LOCUS       sequence-410921-        8029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-opt. increased fidelity 
            Blackjack-evoSpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 
            5'-extended 21nt spacer containing sgRNAs with higher fidelity than 
            evoSpCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8029)
  AUTHORS   Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E
  TITLE     Blackjack mutations improve the on-target activities of increased 
            fidelity variants of SpCas9 with 5'G-extended sgRNAs.
  JOURNAL   Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.
  PUBMED    32144253
REFERENCE   2  (bases 1 to 8029)
  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: "Nat 
            Commun."; date: "2020-03-6"; volume: "11(1):1223. doi"; pages: " 
            10.1038/s41467-020-15021-5"
COMMENT     Sequence Label: B-evoSpCas9
FEATURES             Location/Qualifiers
     source          1..8029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        121..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     rep_origin      complement(478..1066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(558..577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1237..2028,2029..2097))
                     /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     1860..1879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2098..2202)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2270..2288
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2326..2348)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2448..2467
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2484..2939)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2621..2642)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2833..2852
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3014..3154
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3025..3154
                     /label=AAV2 ITR
     polyA_signal    3163..3370
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3359..3376
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3404..3451)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3452..7531)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 variant with improved
                     specificity (Casini et al., 2018)"
                     /label=evoCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERVTNFDKNLPNEKVLPKHSLLNEYF
                     TVYNELTEVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGQLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             complement(7556..7576)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7583..7648)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      7648..7657
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          7669..7896
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 cccccccccc cccgccccca tcgctgcaca aaataattaa aaaataaata aatacaaaat
       61 tgggggtggg gagggggggg agatggggag agtgaagcag aacgtggggc tcacctcgac
      121 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
      181 catgtactgg gcacaatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
      241 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
      301 ccattgacgt caatggaaag tccctattgg cgttactatt gacgtcaatg ggcgggggtc
      361 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgggta cctctagcat
      421 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
      481 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
      541 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
      601 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
      661 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
      721 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
      781 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
      841 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
      901 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
