LOCUS       sequence-410965-        8011 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-opt. increased fidelity 
            Blackjack-SpCas9-HF1 (w/o U6-sgRNA). Cleaves both 20nt and 
            5'-extended 21nt spacer containing sgRNAs with higher fidelity than 
            SpCas9HF1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8011)
  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 8011)
  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-HF1
FEATURES             Location/Qualifiers
     source          1..8011
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        118..403
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     rep_origin      complement(475..1063)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(555..574)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1234..2025,2026..2094))
                     /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     1857..1876
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2095..2199)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2267..2285
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2323..2345)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2445..2464
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2481..2936)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2618..2639)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2830..2849
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3011..3151
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3022..3151
                     /label=AAV2 ITR
     polyA_signal    3160..3367
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3356..3373
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3401..3448)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3449..7528)
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             complement(7553..7573)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7580..7645)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      7645..7654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          7666..7893
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 cccccccccc gcccccatcg ctgcacaaaa taattaaaaa ataaataaat acaaaattgg
       61 gggtggggag gggggggaga tggggagagt gaagcagaac gtggggctca cctcgaccat
      121 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
      181 gtactgggca caatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
      241 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
      301 ttgacgtcaa tggaaagtcc ctattggcgt tactattgac gtcaatgggc gggggtcgtt
      361 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgggtacct ctagcatgtg
      421 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      481 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      541 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      601 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      661 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      721 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      781 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      841 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      901 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      961 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1021 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1081 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1141 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1201 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1261 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1321 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcggcttcc
     1381 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1441 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1501 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1561 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1621 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1681 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1741 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1801 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1861 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     1921 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     1981 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2041 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2101 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2161 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2221 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     2281 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2341 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2401 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     2461 tgtactgaga gtgcaccata aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa
     2521 tttttgttaa atcagctcat tttttaacca atagaccgaa atcggcaaaa tcccttataa
     2581 atcaaaagaa tagcccgaga tagagttgag tgttgttcca gtttggaaca agagtccact
     2641 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2701 actacgtgaa ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2761 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     2821 gagaaaggaa gggaagaaag cgaaaggagc gggcgctaag gcgctggcaa gtgtagcggt
     2881 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtacta
     2941 tggttgcttt gacgtatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
     3001 atcaggcgcc cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
     3061 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
