LOCUS       Exported                7368 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  dCas9, MCP-SoxS(R93A/S101A).
ACCESSION   .
VERSION     .
KEYWORDS    pCD442
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7368)
  AUTHORS   Fontana J, Dong C, Kiattisewee C, Chavali VP, Tickman BI, Carothers 
            JM, Zalatan JG
  TITLE     Effective CRISPRa-mediated control of gene expression in bacteria 
            must overcome strict target site requirements.
  JOURNAL   Nat Commun. 2020 Apr 1;11(1):1618. doi: 10.1038/s41467-020-15454-y.
  PUBMED    32238808
REFERENCE   2  (bases 1 to 7368)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7368
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        complement(209..287)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             583..4689
                     /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      4713..4784
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4800..4827
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4924..4935
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(5703..5720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5837..6382)
                     /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(5917..5936)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      6496..6590
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(6611..7270)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7185..7204
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7271..5)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gatcaacgtc tcattttcgc cagatatcga cgtcttaagt tacgaaatca tcctgtggag
       61 cttagtaggt ttagcaagat ggcagcgcct aaatgtagaa tgataaaagg attaagagat
      121 taatttccct aaaaatgata aaacaagcgt tttgaaagcg cttgtttttt tggtttgcag
      181 tcagagtaga atagaagtat caaaaaaagc accgactcgg tgccactttt tcaagttgat
      241 aacggactag ccttatttta acttgctatg ctgttttgaa tggttccaac aagattattt
      301 tataactttt ataacaaata atcaaggaga aattcaaaga aatttatcag ccataaaaca
      361 atacttaata ctatagaatg ataacaaaat aaactacttt ttaaaagaat tttgtgttat
      421 aatctattta ttattaagta ttgggtaata ttttttgaag agatattttg aaaaagaaaa
      481 attaaagcat attaaactaa tttcggaggt cattaaaact attattgaaa tcatcaaact
      541 cattatggat ttaatttaaa ctttttattt taggaggcaa aaatggataa gaaatactca
      601 ataggcttag ctatcggcac aaatagcgtc ggatgggcgg tgatcactga tgaatataag
      661 gttccgtcta aaaagttcaa ggttctggga aatacagacc gccacagtat caaaaaaaat
      721 cttatagggg ctcttttatt tgacagtgga gagacagcgg aagcgactcg tctcaaacgg
      781 acagctcgta gaaggtatac acgtcggaag aatcgtattt gttatctaca ggagattttt
      841 tcaaatgaga tggcgaaagt agatgatagt ttctttcatc gacttgaaga gtcttttttg
      901 gtggaagaag acaagaagca tgaacgtcat cctatttttg gaaatatagt agatgaagtt
      961 gcttatcatg agaaatatcc aactatctat catctgcgaa aaaaattggt agattctact
     1021 gataaagcgg atttgcgctt aatctatttg gccttagcgc atatgattaa gtttcgtggt
     1081 cattttttga ttgagggaga tttaaatcct gataatagtg atgtggacaa actatttatc
     1141 cagttggtac aaacctacaa tcaattattt gaagaaaacc ctattaacgc aagtggagta
     1201 gatgctaaag cgattctttc tgcacgattg agtaaatcaa gacgattaga aaatctcatt
     1261 gctcagctcc ccggtgagaa gaaaaatggc ttatttggga atctcattgc tttgtcattg
     1321 ggtttgaccc ctaattttaa atcaaatttt gatttggcag aagatgctaa attacagctt
     1381 tcaaaagata cttacgatga tgatttagat aatttattgg cgcaaattgg agatcaatat
     1441 gctgatttgt ttttggcagc taagaattta tcagatgcta ttttactttc agatatccta
     1501 agagtaaata ctgaaataac taaggctccc ctatcagctt caatgattaa acgctacgat
