LOCUS       sequence-273581-        7939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Himar-dCas9 in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7939)
  AUTHORS   Chen SP, Wang HH
  TITLE     An Engineered Cas-Transposon System for Programmable and 
            Site-Directed DNA Transpositions.
  JOURNAL   CRISPR J. 2019 Dec;2(6):376-394. doi: 10.1089/crispr.2019.0030. Epub
            2019 Nov 19.
  PUBMED    31742433
REFERENCE   2  (bases 1 to 7939)
  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"; doi: 
            "10.1089/crispr.2019.0030"; journalName: "CRISPR J"; date: 
            "2019-12"; volume: "2"; issue: "6"; pages: "376-394"
COMMENT     Sequence Label: pHdCas9
FEATURES             Location/Qualifiers
     source          1..7939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..633)
                     /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     538..557
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        649..704
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             722..733
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1832..1858,1859..1861,1862..4348,4349..4351,
                     4352..5938)
                     /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      5962..6008
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(6045..6062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6179..6724)
                     /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(6259..6278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6838..6932)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(6953..7744,7745..7813))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7576..7595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7814..7916)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
      721 taaagaggag aaaggatcta tggaaaaaaa ggaatttcgt gttttgataa aatactgttt
      781 tctgaaggga aaaaatacag tggaagcaaa aacttggctt gataatgagt ttccggactc
      841 tgccccaggg aaatcaacaa taattgattg gtatgcaaaa ttcaagcgtg gtgaaatgag
      901 cacggaggac ggtgaacgca gtggacgccc gaaagaggtg gttaccgacg aaaacatcaa
      961 aaaaatccac aaaatgattt tgaatgaccg taaaatgaag ttgatcgaga tagcagaggc
     1021 cttaaagata tcaaaggaac gtgttggtca tatcattcat caatatttgg atatgcggaa
     1081 gctctgtgcg aaatgggtgc cgcgcgagct cacatttgac caaaaacaac gacgtgttga
     1141 tgattctaag cggtgtttgc agctgttaac tcgtaataca cccgagtttt tccgtcgata
     1201 tgtgacaatg gatgaaacat ggctccatca ctacactcct gagtccaatc gacagtcggc
     1261 tgagtggaca gcgaccggtg aaccgtctcc gaagcgtgga aagactcaaa agtccgctgg
     1321 caaagtaatg gcctctgttt tttgggatgc gcatggaata atttttatcg attatcttga
     1381 gaagggaaaa accatcaaca gtgactatta tatggcgtta ttggagcgtt tgaaggtcga
     1441 aatcgcggca aaacggcccc acatgaagaa gaaaaaagtg ttgttccacc aagacaacgc
     1501 accgtgccac aagtcattga gaacgatggc aaaaattcat gaattgggct tcgaattgct
     1561 tccccacccg ccgtattctc cagatctggc ccccagcgac tttttcttgt tctcagacct
     1621 caaaaggatg ctcgcaggga aaaaatttgg ctgcaatgaa gaggtgatcg ccgaaactga
     1681 ggcctatttt gaggcaaaac cgaaggagta ctaccaaaat ggtatcaaaa aattggaagg
     1741 tcgttataat cgttgtatcg ctcttgaagg gaactatgtt gaaagcggtt ccgaaactcc
     1801 cggtacatca gaaagcgcga cccccgaaag catggataaa aagtattcta ttggtttagc
     1861 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa
     1921 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc
     1981 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag
     2041 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat
     2101 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga
     2161 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga
     2221 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga
     2281 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat
     2341 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca
     2401 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc
     2461 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc
     2521 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc
     2581 taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac
     2641 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt
     2701 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac
     2761 tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca
     2821 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat
     2881 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga
     2941 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt
     3001 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat
     3061 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta
     3121 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta
     3181 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga
     3241 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc
     3301 atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa
     3361 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt
     3421 tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga
     3481 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa
     3541 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc
     3601 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga
     3661 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga
     3721 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca
     3781 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat
     3841 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa
     3901 tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa
     3961 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag
     4021 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt
     4081 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac
     4141 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga
     4201 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa
     4261 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat
     4321 taatcgttta agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga
     4381 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt
     4441 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa
     4501 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga
     4561 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca
     4621 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat
     4681 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt
     4741 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa
     4801 tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta
     4861 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg
     4921 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat
     4981 tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg
     5041 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc
     5101 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat
     5161 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa
     5221 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga
     5281 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga
     5341 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt
     5401 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg
     5461 taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag
     5521 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga
     5581 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat
     5641 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt
     5701 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat
     5761 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac
     5821 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca
     5881 atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactaact
     5941 cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     6001 cgttttatcc gaatacgggt tcttgcatgg atatattccg cttcctcgct cactgactcg
     6061 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc
     6121 ctggaagatg ccaggaagat acttaatagg gaagtgagag ggccgcggca aagccgtttt
     6181 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc
     6241 gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct
     6301 ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc
     6361 attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac
     6421 gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6481 ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt
     6541 agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc
     6601 tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct tcgaaaaacc
     6661 gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc
     6721 tcaagaagat catcttatta atcagataaa atatttctag atttcagtgc aatttatctc
     6781 ttcaaatgta gcacctgaag tcagccccat acgatataag ttgttactag tgcttggatt
     6841 ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc
     6901 tgaggtcatt actggatcta tcaacaggag tccaagcgag ctcgatatca aattaccaat
     6961 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     7021 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     7081 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     7141 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     7201 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     7261 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     7321 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagtt
     7381 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7441 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7501 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7561 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7621 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7681 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7741 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7801 gttgaatact cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacgc
     7861 ccggtagtga tcttatttca ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt
     7921 ctcattttcg ccagatatc
//