      961 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     1021 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     1081 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     1141 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     1201 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     1261 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     1321 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggct
     1381 tccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     1441 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     1501 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     1561 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     1621 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     1681 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     1741 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     1801 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     1861 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     1921 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     1981 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     2041 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     2101 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     2161 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     2221 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     2281 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     2341 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     2401 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
     2461 gattgtactg agagtgcacc ataaaattgt aaacgttaat attttgttaa aattcgcgtt
     2521 aaatttttgt taaatcagct cattttttaa ccaatagacc gaaatcggca aaatccctta
     2581 taaatcaaaa gaatagcccg agatagagtt gagtgttgtt ccagtttgga acaagagtcc
     2641 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2701 cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2761 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2821 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct aaggcgctgg caagtgtagc
     2881 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta
     2941 ctatggttgc tttgacgtat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3001 cgcatcaggc gcccctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggca
     3061 aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga
     3121 gagggagtgg ccaactccat cactaggggt tcctgcggcc gctccccagc atgcctgcta
     3181 ttctcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga
     3241 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
     3301 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta
     3361 gaaggcacag tcgaggctga tcagcgagct ctaggaattc ttactttttc ttttttgcct
     3421 ggccggcctt tttcgtggcc gccggccttt tgtcgcctcc cagctgagac aggtcgatcc
     3481 gtgtctcgta caggccggtg atgctctggt ggatcagggt ggcgtccagc acctctttgg
     3541 tgctggtgta cctcttccgg tcgatggtgg tgtcaaagta cttgaaggcg gcaggggctc
     3601 ccagattggt cagggtaaac aggtggatga tattctcggc ctgctctctg atgggcttat
     3661 cccggtgctt gttgtaggcg gacagcactt tgtccagatt agcgtcggcc aggatcactc
     3721 tcttggagaa ctcgctgatc tgctcgatga tctcgtccag gtagtgcttg tgctgttcca
     3781 caaacagctg tttctgctca ttatcctcgg gggagccctt cagcttctca tagtggctgg
     3841 ccaggtacag gaagttcaca tatttggagg gcagggccag ttcgtttccc ttctgcagtt
     3901 cgccggcaga ggccagcatt ctcttccggc cgttttccag ctcgaacagg gagtacttag
     3961 gcagcttgat gatcaggtcc tttttcactt ctttgtagcc cttggcttcc agaaagtcga
     4021 tgggattctt ctcgaagctg cttctttcca tgatggtgat ccccagcagc tctttcacac
     4081 tcttcagttt cttggacttg cccttttcca ctttggccac caccagcaca gaataggcca
     4141 cggtggggct gtcgaagccg ccgtacttct tagggtccca gtccttcttt ctggcgatca
     4201 gcttatcgct gttcctcttg ggcaggatag actctttgct gaagccgcct gtctgcacct
     4261 cggtcttttt cacgatattc acttggggca tgctcagcac tttccgcacg gtggcaaaat
     4321 cccggccctt atcccacacg atctccccgg tttcgccgtt tgtctcgatc agaggccgct
     4381 tccggatctc gccgttggcc agggtaatct cggtcttgaa aaagttcatg atgttgctgt
     4441 agaagaagta cttggcggta gccttgccga tttcctgctc gctcttggcg atcatcttcc
     4501 gtacatcgta caccttgcca cccagcttag ggtacttttt gatcagggcg gttcccacga
     4561 cggcgttcag gtacgcgtcg tgggcgtggt ggtagttgtt gatctcgcgc actttgtaaa
     4621 actggaaatc cttccggaaa tcggacacca gcttggactt cagggtgatc actttcactt
     4681 cccggatcag cttgtcattc tcgtcgtact tagtgttcat ccgggagtcc aggatctgtg