     1561 gaacatcatc aagacttgac tcttttaaaa gctttagttc gacaacaact tccagaaaag
     1621 tataaagaaa tcttttttga tcaatcaaaa aacggatatg caggttatat tgatggggga
     1681 gctagccaag aagaatttta taaatttatc aaaccaattt tagaaaaaat ggatggtact
     1741 gaggaattat tggtgaaact aaatcgtgaa gatttgctgc gcaagcaacg gacctttgac
     1801 aacggctcta ttccccatca aattcacttg ggtgagctgc atgctatttt gagaagacaa
     1861 gaagactttt atccattttt aaaagacaat cgtgagaaga ttgaaaaaat cttgactttt
     1921 cgaattcctt attatgttgg tccattggcg cgtggcaata gtcgttttgc atggatgact
     1981 cggaagtctg aagaaacaat taccccatgg aattttgaag aagttgtcga taaaggtgct
     2041 tcagctcaat catttattga acgcatgaca aactttgata aaaatcttcc aaatgaaaaa
     2101 gtactaccaa aacatagttt gctttatgag tattttacgg tttataacga attgacaaag
     2161 gtcaaatatg ttactgaagg aatgcgaaaa ccagcatttc tttcaggtga acagaagaaa
     2221 gccattgttg atttactctt caaaacaaat cgaaaagtaa ccgttaagca attaaaagaa
     2281 gattatttca aaaaaataga atgttttgat agtgttgaaa tttcaggagt tgaagataga
     2341 tttaatgctt cattaggtac ctaccatgat ttgctaaaaa ttattaaaga taaagatttt
     2401 ttggataatg aagaaaatga agatatctta gaggatattg ttttaacatt gaccttattt
     2461 gaagataggg agatgattga ggaaagactt aaaacatatg ctcacctctt tgatgataag
     2521 gtgatgaaac agcttaaacg tcgccgttat actggttggg gacgtttgtc tcgaaaattg
     2581 attaatggta ttagggataa gcaatctggc aaaacaatat tagatttttt gaaatcagat
     2641 ggttttgcca atcgcaattt tatgcagctg atccatgatg atagtttgac atttaaagaa
     2701 gacattcaaa aagcacaagt gtctggacaa ggcgatagtt tacatgaaca tattgcaaat
     2761 ttagctggta gccctgctat taaaaaaggt attttacaga ctgtaaaagt tgttgatgaa
     2821 ttggtcaaag taatggggcg gcataagcca gaaaatatcg ttattgaaat ggcacgtgaa
     2881 aatcagacaa ctcaaaaggg ccagaaaaat tcgcgagagc gtatgaaacg aatcgaagaa
     2941 ggtatcaaag aattaggaag tcagattctt aaagagcatc ctgttgaaaa tactcaattg
     3001 caaaatgaaa agctctatct ctattatctc caaaatggaa gagacatgta tgtggaccaa
     3061 gaattagata ttaatcgttt aagtgattat gatgtcgatg ccattgttcc acaaagtttc
     3121 cttaaagacg attcaataga caataaggtc ttaacgcgtt ctgataaaaa tcgtggtaaa
     3181 tcggataacg ttccaagtga agaagtagtc aaaaagatga aaaactattg gagacaactt
     3241 ctaaacgcca agttaatcac tcaacgtaag tttgataatt taacgaaagc tgaacgtgga
     3301 ggtttgagtg aacttgataa agctggtttt atcaaacgcc aattggttga aactcgccaa
     3361 atcactaagc atgtggcaca aattttggat agtcgcatga atactaaata cgatgaaaat
     3421 gataaactta ttcgagaggt taaagtgatt accttaaaat ctaaattagt ttctgacttc
     3481 cgaaaagatt tccaattcta taaagtacgt gagattaaca attaccatca tgcccatgat
     3541 gcgtatctaa atgccgtcgt tggaactgct ttgattaaga aatatccaaa acttgaatcg
     3601 gagtttgtct atggtgatta taaagtttat gatgttcgta aaatgattgc taagtctgag
     3661 caagaaatag gcaaagcaac cgcaaaatat ttcttttact ctaatatcat gaacttcttc
     3721 aaaacagaaa ttacacttgc aaatggagag attcgcaaac gccctctaat cgaaactaat
     3781 ggggaaactg gagaaattgt ctgggataaa gggcgagatt ttgccacagt gcgcaaagta
     3841 ttgtccatgc cccaagtcaa tattgtcaag aaaacagaag tacagacagg cggattctcc
     3901 aaggagtcaa ttttaccaaa aagaaattcg gacaagctta ttgctcgtaa aaaagactgg
     3961 gatccaaaaa aatatggtgg ttttgatagt ccaacggtag cttattcagt cctagtggtt
     4021 gctaaggtgg aaaaagggaa atcgaagaag ttaaaatccg ttaaagagtt actagggatc
     4081 acaattatgg aaagaagttc ctttgaaaaa aatccgattg actttttaga agctaaagga
     4141 tataaggaag ttaaaaaaga cttaatcatt aaactaccta aatatagtct ttttgagtta
     4201 gaaaacggtc gtaaacggat gctggctagt gccggagaat tacaaaaagg aaatgagctg
     4261 gctctgccaa gcaaatatgt gaatttttta tatttagcta gtcattatga aaagttgaag
