LOCUS       sequence-140495-        6932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nicking Cas9 derived from pWT Cas9 (Addgene #44250). Ampicillin 
            resistance marker, the tetracycline repressor (TetR), a ColE1 origin
            of replication and Streptococcus pyogenes Cas9 D10A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6932)
  AUTHORS   Standage-Beier K, Zhang Q, Wang X
  TITLE     Targeted Large-Scale Deletion of Bacterial Genomes Using 
            CRISPR-Nickases.
  JOURNAL   ACS Synth Biol. 2015 Nov 20;4(11):1217-25. doi: 
            10.1021/acssynbio.5b00132. Epub 2015 Oct 25.
  PUBMED    26451892
REFERENCE   2  (bases 1 to 6932)
  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: 
            "10.1021/acssynbio.5b00132"; journalName: "ACS Synth Biol"; date: 
            "2015-11-20- 20"; volume: "4"; issue: "11"; pages: "1217-25"
COMMENT     Sequence Label: pCas9(D10A)
FEATURES             Location/Qualifiers
     source          1..6932
                     /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(740..766,767..769,770..4846)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4870..4941
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4957..4984
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(5151..5739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5231..5250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5827..5921)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5951..6742,6743..6811))
                     /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     6574..6593
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6812..6916)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcatgcttaa 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 tggataagaa atactcaata ggcttagcta tcggcacaaa
      781 tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt
      841 tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga
      901 cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg
      961 tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga
     1021 tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga
     1081 acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac
     1141 tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat
     1201 ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt
     1261 aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca
     1321 attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc
     1381 acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa
     1441 aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc
     1501 aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga
     1561 tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa
     1621 gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa
     1681 ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct
     1741 tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca
     1801 atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa
     1861 atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa
     1921 tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat
     1981 tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa
     2041 agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc
     2101 attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac
     2161 cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg
     2221 catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct
     2281 ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat
     2341 gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa
     2401 aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg
     2461 ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta
     2521 ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga
     2581 tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga
     2641 aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg
     2701 ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca
     2761 atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat
     2821 gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc
     2881 tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa
     2941 aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca
     3001 taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca
     3061 gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca
     3121 gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta
     3181 ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag
     3241 tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa
     3301 taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga
     3361 agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca
     3421 acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc
     3481 tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat
     3541 tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa
     3601 agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa
     3661 agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg
     3721 aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa
     3781 agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc
     3841 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa
     3901 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg
     3961 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat
     4021 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag
     4081 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt
     4141 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc
     4201 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt
     4261 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt
     4321 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct
     4381 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa
     4441 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca
     4501 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag
     4561 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata
     4621 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac
     4681 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa
     4741 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg
     4801 tctttatgaa acacgcattg atttgagtca gctaggaggt gactaactcg agtaaggatc
     4861 tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
     4921 tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc
     4981 tgcgtttata cctagggaga cgtcggcgtt cggctgcggc gagcggtatc agctcactca
     5041 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5101 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5161 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5221 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5281 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5341 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5401 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5461 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5521 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5581 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5641 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5701 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5761 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgactagt
     5821 gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag
     5881 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tcgtaaactt
     5941 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6001 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6061 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6121 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6181 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6241 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6301 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6361 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6421 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6481 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6541 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6601 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6661 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6721 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6781 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6841 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6901 aaataggggt tccgcgcaca tttccccgaa aa
//
