LOCUS       Exported                8062 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  AraC-pBAD-dCas9, MCP-SoxS_R93A.
ACCESSION   .
VERSION     .
KEYWORDS    pCD227
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8062)
  AUTHORS   Dong C, Fontana J, Patel A, Carothers JM, Zalatan JG
  TITLE     Synthetic CRISPR-Cas gene activators for transcriptional 
            reprogramming in bacteria.
  JOURNAL   Nat Commun. 2018 Jun 27;9(1):2489. doi: 10.1038/s41467-018-04901-6.
  PUBMED    29950558
REFERENCE   2  (bases 1 to 8062)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8062
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(12..890)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        917..1201
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1124..1143
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             1231..1242
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1249..5355
                     /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      5379..5450
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5466..5493
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             5590..5601
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(6369..6386)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6503..7048)
                     /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(6583..6602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      7162..7256
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(7277..7936)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7851..7870
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7937..8039)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gttatgacaa cttgacggct acatcattca ctttttcttc acaaccggca
       61 cggaactcgc tcgggctggc cccggtgcat tttttaaata cccgcgagaa atagagttga
      121 tcgtcaaaac caacattgcg accgacggtg gcgataggca tccgggtggt gctcaaaagc
      181 agcttcgcct ggctgatacg ttggtcctcg cgccagctta agacgctaat ccctaactgc
      241 tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa catgctgtgc gacgctggcg
      301 atatcaaaat tgctgtctgc caggtgatcg ctgatgtact gacaagcctc gcgtacccga
      361 ttatccatcg gtggatggag cgactcgtta atcgcttcca tgcgccgcag taacaattgc
      421 tcaagcagat ttatcgccag cagctccgaa tagcgccctt ccccttgccc ggcgttaatg
      481 atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt catccgggcg aaagaacccc
      541 gtattggcaa atattgacgg ccagttaagc cattcatgcc agtaggcgcg cggacgaaag
      601 taaacccact ggtgatacca ttcgcgagcc tccggatgac gaccgtagtg atgaatctct
      661 cctggcggga acagcaaaat atcacccggt cggcaaacaa attctcgtcc ctgatttttc
      721 accaccccct gaccgcgaat ggtgagattg agaatataac ctttcattcc cagcggtcgg
      781 tcgataaaaa aatcgagata accgttggcc tcaatcggcg ttaaacccgc caccagatgg
      841 gcattaaacg agtatcccgg cagcagggga tcattttgcg cttcagccat acttttcata
      901 ctcccgccat tcagagaaga aaccaattgt ccatattgca tcagacattg ccgtcactgc
      961 gtcttttact ggctcttctc gctaaccaaa ccggtaaccc cgcttattaa aagcattctg
     1021 taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca aaagtgtcta taatcacggc
     1081 agaaaagtcc acattgatta tttgcacggc gtcacacttt gctatgccat agcattttta
     1141 tccataagat tagcggattc tacctgacgc tttttatcgc aactctctac tgtttctcca
     1201 tacccgtttt tttgggaatt caaaagatct aaagaggaga aaggatctat ggataagaaa
     1261 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
     1321 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
     1381 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
     1441 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
     1501 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
     1561 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
     1621 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
     1681 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
     1741 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
     1801 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     1861 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     1921 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     1981 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     2041 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     2101 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     2161 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     2221 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     2281 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     2341 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     2401 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     2461 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     2521 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     2581 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     2641 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     2701 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     2761 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     2821 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     2881 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     2941 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
     3001 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     3061 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     3121 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     3181 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     3241 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     3301 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     3361 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     3421 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     3481 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     3541 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     3601 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     3661 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     3721 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
     3781 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     3841 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     3901 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     3961 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     4021 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     4081 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     4141 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     4201 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     4261 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     4321 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     4381 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     4441 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     4501 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     4561 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     4621 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     4681 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     4741 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     4801 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     4861 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     4921 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     4981 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     5041 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     5101 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     5161 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     5221 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     5281 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     5341 ctaggaggtg actaactcga gtaaggatct ccaggcatca aataaaacga aaggctcagt
     5401 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     5461 acactggctc accttcgggt gggcctttct gcgtttatac ctagagcgcc cggaagagag
     5521 tcaattcagg gtggtgaatc tgcagtttac ggctagctca gccctaggta ttatgctagc
     5581 gaattcatta aagaggagaa aggtaccatg gggcccgctt ctaactttac tcagttcgtt
     5641 ctcgtcgaca atggcggaac tggcgacgtg actgtcgccc caagcaactt cgctaacggg
     5701 atcgctgaat ggatcagctc taactcgcgt tcacaggctt acaaagtaac ctgtagcgtt
     5761 cgtcagagct ctgcgcagaa tcgcaaatac accatcaaag tcgaggtgcc taaaggcgcc
     5821 tggcgttcgt acttaaatat ggaactaacc attccaattt tcgccacgaa ttccgactgc
     5881 gagcttattg ttaaggcaat gcaaggtctc ctaaaagatg gaaacccgat tccctcagca
     5941 atcgcagcaa actccggcat ctacggtggc ggaggtagca tgtcccatca gaaaattatt
     6001 caggatctta tcgcatggat tgacgagcat attgaccagc cgcttaacat tgatgtagtc
     6061 gcaaaaaaat caggctattc aaagtggtac ttgcaacgaa tgttccgcac ggtgacgcat
     6121 cagacgcttg gcgattacat tcgccaacgc cgcctgttac tggccgccgt tgagttgcgc
     6181 accaccgagc gtccgatttt tgatatcgca atggacctgg gttatgtctc gcagcagacc
     6241 ttctcccgcg ttttcgcgcg gcagtttgat cgcactccca gcgattatcg ccaccgcctg
     6301 taagcggccg ccacgcaaaa aaccccgctt cggcggggtt ttttcgccct agggatatat
     6361 tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg
     6421 gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg
     6481 agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa
     6541 tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6601 cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc
     6661 cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt
     6721 cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc
     6781 ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc
     6841 actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga
     6901 aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt
     6961 agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga
     7021 gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt
     7081 ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata
     7141 taagttgtta ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt
     7201 tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag
     7261 cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat
     7321 taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg
     7381 gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga
     7441 agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg
     7501 agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac
     7561 acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc
     7621 agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat
     7681 cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca
     7741 ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct
     7801 ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact
     7861 gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag
     7921 tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata
     7981 cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa
     8041 cgtctcattt tcgccagata tc
//
