LOCUS       sequence-250946-        4960 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  dcas9 generator and reporter circuit (PpspA-2G6 with sfgfp::ASV).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4960)
  AUTHORS   Liu Y, Wan X, Wang B
  TITLE     Engineered CRISPRa enables programmable eukaryote-like gene 
            activation in bacteria.
  JOURNAL   Nat Commun. 2019 Aug 26;10(1):3693. doi: 10.1038/s41467-019-11479-0.
  PUBMED    31451697
REFERENCE   2  (bases 1 to 4960)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2019-08-26"; volume: "10(1):3693. doi"; pages: " 
            10.1038/s41467-019-11479-0"
COMMENT     Sequence Label: pLY152
FEATURES             Location/Qualifiers
     source          1..4960
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(join(64..1650,1651..1653,1654..4140,4141..4143,
                     4144..4170))
                     /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"
     RBS             4177..4188
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        4206..4261
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    4207..4225
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             4277..4903
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4353..4372)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             4918..4929
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 gggagaccag aaacaaaaaa aggggagcgg tttcccgctc cccttcaata attggctcta
       61 gtattagtca cctcctagct gactcaaatc aatgcgtgtt tcataaagac cagtgatgga
      121 ttgatggata agagtggcat ctaaaacttc ttttgtagac gtatatcgtt tacgatcaat
      181 tgttgtatca aaatatttaa aagcagcggg agctccaaga ttcgtcaacg taaataaatg
      241 aataatattt tctgcttgtt cacgtattgg tttgtctcta tgtttgttat atgcactaag
      301 aactttatct aaattggcat ctgctaaaat aacacgctta gaaaattcac tgatttgctc
      361 aataatctca tctaaataat gcttatgctg ctccacaaac aattgttttt gttcgttatc
      421 ttctggacta cccttcaact tttcataatg actagctaaa tataaaaaat tcacatattt
      481 gcttggcaga gccagctcat ttcctttttg taattctccg gcactagcca gcatccgttt
      541 acgaccgttt tctaactcaa aaagactata tttaggtagt ttaatgatta agtctttttt
      601 aacttcctta tatcctttag cttctaaaaa gtcaatcgga tttttttcaa aggaacttct
      661 ttccataatt gtgatcccta gtaactcttt aacggatttt aacttcttcg atttcccttt
      721 ttccacctta gcaaccacta ggactgaata agctaccgtt ggactatcaa aaccaccata
      781 tttttttgga tcccagtctt ttttacgagc aataagcttg tccgaatttc tttttggtaa
      841 aattgactcc ttggagaatc cgcctgtctg tacttctgtt ttcttgacaa tattgacttg
      901 gggcatggac aatactttgc gcactgtggc aaaatctcgc cctttatccc agacaatttc
      961 tccagtttcc ccattagttt cgattagagg gcgtttgcga atctctccat ttgcaagtgt
     1021 aatttctgtt ttgaagaagt tcatgatatt agagtaaaag aaatattttg cggttgcttt
     1081 gcctatttct tgctcagact tagcaatcat tttacgaaca tcataaactt tataatcacc
     1141 atagacaaac tccgattcaa gttttggata tttcttaatc aaagcagttc caacgacggc
     1201 atttagatac gcatcatggg catgatggta attgttaatc tcacgtactt tatagaattg
     1261 gaaatctttt cggaagtcag aaactaattt agattttaag gtaatcactt taacctctcg
     1321 aataagttta tcattttcat cgtatttagt attcatgcga ctatccaaaa tttgtgccac
     1381 atgcttagtg atttggcgag tttcaaccaa ttggcgtttg ataaaaccag ctttatcaag
     1441 ttcactcaaa cctccacgtt cagctttcgt taaattatca aacttacgtt gagtgattaa
     1501 cttggcgttt agaagttgtc tccaatagtt tttcatcttt ttgactactt cttcacttgg
     1561 aacgttatcc gatttaccac gatttttatc agaacgcgtt aagaccttat tgtctattga
     1621 atcgtcttta aggaaacttt gtggaacaat ggcatcgaca tcataatcac ttaaacgatt
     1681 aatatctaat tcttggtcca catacatgtc tcttccattt tggagataat agagatagag
     1741 cttttcattt tgcaattgag tattttcaac aggatgctct ttaagaatct gacttcctaa
     1801 ttctttgata ccttcttcga ttcgtttcat acgctctcgc gaatttttct ggcccttttg
     1861 agttgtctga ttttcacgtg ccatttcaat aacgatattt tctggcttat gccgccccat
