taaacggatg ctggctagtg ccggagaatt 3661 acaaaaagga aatgagctgg ctctgccaag caaatatgtg aattttttat atttagctag 3721 tcattatgaa aagttgaagg gtagtccaga agataacgaa caaaaacaat tgtttgtgga 3781 gcagcataag cattatttag atgagattat tgagcaaatc agtgaatttt ctaagcgtgt 3841 tattttagca gatgccaatt tagataaagt tcttagtgca tataacaaac atagagacaa 3901 accaatacgt gaacaagcag aaaatattat tcatttattt acgttgacga atcttggagc 3961 tcccgctgct tttaaatatt ttgatacaac aattgatcgt aaacgatata cgtctacaaa 4021 agaagtttta gatgccactc ttatccatca atccatcact ggtctttatg aaacacgcat 4081 tgatttgagt cagctaggag gtgactaagg tagagaccta ctagtagcgg ccgctgcagg 4141 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 4201 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 4261 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 4321 acaggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 4381 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 4441 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 4501 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 4561 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 4621 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 4681 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 4741 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 4801 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 4861 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 4921 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 4981 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 5041 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 5101 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 5161 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgcgacc 5221 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 5281 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 5341 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 5401 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 5461 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 5521 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 5581 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 5641 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 5701 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 5761 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 5821 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 5881 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 5941 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 6001 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 6061 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 6121 cgaggcagaa tttcagataa aaaaaatcct tagctttcgc taaggatgat ttctggaatt 6181 cgcggccgct tctagagact agtgggtctc a //