LOCUS       sequence-107969-        6777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-dCas9 in E. coli in a tetracycline-dependent manner
            for enChIP analysis to purify specific genomic regions of interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6777)
  AUTHORS   Fujita T, Yuno M, Fujii H
  TITLE     An enChIP system for the analysis of bacterial genome functions.
  JOURNAL   BMC Res Notes. 2018 Jun 14;11(1):387. doi: 
            10.1186/s13104-018-3486-3.
  PUBMED    29898790
REFERENCE   2  (bases 1 to 6777)
  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: "BMC Res 
            Notes."; date: "2018-06-14"; volume: "11(1):387. doi"; pages: " 
            10.1186/s13104-018-3486-3"
COMMENT     Sequence Label: 3xFLAG-dCas9/p-bacteria
FEATURES             Location/Qualifiers
     source          1..6777
                     /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             743..808
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(812..838,839..841,842..3328,3329..3331,3332..4918)
                     /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      4942..5013
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5029..5056
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5084..5101)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5218..5763)
                     /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(5298..5317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5877..5971)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5992..6651)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6566..6585
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6652..6754)
                     /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 tggactacaa agaccatgac ggtgattata aagatcatga
      781 catcgattac aaggatgacg atgacaagct catggataag aaatactcaa taggcttagc
      841 tatcggcaca aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa
      901 aaagttcaag gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc
      961 tcttttattt gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag
     1021 aaggtataca cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat
     1081 ggcgaaagta gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga
     1141 caagaagcat gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga
     1201 gaaatatcca actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga
     1261 tttgcgctta atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat
     1321 tgagggagat ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca
     1381 aacctacaat caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc
     1441 gattctttct gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc
     1501 cggtgagaag aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc
     1561 taattttaaa tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac
     1621 ttacgatgat gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt
     1681 tttggcagct aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac
     1741 tgaaataact aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca
     1801 agacttgact cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat
     1861 cttttttgat caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga
     1921 agaattttat aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt
     1981 ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat
     2041 tccccatcaa attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta
     2101 tccattttta aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta
     2161 ttatgttggt ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga
     2221 agaaacaatt accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc
     2281 atttattgaa cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa
     2341 acatagtttg ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt
     2401 tactgaagga atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga
     2461 tttactcttc aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa
     2521 aaaaatagaa tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc
     2581 attaggtacc taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga
     2641 agaaaatgaa gatatcttag aggatattgt tttaacattg accttatttg aagataggga
     2701 gatgattgag gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca
     2761 gcttaaacgt cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat
     2821 tagggataag caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa
     2881 tcgcaatttt atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa
     2941 agcacaagtg tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag
     3001 ccctgctatt aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt
     3061 aatggggcgg cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac
     3121 tcaaaagggc cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga
     3181 attaggaagt cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa
     3241 gctctatctc tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat
     3301 taatcgttta agtgattatg atgtcgatgc cattgttcca caaagtttcc ttaaagacga
     3361 ttcaatagac aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt
     3421 tccaagtgaa gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa
     3481 gttaatcact caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga
     3541 acttgataaa gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca
     3601 tgtggcacaa attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat
     3661 tcgagaggtt aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt
     3721 ccaattctat aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa
     3781 tgccgtcgtt ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta
     3841 tggtgattat aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg
     3901 caaagcaacc gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat
     3961 tacacttgca aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg
     4021 agaaattgtc tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc
     4081 ccaagtcaat attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat
     4141 tttaccaaaa agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa
     4201 atatggtggt tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga
     4261 aaaagggaaa tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga
     4321 aagaagttcc tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt
     4381 taaaaaagac ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg
     4441 taaacggatg ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag
     4501 caaatatgtg aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga
     4561 agataacgaa caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat
     4621 tgagcaaatc agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt
     4681 tcttagtgca tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat
     4741 tcatttattt acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac
     4801 aattgatcgt aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca
     4861 atccatcact ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactaact
     4921 cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     4981 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
     5041 ggtgggcctt tctgcgttta tacctaggga tatattccgc ttcctcgctc actgactcgc
     5101 tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc
     5161 tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt
     5221 ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg
     5281 aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc
     5341 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca
     5401 ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg
     5461 aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5521 cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta
     5581 gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct
     5641 cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg
     5701 ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct
     5761 caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca atttatctct
     5821 tcaaatgtag cacctgaagt cagccccata cgatataagt tgttactagt gcttggattc
     5881 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     5941 gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgatatcaa attacgcccc
     6001 gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga catggaagcc
     6061 atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
     6121 ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg ccacgtttaa
     6181 atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat tctcaataaa
     6241 ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg aatatatgtg
     6301 tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg tttcagtttg
     6361 ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct caccgtcttt
     6421 cattgccata cgaaattccg gatgagcatt catcaggcgg gcaagaatgt gaataaaggc
     6481 cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa tatccagctg
     6541 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     6601 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     6661 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     6721 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc agatatc
//