     4741 ccacgtgctt tgtgatctgc cgggtttcca ccagctgtct cttgatgaag ccggccttat
     4801 ccagttcgct caggccgcct ctctcggcct tggtcagatt gtcgaacttt ctctgggtaa
     4861 tcagcttggc gttcagcagc tgccgccagt agttcttcat cttcttcacg acctcttcgg
     4921 agggcacgtt gtcgctcttg ccccggttct tgtcgcttct ggtcagcacc ttgttgtcga
     4981 tggagtcgtc cttcagaaag ctctgaggca cgatatggtc cacatcgtag tcggacagcc
     5041 ggttgatgtc cagttcctgg tccacgtaca tatcccgccc attctgcagg tagtacaggt
     5101 acagcttctc gttctgcagc tgggtgtttt ccacggggtg ttctttcagg atctggctgc
     5161 ccagctcttt gatgccctct tcgatccgct tcattctctc gcggctgttc ttctgtccct
     5221 tctgggtggt ctggttctct ctggccattt cgatcacgat gttctcgggc ttgtgccggc
     5281 ccatcacttt cacgagctcg tccaccacct tcactgtctg caggatgccc ttcttaatgg
     5341 cggggctgcc ggccagattg gcaatgtgct cgtgcaggct atcgccctgg ccggacacct
     5401 gggctttctg gatgtcctct ttaaaggtca ggctgtcgtc gtggatcagc tgcatgaagt
     5461 ttctgttggc gaagccgtcg gacttcagga aatccaggat tgtcttgccg gactgcttgt
     5521 cccggatgcc gttgatcagc ttccggctca gctggcccca gccggtgtat ctccgccgct
     5581 tcagctgctt catcactttg tcgtcgaaca ggtgggcata ggttttcagc cgttcctcga
     5641 tcatctctct gtcctcaaac agtgtcaggg tcagcacgat atcttccaga atgtcctcgt
     5701 tttcctcatt gtccaggaag tccttgtcct tgataatttt cagcagatcg tggtatgtgc
     5761 ccagggaggc gttgaaccga tcttccacgc cggagatttc cacggagtcg aagcactcga
     5821 ttttcttgaa gtagtcctct ttcagctgct tcacggtcac tttccggttg gtcttgaaca
     5881 gcaggtccac gatggccttt ttctgctcgc cgctcaggaa ggcgggcttt ctcattccct
     5941 cggtcacgta tttcacttcg gtcagctcgt tatacacggt gaagtactcg ttcagcaggc
     6001 tgtgcttggg cagcaccttc tcgttgggca ggttcttatc gaagttggtc acccgctcga
     6061 tgaagctctg ggcggaagcg cccttgtcca ccacttcctc gaagttccag ggggtgatgg
     6121 tttcctcgct ctttctggtc atccaggcga atctgctgtt tcccctggcc agagggccca
     6181 cgtagtaggg gatgcggaag gtcaggatct tctcgatctt ttcccggttg tccttcagga
     6241 atgggtaaaa atcttcctgc cgccgcagaa tggcgtgcag ctctcccagg tggatctggt
     6301 gggggatgct gccgttgtcg aaggtccgct gcttccgcag caggtcctct ctgttcagct
     6361 tcacgagcag ttcctcggtg ccgtccatct tttccaggat gggcttgatg aacttgtaga
     6421 actcttcctg gctggctccg ccgtcaatgt agccggcgta gccgttcttg ctctggtcga
     6481 agaaaatctc tttgtacttc tcaggcagct gctgccgcac gagagctttc agcagggtca
     6541 ggtcctggtg gtgctcgtcg tatctcttga tcatagaggc gctcaggggg gccttggtga
     6601 tctcggtgtt cactctcagg atgtcgctca gcaggatggc gtcggacagg ttcttggcgg
     6661 ccagaaacag gtcggcgtac tggtcgccga tctgggccag caggttgtcc aggtcgtcgt
     6721 cgtaggtgtc cttgctcagc tgcagtttgg catcctcggc caggtcgaag ttgctcttga
     6781 agttgggggt caggcccagg ctcagggcaa tcaggtttcc gaacaggcca ttcttcttct
     6841 cgccgggcag ctgggcgatc agattttcca gccgtctgct cttgctcagt ctggcagaca
     6901 ggatggcctt ggcgtccacg ccgctggcgt tgatggggtt ttcctcgaac agctggttgt
     6961 aggtctgcac cagctggatg aacagcttgt ccacgtcgct gttgtcgggg ttcaggtcgc
     7021 cctcgatcag gaagtggccc cggaacttga tcatgtgggc cagggccaga tagatcagcc
     7081 gcaggtcggc cttgtcggtg ctgtccacca gtttctttct caggtggtag atggtggggt
     7141 acttctcgtg gtaggccacc tcgtccacga tgttgccgaa gatggggtgc cgctcgtgct
     7201 tcttatcctc ttccaccagg aaggactctt ccagtctgtg gaagaagctg tcgtccacct
     7261 tggccatctc gttgctgaag atctcttgca gatagcagat ccggttcttc cgtctggtgt
     7321 atcttcttct ggcggttctc ttcagccggg tggcctcggc tgtttcgccg ctgtcgaaca
     7381 gcagggctcc gatcaggttc ttcttgatgc tgtgccggtc ggtgttgccc agcaccttga
     7441 atttcttgct gggcaccttg tactcgtcgg tgatcacggc ccagcccaca gagttggtgc
     7501 cgatgtccag gccgatgctg tacttcttgt cggctgctgg gactccgtgg ataccgacct
     7561 tccgcttctt ctttggggcc atcttatcgt catcgtcttt gtaatcaata tcatgatcct
     7621 tgtagtctcc gtcgtggtcc ttatagtcca tggtggcacc ggtccaacct gaaaaaaagt
     7681 gatttcaggc aggtgctcca ggtaattaaa cattaatacc ccaccaacca accatccctt
     7741 aaacccttac ctcttgctca gctaattaca gcccggagga gaagggccgt cccgcccgct
     7801 cacctgtggg agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg cggcggcgga
     7861 gcggggcacg gggcgaaggc agcgcgcagc gactcccgcc cgccgcgcgc ttcgcttttt
     7921 atagggccgc cgccgccgcc gcctcgccat aaaaggaaac tttcggagcg cgccgctctg
     7981 attggctgcc gccgcacctc tccgcctcgc cccgccccgc ccctcgccc
//
