LOCUS       Exported                7011 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expressing CasTA of dCas9 fused AsiA.
ACCESSION   .
VERSION     .
KEYWORDS    pdCas9-AsiA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7011)
  AUTHORS   Ho HI, Fang JR, Cheung J, Wang HH
  TITLE     Programmable CRISPR-Cas transcriptional activation in bacteria.
  JOURNAL   Mol Syst Biol. 2020 Jul;16(7):e9427. doi: 10.15252/msb.20199427.
  PUBMED    32657546
REFERENCE   2  (bases 1 to 7011)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7011
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..56
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    37..55
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             74..85
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             92..4195
                     /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      4528..4599
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4615..4642
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4670..4687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4804..5349)
                     /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(4884..4903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      5463..5557
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5578..6237)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6152..6171
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6238..6340)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6370..6993)
                     /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     6901..6920
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 gttgacactc tatcgttgat agagttattt taccactccc tatcagtgat agagaaaaga
       61 attcaaaaga tctaaagagg agaaaggatc tatggataag aaatactcaa taggcttagc
      121 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa
      181 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc
      241 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag
      301 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat
      361 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga
      421 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga
      481 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga
      541 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat
      601 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca
      661 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc
      721 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc
      781 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc
      841 taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac
      901 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt
      961 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac
     1021 tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca
     1081 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat
     1141 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga
     1201 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt
     1261 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat
     1321 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta
     1381 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta
     1441 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga
     1501 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc
     1561 atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa
     1621 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt
     1681 tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga
     1741 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa
     1801 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc
     1861 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga
     1921 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga
     1981 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca
     2041 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat
     2101 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa
     2161 tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa
     2221 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag
     2281 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt
     2341 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac
     2401 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga
     2461 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa
     2521 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat
     2581 taatcgttta agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga
     2641 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt
     2701 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa
     2761 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga
     2821 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca
     2881 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat
     2941 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt
     3001 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa
     3061 tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta
     3121 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg
     3181 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat
     3241 tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg
     3301 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc
     3361 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat
     3421 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa
     3481 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga
     3541 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga
     3601 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt
     3661 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg
     3721 taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag
     3781 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga
     3841 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat
     3901 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt
     3961 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat
     4021 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac
     4081 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca
     4141 atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgacagcgc
     4201 aggtggtggc ggttctggtg gtggtggcag catgaacaag aacatcgaca cggtccgtga
     4261 aatcattaca gtagcttcca ttctgatcaa gttttcccgc gaggatatcg ttgaaaaccg
     4321 tgcgaacttt attgccttct tgaacgaaat tggagttact cacgaggggc gcaagttgaa
     4381 tcagaactcg tttcgcaaga ttgtaagcga actgacgcag gaagacaaaa aaactttaat
     4441 tgatgagttc aacgagggat ttgagggcgt ttatcgttat ttggaaatgt ataccaataa
     4501 gtaacccgag taaggatctc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     4561 gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca
     4621 ccttcgggtg ggcctttctg cgtttatacc tagggatata ttccgcttcc tcgctcactg
     4681 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
     4741 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
     4801 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
     4861 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
     4921 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
     4981 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
     5041 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5101 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
     5161 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
     5221 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
     5281 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
     5341 cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt
     5401 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagtgctt
     5461 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     5521 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta
     5581 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg
     5641 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc
     5701 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac
     5761 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc
     5821 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
     5881 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc
     5941 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc
     6001 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat
     6061 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc
     6121 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc
     6181 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt
     6241 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat
     6301 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat
     6361 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
     6421 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
     6481 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
     6541 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
     6601 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
     6661 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     6721 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     6781 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     6841 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     6901 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     6961 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt t
//
