LOCUS       sequence-194252-        7614 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Human expression plasmid for SpCas9 Cluster 4 variant: 
            CMV-T7-hSpCas9-Cluster4(F491A, M495A, T496A, 
            N497A)-NLS(SV40)-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7614)
  AUTHORS   Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington 
            LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA
  TITLE     Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
  JOURNAL   Nature. 2017 Sep 20. doi: 10.1038/nature24268.
  PUBMED    28931002
REFERENCE   2  (bases 1 to 7614)
  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: "Nature. 
            2017 Sep 20. doi: 10.1038/nature24268."
COMMENT     Sequence Label: MMW3770 - human expression plasmid for SpCas9
            ...
FEATURES             Location/Qualifiers
     source          1..7614
                     /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             join(409..1896,1897..1899,1900..2388,2389..2391,2392..2490,
                     2491..2493,2494..3183,3184..3186,3187..4512)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSAIERAAAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4519..4539
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4546..4611
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4629..4646
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4669..4686)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4675..4899
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4970..4986)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4970..4986)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4983..5005)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4994..5010
                     /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(5018..5024,5025..5042,5043..5048))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5063..5084
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5201..5218)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5372..5960)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5452..5471)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6131..6922,6923..6991))
                     /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     6754..6773
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6992..7096)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7175..7194)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7366..7614,1..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 aatcccgacg gctagaaaac
     1081 ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc
     1141 tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg
     1201 cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat
     1261 cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac
     1321 atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg
     1381 tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct
     1441 gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac
     1501 ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat
     1561 gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact
     1621 ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga
     1681 aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta
     1741 acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg
     1801 atgacaagaa agtccgaaga aacgattact ccctggaatt ttgaggaagt tgtcgataaa
     1861 ggtgcgtcag ctcaatcggc catcgagagg gcagccgcct ttgacaagaa tttaccgaac
     1921 gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc
     1981 acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag
     2041 aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg
     2101 aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa
     2161 gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag
     2221 gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc
     2281 ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac
     2341 gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg
     2401 aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag
     2461 agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc
     2521 aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt
     2581 gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg
     2641 gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca
     2701 cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata
     2761 gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc
     2821 caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt
     2881 gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa
     2941 tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga
     3001 gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg
     3061 cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag
     3121 aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc
     3181 cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac
     3241 gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg
     3301 gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg
     3361 cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta
     3421 gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa
     3481 agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat
     3541 ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa
     3601 accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga
     3661 aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg
     3721 ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag
     3781 gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta
     3841 gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg
     3901 gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg
     3961 aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt
     4021 gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac
     4081 gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag
     4141 ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat
     4201 tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat
     4261 gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag
     4321 caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc
     4381 aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac
     4441 gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag
     4501 cttgggggtg acggatcccc caagaagaag aggaaagtct cgagcgacta caaagaccat
     4561 gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga
     4621 tgaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
     4681 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     4741 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     4801 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     4861 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     4921 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4981 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5041 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5161 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5221 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5281 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5341 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5401 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5461 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5521 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5581 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5641 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5701 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5761 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5821 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5881 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5941 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6001 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6061 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6121 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6181 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6241 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6301 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6361 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6421 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6481 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6541 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6601 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6661 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6721 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6781 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6841 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6901 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6961 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7021 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7081 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     7141 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
     7201 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     7261 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     7321 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     7381 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     7441 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     7501 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     7561 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatc
//
