LOCUS       sequence-107551-        8850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses intein-Cas9(S219-G521R) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8850)
  AUTHORS   Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR
  TITLE     Small molecule-triggered Cas9 protein with improved genome-editing 
            specificity.
  JOURNAL   Nat Chem Biol. 2015 May;11(5):316-8. doi: 10.1038/nchembio.1793. 
            Epub 2015 Apr 6.
  PUBMED    25848930
REFERENCE   2  (bases 1 to 8850)
  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: 
            "10.1038/nchembio.1793"; journalName: "Nat Chem Biol"; date: 
            "2015-05"; volume: "11"; issue: "5"; pages: "316-8"
COMMENT     Sequence Label: pKMD111e - intein-Cas9(S219-G521R)
FEATURES             Location/Qualifiers
     source          1..8850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3364..5748
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED
                     IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK
                     TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG
                     ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQI
                     LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN
                     KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK
                     AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF
                     YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK
                     ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMP
                     QVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV
                     EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEN
                     GRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD
                     EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY
                     FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD"
     CDS             5755..5775
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5782..5847
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5865..5882
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5905..5922)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5911..6135
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6206..6222)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6206..6222)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6219..6241)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6230..6246
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6254..6260,6261..6278,6279..6284))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6299..6320
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6437..6454)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6608..7196)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6688..6707)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7367..8158,8159..8227))
                     /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     7990..8009
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8228..8332)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8411..8430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(8602..8850,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag
      421 tattctattg gtttagacat cggcactaat tccgttggat gggctgtcat aaccgatgaa
      481 tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa
      541 aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg
      601 aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa
      661 atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc
      721 ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat
      781 gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac
      841 tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc
      901 cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg
      961 ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt
     1021 ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatgccttgc cgagggtacc
     1081 cgaatcttcg atccggtcac tggtacaacg catcgcatcg aggatgttgt cgatgggcgc
     1141 aagcctattc atgtcgtggc tgctgccaag gacggaacgc tgctcgcgcg gcccgtggtg
     1201 tcctggttcg accagggaac gcgggatgtg atcgggttgc ggatcgccgg tggcgccatc
     1261 gtgtgggcga cacccgatca caaggtgctg acagagtacg gctggcgtgc cgccggggaa
     1321 ctccgcaagg gagacagggt ggccggaccg ggtggttctg gtaacagcct ggccttgtcc
