LOCUS Exported 7830 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION use as an auxiliary temperature-sensitive plasmid to successfully transform pdCas9_CL plasmid. ACCESSION . VERSION . KEYWORDS pAUX_OL SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7830) 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 7830) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7830 /organism="synthetic DNA construct" /mol_type="other DNA" misc_RNA 81..156 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 211..232 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" RBS 276..287 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 294..4400 /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 4424..4495 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4511..4538 /label=T7Te terminator /note="phage T7 early transcription terminator" terminator 4556..4800 /label=-lambda tL3 terminator /note="transcription terminator tL3 from phage lambda" CDS complement(4923..5873) /codon_start=1 /gene="rep101 encoding an A56V mutation" /product="temperature-sensitive version of the RepA protein needed for replication with the pSC101 origin (Armstrong et al., 1984)" /label=Rep101(Ts) /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 5921..6143 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" CDS complement(6765..7625) /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 7388..7407 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7626..7730) /gene="bla" /label=AmpR promoter primer_bind 7817..5 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 gaggcagggc gcgccaaaaa gagtattgac ttcgcatctt tttgtaccta taatagattc 61 agtattgact attaatcatc gttttagagc tagaaatagc aagttaaaat aaggctagtc 121 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttccaatta ttgaacaccc 181 taacgggtgt tttttttttt ttggtctccc gaattcgcgg ccgcttctag agttgacagc 241 tagctcagtc ctagggacta tgctagctac tagagaaaga ggagaaagga tctatggata 301 agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg gtgatcactg 361 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta 421 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc 481 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac 541 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag 601 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag 661 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg 721 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta 781 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca 841 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg 901 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag 961 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg 1021 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta 1081 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg 1141 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt 1201 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta 1261 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac 1321 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata 1381 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa 1441 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac 1501 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt 1561 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa 1621 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg 1681 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg 1741 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc 1801 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg 1861 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg 1921 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc 1981 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag 2041 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag 2101 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat 2161 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct 2221 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt 2281 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt 2341 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga 2401 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac 2461 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag 2521 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa 2581 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac 2641 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa 2701 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt 2761 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat gccattgttc 2821 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa 2881 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt 2941 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag 3001 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg 3061 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat 3121 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag 3181 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc 3241 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa 3301 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg 3361 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca 3421 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa 3481 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag 3541 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag 3601 gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta 3661 aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag 3721 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt 3781 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag 3841 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc 3901 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag 3961 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg 4021 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata 4081 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag 4141 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac 4201 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg 4261 cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt 4321 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga 4381 gtcagctagg aggtgactaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc 4441 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta 4501 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataaatggc gatgacgcat 4561 cctcacgata atatccgggt aggcgcaatc actttcgtct actccgttac aaagcgaggc 4621 tgggtatttc ccggcctttc tgttatccga aatccactga aagcacagcg gctggctgag 4681 gagataaata ataaacgagg ggctgtatgc acaaagcatc ttctgttgag ttaagaacga 4741 gtatcgagat ggcacatagc cttgctcaaa ttggaatcag gtttgtgcca ataccagtag 4801 aaacagacga agaatccatg ggtatggaca gttttccctt tgatatgtaa cggtgaacag 4861 ttgttctact tttgtttgtt agtcttgatg cttcactgat agatacaaga gccataagaa 4921 cctcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg tttttgcgtg 4981 agccatgaga acgaaccatt gagatcatac ttactttgca tgtcactcaa aaattttgcc 5041 tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt tcttagtccg 5101 ttatgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt catttttatc 5161 tggttgttct caagttcggt tacgagatcc atttgtctat ctagttcaac ttggaaaatc 5221 aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct gtaagtgttt 5281 aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc atggtagtta 5341 ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt tgccttgtga 5401 gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga gtatttgttt 5461 tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg gaaaagataa 5521 ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt ggcatagttt 5581 gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac agttctcgtc 5641 atcagctctc tggttgcttt agctaataca ccataagcat tttccctact gatgttcatc 5701 atctgaacgt attggttata agtgaacgat accgtccgtt ctttccttgt agggttttca 5761 atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg ctccgttaag 5821 tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga catacatctc 5881 aattggtcta ggtgatttta atcactatac caattgagat gggctagtca atgataatta 5941 ctagtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt tgctggaaaa 6001 cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt tttttttgtt 6061 tatattcaag tggttataat ttatagaata aagaaagaat aaaaaaagat aaaaagaata 6121 gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta caaaaggatg 6181 tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg cttaagtagc 6241 accctcgcaa gctcggttgc ggccgcaatc gggcaaatcg ctgaatattc cttttgtctc 6301 cgaccatcag gcacctgagt cgctgtcttt ttcgtgacat tcagttcgct gcgctcacgg 6361 ctctggcagt gaatgggggt aaatggcact acaggcgcct tttatggatt catgcaagga 6421 aactacccat aatacaagaa aagcccgtca cgggcttctc agggcgtttt atggcgggtc 6481 tgctatgtgg tgctatctga ctttttgctg ttcagcagtt cctgccctct gattttccag 6541 tctgaccact tcggattatc ccgtgacagg tcattcagac tggctaatgc acccagtaag 6601 gcagcggtat catcaacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6661 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6721 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 6781 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6841 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6901 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6961 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 7021 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 7081 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 7141 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 7201 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 7261 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 7321 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 7381 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 7441 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 7501 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 7561 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7621 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 7681 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 7741 cgaaaagtgc cacctgcatc gatttatgcc acctgacgtc taagaaacca ttattatcat 7801 gacattaacc tataaaaata ggcgtatcac //