LOCUS       sequence-261519-        8052 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized Blackjack-SpCas9 (w/o 
            U6-sgRNA). Cleaves both 20nt and 5'-extended 21nt spacer containing 
            sgRNAs with higher fidelity than that of WT SpCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8052)
  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 8052)
  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-SpCas9
FEATURES             Location/Qualifiers
     source          1..8052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          159..386
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      398..407
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             407..472
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             479..499
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             524..4603
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             4604..4651
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4679..4696)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4685..4892
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4901..5041
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4901..5030
                     /label=AAV2 ITR
     rep_origin      5116..5571
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5203..5222)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5413..5434
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5588..5607)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5707..5729
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5767..5785)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5853..5957
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5958..6026,6027..6818)
                     /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     complement(6176..6195)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6989..7577
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7478..7497
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7649..7934
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        join(7936..8052,1..158)
                     /label=chicken beta-actin promoter
ORIGIN
        1 kgcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg
       61 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
      121 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
      181 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
      241 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
      301 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
      361 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
      421 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
      481 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
      541 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
      601 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
      661 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
      721 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
      781 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
      841 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
      901 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
      961 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     1021 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     1081 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     1141 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     1201 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     1261 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     1321 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     1381 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     1441 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     1501 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     1561 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     1621 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     1681 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     1741 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     1801 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     1861 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     1921 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     1981 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     2041 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     2101 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     2161 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     2221 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     2281 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     2341 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     2401 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     2461 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     2521 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     2581 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     2641 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     2701 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     2761 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     2821 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     2881 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     2941 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     3001 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     3061 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     3121 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     3181 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     3241 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     3301 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     3361 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     3421 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     3481 gtacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tgggtggcaa
     3541 ggtgtacgat gtacggaaga tgatcgccaa gagcgagcag gaaatcggca aggctaccgc
     3601 caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta ccctggccaa
     3661 cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg agatcgtgtg
     3721 ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc aagtgaatat
     3781 cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc tgcccaagag
     3841 gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt acggcggctt
     3901 cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa agggcaagtc
     3961 caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa gaagcagctt
     4021 cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga aaaaggacct
     4081 gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga agagaatgct
     4141 ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca aatatgtgaa
     4201 cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg ataatgagca
     4261 gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg agcagatcag
     4321 cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc tgtccgccta
     4381 caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc acctgtttac
     4441 cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca tcgaccggaa
     4501 gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga gcatcaccgg
     4561 cctgtacgag acacggatcg acctgtctca gctgggaggc gacaaaaggc cggcggccac
     4621 gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc ctagagctcg ctgatcagcc
     4681 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4741 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4801 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4861 gattgggaag agaatagcag gcatgctggg gagcggccgc aggaacccct agtgatggag
     4921 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4981 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     5041 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     5101 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5161 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     5221 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5281 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     5341 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5401 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     5461 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     5521 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     5581 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     5641 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5701 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5761 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5821 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5881 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5941 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6001 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6061 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6121 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6181 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     6241 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6301 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6361 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6421 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6481 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6541 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6601 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6661 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6721 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6781 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6841 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6901 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6961 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7021 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     7081 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     7141 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7201 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7261 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7321 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7381 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7441 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7501 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7561 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7621 ggccttttgc tcacatgcta gaggtacccg ttacataact tacggtaaat ggcccgcctg
     7681 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
     7741 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     7801 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
     7861 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
     7921 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
     7981 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
     8041 cggggggggg gg
//