     1381 ctgacggccg accagatggt cagtgccttg ttggatgctg agccccccat actctattcc
     1441 gagtatgatc ctaccagtcc cttcagtgaa gcttcgatga tgggcttact gaccaacctg
     1501 gcagacaggg agctggttca catgatcaac tgggcgaaga gggtgccagg ctttgtggat
     1561 ttgaccctcc atgatcaggc ccaccttcta gaacgtgcct ggctagagat cctgatgatt
     1621 ggtctcgtct ggcgctccat ggagcaccca gggaagctac tgtttgctcc taacttgctc
     1681 ttggacagga accagggaaa atgtgtagag ggcatggtgg agatcttcga catgctgctg
     1741 gctacatcat ctcggttccg catgatgaat ctgcagggag aggagtttgt gtgcctcaaa
     1801 tctattattt tgcttaattc tggagtgtac acatttctgt ccagcaccct gaagtctctg
     1861 gaagagaagg accatatcca ccgagccctg gacaagatca cggacacttt gatccacctg
     1921 atggccaagg caggcctgac cctgcagcag cagcaccagc ggctggccca gctcctcctc
     1981 atcctctccc acatcaggca catgagtaac aaaagaatgg agcatctgta cagcatgaag
     2041 tacaagaacg tggtgcccct ctatgacctg ctgctggaga tgctggacgc ccaccgccta
     2101 catgcgggtg gttctggtgc tagccgcgtg caggcgttcg cggatgccct ggatgacaaa
     2161 ttcctgcacg acatgctggc ggaaggactc cgctattccg tgatccgaga agtgctgcca
     2221 acgcggcggg cacgaacgtt cgacctcgag gtcgaggaac tgcacaccct cgtcgccgaa
     2281 ggggttgtcg tgcacaactg ccgacggcta gaaaacctga tcgcacaatt acccggagag
     2341 aagaaaaatg ggttgttcgg taaccttata gcgctctcac taggcctgac accaaatttt
     2401 aagtcgaact tcgacttagc tgaagatgcc aaattgcagc ttagtaagga cacgtacgat
     2461 gacgatctcg acaatctact ggcacaaatt ggagatcagt atgcggactt atttttggct
     2521 gccaaaaacc ttagcgatgc aatcctccta tctgacatac tgagagttaa tactgagatt
     2581 accaaggcgc cgttatccgc ttcaatgatc aaaaggtacg atgaacatca ccaagacttg
     2641 acacttctca aggccctagt ccgtcagcaa ctgcctgaga aatataagga aatattcttt
     2701 gatcagtcga aaaacgggta cgcaggttat attgacggcg gagcgagtca agaggaattc
     2761 tacaagttta tcaaacccat attagagaag atggatggga cggaagagtt gcttgtaaaa
     2821 ctcaatcgcg aagatctact gcgaaagcag cggactttcg acaacggtag cattccacat
     2881 caaatccact taggcgaatt gcatgctata cttagaaggc aggaggattt ttatccgttc
     2941 ctcaaagaca atcgtgaaaa gattgagaaa atcctaacct ttcgcatacc ttactatgtg
     3001 ggacccctgg cccgagggaa ctctcggttc gcatggatga caagaaagtc cgaagaaacg
     3061 attactccat ggaattttga ggaagttgtc gataaaggtg cgtcagctca atcgttcatc
     3121 gagaggatga ccaactttga caagaattta ccgaacgaaa aagtattgcc taagcacagt
     3181 ttactttacg agtatttcac agtgtacaat gaactcacga aagttaagta tgtcactgag
     3241 ggcatgcgta aacccgcctt tctaagcgga gaacagaaga aagcaatagt agatctgtta
     3301 ttcaagacca accgcaaagt gacagttaag caattgaaag aggactactt taagaaaatt
     3361 gaatgcttcg attctgtcga gatctccggg gtagaagatc gatttaatgc gtcacttggt
     3421 acgtatcatg acctcctaaa gataattaaa gataaggact tcctggataa cgaagagaat
     3481 gaagatatct tagaagatat agtgttgact cttaccctct ttgaagatcg ggaaatgatt
     3541 gaggaaagac taaaaacata cgctcacctg ttcgacgata aggttatgaa acagttaaag
     3601 aggcgtcgct atacgggctg gggacgattg tcgcggaaac ttatcaacgg gataagagac
     3661 aagcaaagtg gtaaaactat tctcgatttt ctaaagagcg acggcttcgc caataggaac
     3721 tttatgcagc tgatccatga tgactcttta accttcaaag aggatataca aaaggcacag
     3781 gtttccggac aaggggactc attgcacgaa catattgcga atcttgctgg ttcgccagcc
     3841 atcaaaaagg gcatactcca gacagtcaaa gtagtggatg agctagttaa ggtcatggga
     3901 cgtcacaaac cggaaaacat tgtaatcgag atggcacgcg aaaatcaaac gactcagaag
     3961 gggcaaaaaa acagtcgaga gcggatgaag agaatagaag agggtattaa agaactgggc
     4021 agccagatct taaaggagca tcctgtggaa aatacccaat tgcagaacga gaaactttac
     4081 ctctattacc tacaaaatgg aagggacatg tatgttgatc aggaactgga cataaaccgt
     4141 ttatctgatt acgacgtcga tcacattgta ccccaatcct ttttgaagga cgattcaatc
     4201 gacaataaag tgcttacacg ctcggataag aaccgaggga aaagtgacaa tgttccaagc
     4261 gaggaagtcg taaagaaaat gaagaactat tggcggcagc tcctaaatgc gaaactgata
     4321 acgcaaagaa agttcgataa cttaactaaa gctgagaggg gtggcttgtc tgaacttgac
     4381 aaggccggat ttattaaacg tcagctcgtg gaaacccgcc aaatcacaaa gcatgttgca
     4441 cagatactag attcccgaat gaatacgaaa tacgacgaga acgataagct gattcgggaa
     4501 gtcaaagtaa tcactttaaa gtcaaaattg gtgtcggact tcagaaagga ttttcaattc
     4561 tataaagtta gggagataaa taactaccac catgcgcacg acgcttatct taatgccgtc
     4621 gtagggaccg cactcattaa gaaatacccg aagctagaaa gtgagtttgt gtatggtgat
     4681 tacaaagttt atgacgtccg taagatgatc gcgaaaagcg aacaggagat aggcaaggct
     4741 acagccaaat acttctttta ttctaacatt atgaatttct ttaagacgga aatcactctg
     4801 gcaaacggag agatacgcaa acgaccttta attgaaacca atggggagac aggtgaaatc
