LOCUS       Exported                8033 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Sp.pCas9-dCas9, pTet-MCP-SoxS_R93A.
ACCESSION   .
VERSION     .
KEYWORDS    pJF093
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8033)
  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 8033)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8033
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      3705..3776
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3792..3819
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3872..4495)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     4403..4422
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        4514..4569
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    4550..4568
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             4581..4592
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(5360..5377)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5494..6039)
                     /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(5574..5593)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      6153..6247
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6268..6927)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6842..6861
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6928..7030)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_RNA        complement(7234..7312)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             7608..3681
                     /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"
ORIGIN
        1 gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc gcatatgatt
       61 aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag tgatgtggac
      121 aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa ccctattaac
      181 gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc aagacgatta
      241 gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg gaatctcatt
      301 gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc agaagatgct
      361 aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt ggcgcaaatt
      421 ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc tattttactt
      481 tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc ttcaatgatt
      541 aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt tcgacaacaa
      601 cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata tgcaggttat
      661 attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat tttagaaaaa
      721 atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa
      781 cggacctttg acaacggctc tattccccat caaattcact tgggtgagct gcatgctatt
      841 ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa
      901 atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt
      961 gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga agaagttgtc
     1021 gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga taaaaatctt
     1081 ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac ggtttataac
     1141 gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt
     1201 gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag
     1261 caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga aatttcagga
     1321 gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa aattattaaa
     1381 gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat tgttttaaca
     1441 ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata tgctcacctc
     1501 tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg gggacgtttg
     1561 tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat attagatttt
     1621 ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga tgatagtttg
     1681 acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag tttacatgaa
     1741 catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca gactgtaaaa
     1801 gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat cgttattgaa
     1861 atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa
     1921 cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca tcctgttgaa
     1981 aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg aagagacatg
     2041 tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga tgccattgtt
     2101 ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg ttctgataaa
     2161 aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat gaaaaactat
     2221 tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa tttaacgaaa
     2281 gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg ccaattggtt
     2341 gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat gaatactaaa
     2401 tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa atctaaatta
     2461 gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa caattaccat
     2521 catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa gaaatatcca
     2581 aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg taaaatgatt
     2641 gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta ctctaatatc
     2701 atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa acgccctcta
     2761 atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga ttttgccaca
     2821 gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga agtacagaca
     2881 ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct tattgctcgt
     2941 aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt agcttattca
     3001 gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag
     3061 ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat tgacttttta
     3121 gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc taaatatagt
     3181 ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga attacaaaaa
     3241 ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc tagtcattat
     3301 gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt ggagcagcat
     3361 aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg tgttatttta
     3421 gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga caaaccaata
     3481 cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg agctcccgct
     3541 gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac aaaagaagtt
     3601 ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg cattgatttg
     3661 agtcagctag gaggtgacta actcgagtaa ggatctccag gcatcaaata aaacgaaagg
     3721 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     3781 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatacctag agcgcccgga
     3841 agagagtcaa ttcagggtgg tgaatctgca gttaagaccc actttcacat ttaagttgtt
     3901 tttctaatcc gcatatgatc aattcaaggc cgaataagaa ggctggctct gcaccttggt
     3961 gatcaaataa ttcgatagct tgtcgtaata atggcggcat actatcagta gtaggtgttt
     4021 ccctttcttc tttagcgact tgatgctctt gatcttccaa tacgcaacct aaagtaaaat
     4081 gccccacagc gctgagtgca tataatgcat tctctagtga aaaaccttgt tggcataaaa
     4141 aggctaattg attttcgaga gtttcatact gtttttctgt aggccgtgta cctaaatgta
     4201 cttttgctcc atcgcgatga cttagtaaag cacatctaaa acttttagcg ttattacgta
     4261 aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc ctatctaaca
     4321 tctcaatggc taaggcgtcg agcaaagccc gcttattttt tacatgccaa tacaatgtag
     4381 gctgctctac acctagcttc tgggcgagtt tacgggttgt taaaccttcg attccgacct
     4441 cattaagcag ctctaatgcg ctgttaatca ctttactttt atctaatcta gacatcatta
     4501 attcctaatt tttgttgaca ctctatcgtt gatagagtta ttttaccact ccctatcagt
     4561 gatagagaaa agaattcatt aaagaggaga aaggtaccat ggggcccgct tctaacttta
     4621 ctcagttcgt tctcgtcgac aatggcggaa ctggcgacgt gactgtcgcc ccaagcaact
     4681 tcgctaacgg gatcgctgaa tggatcagct ctaactcgcg ttcacaggct tacaaagtaa
     4741 cctgtagcgt tcgtcagagc tctgcgcaga atcgcaaata caccatcaaa gtcgaggtgc
     4801 ctaaaggcgc ctggcgttcg tacttaaata tggaactaac cattccaatt ttcgccacga
     4861 attccgactg cgagcttatt gttaaggcaa tgcaaggtct cctaaaagat ggaaacccga
     4921 ttccctcagc aatcgcagca aactccggca tctacggtgg cggaggtagc atgtcccatc
     4981 agaaaattat tcaggatctt atcgcatgga ttgacgagca tattgaccag ccgcttaaca
     5041 ttgatgtagt cgcaaaaaaa tcaggctatt caaagtggta cttgcaacga atgttccgca
     5101 cggtgacgca tcagacgctt ggcgattaca ttcgccaacg ccgcctgtta ctggccgccg
     5161 ttgagttgcg caccaccgag cgtccgattt ttgatatcgc aatggacctg ggttatgtct
     5221 cgcagcagac cttctcccgc gttttcgcgc ggcagtttga tcgcactccc agcgattatc
     5281 gccaccgcct gtaagcggcc gccacgcaaa aaaccccgct tcggcggggt tttttcgccc
     5341 tagggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc
     5401 gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta
     5461 acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa
     5521 gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata
     5581 ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac
     5641 cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg
     5701 ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct
     5761 gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac
     5821 tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa
     5881 ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc
     5941 aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt
     6001 tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag
     6061 ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     6121 cccatacgat ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg
     6181 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     6241 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg
     6301 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     6361 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     6421 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     6481 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     6541 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     6601 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     6661 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     6721 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     6781 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     6841 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     6901 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     6961 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     7021 gtgccgatca acgtctcatt ttcgccagat atcgacgtct taagttacga aatcatcctg
     7081 tggagcttag taggtttagc aagatggcag cgcctaaatg tagaatgata aaaggattaa
     7141 gagattaatt tccctaaaaa tgataaaaca agcgttttga aagcgcttgt ttttttggtt
     7201 tgcagtcaga gtagaataga agtatcaaaa aaagcaccga ctcggtgcca ctttttcaag
     7261 ttgataacgg actagcctta ttttaacttg ctatgctgtt ttgaatggtt ccaacaagat
     7321 tattttataa cttttataac aaataatcaa ggagaaattc aaagaaattt atcagccata
     7381 aaacaatact taatactata gaatgataac aaaataaact actttttaaa agaattttgt
     7441 gttataatct atttattatt aagtattggg taatattttt tgaagagata ttttgaaaaa
     7501 gaaaaattaa agcatattaa actaatttcg gaggtcatta aaactattat tgaaatcatc
     7561 aaactcatta tggatttaat ttaaactttt tattttagga ggcaaaaatg gataagaaat
     7621 actcaatagg cttagctatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat
     7681 ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa
     7741 aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca
     7801 aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga
     7861 ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt
     7921 ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg
     7981 aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttg
//