     4321 ggtagtccag aagataacga acaaaaacaa ttgtttgtgg agcagcataa gcattattta
     4381 gatgagatta ttgagcaaat cagtgaattt tctaagcgtg ttattttagc agatgccaat
     4441 ttagataaag ttcttagtgc atataacaaa catagagaca aaccaatacg tgaacaagca
     4501 gaaaatatta ttcatttatt tacgttgacg aatcttggag ctcccgctgc ttttaaatat
     4561 tttgatacaa caattgatcg taaacgatat acgtctacaa aagaagtttt agatgccact
     4621 cttatccatc aatccatcac tggtctttat gaaacacgca ttgatttgag tcagctagga
     4681 ggtgactaac tcgagtaagg atctccaggc atcaaataaa acgaaaggct cagtcgaaag
     4741 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     4801 gctcaccttc gggtgggcct ttctgcgttt atacctagag cgcccggaag agagtcaatt
     4861 cagggtggtg aatctgcagt ttacggctag ctcagcccta ggtattatgc tagcgaattc
     4921 attaaagagg agaaaggtac catggggccc gcttctaact ttactcagtt cgttctcgtc
     4981 gacaatggcg gaactggcga cgtgactgtc gccccaagca acttcgctaa cgggatcgct
     5041 gaatggatca gctctaactc gcgttcacag gcttacaaag taacctgtag cgttcgtcag
     5101 agctctgcgc agaatcgcaa atacaccatc aaagtcgagg tgcctaaagg cgcctggcgt
     5161 tcgtacttaa atatggaact aaccattcca attttcgcca cgaattccga ctgcgagctt
     5221 attgttaagg caatgcaagg tctcctaaaa gatggaaacc cgattccctc agcaatcgca
     5281 gcaaactccg gcatctacgg tggcggaggt agcatgtccc atcagaaaat tattcaggat
     5341 cttatcgcat ggattgacga gcatattgac cagccgctta acattgatgt agtcgcaaaa
     5401 aaatcaggct attcaaagtg gtacttgcaa cgaatgttcc gcacggtgac gcatcagacg
     5461 cttggcgatt acattcgcca acgccgcctg ttactggccg ccgttgagtt gcgcaccacc
     5521 gagcgtccga tttttgatat cgcaatggac ctgggttatg tctcgcagca gaccttctcc
     5581 cgcgttttcg cgcggcagtt tgatcgcact cccgcggatt atcgccaccg cctgtaagcg
     5641 gccgccacgc aaaaaacccc gcttcggcgg ggttttttcg ccctagggat atattccgct
     5701 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
     5761 gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     5821 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     5881 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5941 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
     6001 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
     6061 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
     6121 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
     6181 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
     6241 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
     6301 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
     6361 gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat atttctagat
     6421 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     6481 gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga gcgttctgaa
     6541 caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc caagcgagct
     6601 cgatatcaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca
     6661 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     6721 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     6781 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     6841 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     6901 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     6961 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     7021 tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg
     7081 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     7141 aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg
     7201 cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt
     7261 ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     7321 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgcc
//