     1921 tactttgacc aattcatcaa caacttttac agtctgtaaa ataccttttt taatagcagg
     1981 gctaccagct aaatttgcaa tatgttcatg taaactatcg ccttgtccag acacttgtgc
     2041 tttttgaatg tcttctttaa atgtcaaact atcatcatgg atcagctgca taaaattgcg
     2101 attggcaaaa ccatctgatt tcaaaaaatc taatattgtt ttgccagatt gcttatccct
     2161 aataccatta atcaattttc gagacaaacg tccccaacca gtataacggc gacgtttaag
     2221 ctgtttcatc accttatcat caaagaggtg agcatatgtt ttaagtcttt cctcaatcat
     2281 ctccctatct tcaaataagg tcaatgttaa aacaatatcc tctaagatat cttcattttc
     2341 ttcattatcc aaaaaatctt tatctttaat aatttttagc aaatcatggt aggtacctaa
     2401 tgaagcatta aatctatctt caactcctga aatttcaaca ctatcaaaac attctatttt
     2461 tttgaaataa tcttctttta attgcttaac ggttactttt cgatttgttt tgaagagtaa
     2521 atcaacaatg gctttcttct gttcacctga aagaaatgct ggttttcgca ttccttcagt
     2581 aacatatttg acctttgtca attcgttata aaccgtaaaa tactcataaa gcaaactatg
     2641 ttttggtagt actttttcat ttggaagatt tttatcaaag tttgtcatgc gttcaataaa
     2701 tgattgagct gaagcacctt tatcgacaac ttcttcaaaa ttccatgggg taattgtttc
     2761 ttcagacttc cgagtcatcc atgcaaaacg actattgcca cgcgccaatg gaccaacata
     2821 ataaggaata cgaaaagtca agattttttc aatcttctca cgattgtctt ttaaaaatgg
     2881 ataaaagtct tcttgtcttc tcaaaatagc atgcagctca cccaagtgaa tttgatgggg
     2941 aatagagccg ttgtcaaagg tccgttgctt gcgcagcaaa tcttcacgat ttagtttcac
     3001 caataattcc tcagtaccat ccattttttc taaaattggt ttgataaatt tataaaattc
     3061 ttcttggcta gctcccccat caatataacc tgcatatccg ttttttgatt gatcaaaaaa
     3121 gatttcttta tacttttctg gaagttgttg tcgaactaaa gcttttaaaa gagtcaagtc
     3181 ttgatgatgt tcatcgtagc gtttaatcat tgaagctgat aggggagcct tagttatttc
     3241 agtatttact cttaggatat ctgaaagtaa aatagcatct gataaattct tagctgccaa
     3301 aaacaaatca gcatattgat ctccaatttg cgccaataaa ttatctaaat catcatcgta
     3361 agtatctttt gaaagctgta atttagcatc ttctgccaaa tcaaaatttg atttaaaatt
     3421 aggggtcaaa cccaatgaca aagcaatgag attcccaaat aagccatttt tcttctcacc
     3481 ggggagctga gcaatgagat tttctaatcg tcttgattta ctcaatcgtg cagaaagaat
     3541 cgctttagca tctactccac ttgcgttaat agggttttct tcaaataatt gattgtaggt
     3601 ttgtaccaac tggataaata gtttgtccac atcactatta tcaggattta aatctccctc
     3661 aatcaaaaaa tgaccacgaa acttaatcat atgcgctaag gccaaataga ttaagcgcaa
     3721 atccgcttta tcagtagaat ctaccaattt ttttcgcaga tgatagatag ttggatattt
     3781 ctcatgataa gcaacttcat ctactatatt tccaaaaata ggatgacgtt catgcttctt
     3841 gtcttcttcc accaaaaaag actcttcaag tcgatgaaag aaactatcat ctactttcgc
     3901 catctcattt gaaaaaatct cctgtagata acaaatacga ttcttccgac gtgtatacct
     3961 tctacgagct gtccgtttga ggcgagtcgc ttccgctgtc tctccactgt caaataaaag
     4021 agcccctata agattttttt tgatactgtg gcggtctgta tttcccagaa ccttgaactt
     4081 tttagacgga accttatatt catcagtgat caccgcccat ccgacgctat ttgtgccgat
     4141 agctaagcct attgagtatt tcttatccat agatcctttc tcctctttag atcttttcaa
     4201 ttcttttctc tatcactgat agggagtggt aaaataactc tatcaacgat agagtgtcaa
     4261 caaaaattag gaattaatga tgtcaagatt agataaaagt aaagtgatta acagcgcatt
     4321 agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct
     4381 aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc
     4441 cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag
     4501 ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg
     4561 cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga
     4621 aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact
     4681 cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc
     4741 taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac gacaagctat
     4801 cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat
     4861 atgcggatta gaaaaacaac ttaaatgtga aagtgggtct taatactaga gccaattatt
     4921 gaaggccgct aacgcggcct ttttttgttt ctggtctccc
//