     4861 gtatgggata agggccggga cttcgcgacg gtgagaaaag ttttgtccat gccccaagtc
     4921 aacatagtaa agaaaactga ggtgcagacc ggagggtttt caaaggaatc gattcttcca
     4981 aaaaggaata gtgataagct catcgctcgt aaaaaggact gggacccgaa aaagtacggt
     5041 ggcttcgata gccctacagt tgcctattct gtcctagtag tggcaaaagt tgagaaggga
     5101 aaatccaaga aactgaagtc agtcaaagaa ttattgggga taacgattat ggagcgctcg
     5161 tcttttgaaa agaaccccat cgacttcctt gaggcgaaag gttacaagga agtaaaaaag
     5221 gatctcataa ttaaactacc aaagtatagt ctgtttgagt tagaaaatgg ccgaaaacgg
     5281 atgttggcta gcgccggaga gcttcaaaag gggaacgaac tcgcactacc gtctaaatac
     5341 gtgaatttcc tgtatttagc gtcccattac gagaagttga aaggttcacc tgaagataac
     5401 gaacagaagc aactttttgt tgagcagcac aaacattatc tcgacgaaat catagagcaa
     5461 atttcggaat tcagtaagag agtcatccta gctgatgcca atctggacaa agtattaagc
     5521 gcatacaaca agcacaggga taaacccata cgtgagcagg cggaaaatat tatccatttg
     5581 tttactctta ccaacctcgg cgctccagcc gcattcaagt attttgacac aacgatagat
     5641 cgcaaacgat acacttctac caaggaggtg ctagacgcga cactgattca ccaatccatc
     5701 acgggattat atgaaactcg gatagatttg tcacagcttg ggggtgacgg atcccccaag
     5761 aagaagagga aagtctcgag cgactacaaa gaccatgacg gtgattataa agatcatgac
     5821 atcgattaca aggatgacga tgacaaggct gcaggatgac cggtcatcat caccatcacc
     5881 attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     5941 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     6001 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     6061 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     6121 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct
     6181 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6241 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat
     6301 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6361 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6421 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6481 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6541 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     6601 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     6661 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     6721 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     6781 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg
     6841 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6901 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6961 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     7021 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     7081 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     7141 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     7201 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     7261 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     7321 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     7381 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     7441 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     7501 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     7561 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     7621 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     7681 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     7741 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     7801 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7861 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7921 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7981 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     8041 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     8101 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     8161 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     8221 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     8281 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     8341 cccgaaaagt gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt
     8401 cgactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     8461 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     8521 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     8581 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     8641 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     8701 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     8761 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     8821 ctgcccactt ggcagtacat caagtgtatc
//