     3121 ggagtggcca actccatcac taggggttcc tgcggccgct ccccagcatg cctgctattc
     3181 tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac
     3241 ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca
     3301 ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa
     3361 ggcacagtcg aggctgatca gcgagctcta ggaattctta ctttttcttt tttgcctggc
     3421 cggccttttt cgtggccgcc ggccttttgt cgcctcccag ctgagacagg tcgatccgtg
     3481 tctcgtacag gccggtgatg ctctggtgga tcagggtggc gtccagcacc tctttggtgc
     3541 tggtgtacct cttccggtcg atggtggtgt caaagtactt gaaggcggca ggggctccca
     3601 gattggtcag ggtaaacagg tggatgatat tctcggcctg ctctctgatg ggcttatccc
     3661 ggtgcttgtt gtaggcggac agcactttgt ccagattagc gtcggccagg atcactctct
     3721 tggagaactc gctgatctgc tcgatgatct cgtccaggta gtgcttgtgc tgttccacaa
     3781 acagctgttt ctgctcatta tcctcggggg agcccttcag cttctcatag tggctggcca
     3841 ggtacaggaa gttcacatat ttggagggca gggccagttc gtttcccttc tgcagttcgc
     3901 cggcagaggc cagcattctc ttccggccgt tttccagctc gaagagcgag tacttaggca
     3961 gcttgatgat caggtccttt ttcacttctt tgtagccctt ggcttccaga aagtcgatgg
     4021 gattcttctc gaagctgctt ctttccatga tggtgatccc cagcagctct ttcacactct
     4081 taagcttctt ggacttgccc ttttccactt tggccaccac cagcacagaa taggccacgg
     4141 tggggctgtc gaagccgccg tacttcttag gatcccagtc cttctttctg gcgatcagct
     4201 tatcgctgtt cctcttgggc aggatagact ctttgctgaa gccgcctgtc tgcacctcgg
     4261 tctttttcac gatattcact tggggcatgc tcagcacttt ccgcacggtg gcaaaatccc
     4321 ggcccttatc ccacacgatc tccccggttt cgccgtttgt ctcgatcaga ggccgcttcc
     4381 ggatctcgcc gttggccagg gtaatctcgg tcttgaaaaa gttcatgatg ttgctgtaga
     4441 agaagtactt ggcggtagcc ttgccgattt cctgctcgct cttggcgatc atcttccgta
     4501 catcgtacac cttgccaccc agcttagggt actttttgat cagggcggtt cccacgacgg
     4561 cgttcaggta cgcgtcgtgg gcgtggtggt agttgttgat ctcgcgcact ttgtaaaact
     4621 ggaaatcctt ccggaaatcg gacaccagct tggacttcag ggtgatcact ttcacttccc
     4681 ggatcagctt gtcattctcg tcgtacttag tgttcatccg ggagtccagg atctgtgcca
     4741 cgtgctttgt gatcgcccgg gtttccacca gctgtctctt gatgaagccg gccttatcca
     4801 gttcgctcag gccgcctctc tcggccttgg tcagattgtc gaactttctc tgggtaatca
     4861 gcttggcgtt cagcagctgc cgccagtagt tcttcatctt cttcacgacc tcttcggagg
     4921 gcacgttgtc gctcttgccc cggttcttgt cgcttctggt cagcaccttg ttgtcgatgg
     4981 agtcgtcctt aagaaagctc tgaggcacga tatggtccac atcgtagtcg gacagccggt
     5041 tgatgtccag ttcctggtcc acgtacatat cccgcccatt ctggaggtag tacaggtaca
     5101 gcttctcgtt ctggagctgg gtgttttcca cggggtgttc tttcaggatc tggctgccca
     5161 gctctttgat gccctcttcg atccgcttca ttctctcgcg actgttcttc tgtcccttct
     5221 gggtggtctg gttctctctg gccatttcga tcacgatgtt ctcgggcttg tgccggccca
     5281 tcactttcac cagctcgtcc accaccttca ctgtctggag gatgcccttc ttaatggcgg
     5341 ggctgccggc gagattggca atgtgctcgt gcaggctatc gccctggccg gacacctggg
     5401 ctttctggat gtcctcttta aaggtcaggc tgtcgtcgtg gatcagcgcc atgaagtttc
     5461 tgttggcgaa gccgtcggac ttcaggaaat ccaggattgt cttgccggac tgcttgtcgc
     5521 ggatgccgtt gatcagcttc cggctcagcg cgccccagcc ggtgtatctc cgccgcttca
     5581 gctgcttcat cactttgtcg tcgaacaggt gggcataggt tttcagccgt tcctcgatca
     5641 tctctctgtc ctcaaacagt gtcagggtca gcacgatatc ttccagaatg tcctcgtttt
     5701 cctcattgtc caggaagtcc ttgtccttga taattttcag cagatcgtgg tatgtgccca
     5761 gggaggcgtt gaaccgatct tccacgccgg agatttccac ggagtcgaag cactcgattt
     5821 tcttgaagta gtcctctttc agctgcttca cggtcacttt ccggttggtc ttgaacagca
     5881 ggtccacgat ggcctttttc tgctcgccgc tcaggaaggc gggctttctc attccctcgg
     5941 tcacgtattt cactttggtc agctcgttat acacggtgaa gtactcgtac agcaggctgt
     6001 gcttgggcag caccttctcg ttgggcaggt tcttatcgaa ggcggtcatc cgctcgatga
     6061 agctctgggc ggaagcgccc ttgtccacca cttcctcgaa gttccagggg gtgatggttt
     6121 cctcgctctt tctggtcatc caggcgaatc tgctgtttcc cctggccaga gggcccacgt
     6181 agtaggggat gcggaaggtc aggatcttct cgatcttttc ccggttgtcc ttcaggaatg
     6241 ggtaaaaatc ttcctgccgc cgcagaatgg cgtgcagctc tcccaggtgg atctggtggg
     6301 ggatgctgcc gttgtcgaag gtccgctgct tccgcagcag gtcctctctg ttcagcttca
     6361 cgagcagttc ctcggtgccg tccatctttt ccaggatggg cttgatgaac ttgtagaact
     6421 cttcctggct ggctccgccg tcaatgtagc cggcgtagcc gttcttgctc tggtcgaaga
     6481 aaatctcttt gtacttctca ggcagctgct gccgcacgag agctttcagc agggtcaggt
     6541 cctggtggtg ctcgtcgtat ctcttgatca tagaggcgct caggggggcc ttggtgatct
     6601 cggtgttcac tctcaggatg tcgctcagca ggatggcgtc ggacaggttc ttggcggcca
     6661 gaaacaggtc ggcgtactgg tcgccgatct gggccagcag gttgtccagg tcgtcgtcgt
     6721 aggtgtcctt gctcagctgc agtttggcat cctcggccag gtcgaagttg ctcttgaagt
     6781 tgggggtcag gcccaggctc agggcaatca ggtttccgaa caggccattc ttcttctcgc
     6841 cgggcagctg ggcgatcaga ttttccagcc gtctgctctt gctcagtctg gcagacagga
     6901 tggccttggc gtccacgccg ctggcgttga tggggttttc ctcgaacagc tggttgtagg
     6961 tctgcaccag ctggatgaac agcttgtcca cgtcgctgtt gtcggggttc aggtcgccct
     7021 cgatcaggaa gtggccccgg aacttgatca tgtgggccag ggccagatag atcagccgca
     7081 ggtcggcctt gtcggtgctg tccaccagtt tctttctcag gtggtagatg gtggggtact
     7141 tctcgtggta ggccacctcg tccacgatgt tgccgaagat ggggtgccgc tcgtgcttct
     7201 tatcctcttc caccaggaag gactcttcca gtctgtggaa gaagctgtcg tccaccttgg
     7261 ccatctcgtt gctgaagatc tcttgcagat agcagatccg gttcttccgt ctggtgtatc
     7321 ttcttctggc ggttctcttc agccgggtgg cctcggctgt ttcgccgctg tcgaacagca
     7381 gggctccgat caggttcttc ttgatgctgt gccggtcggt gttgcccagc accttgaatt
     7441 tcttgctggg caccttgtac tcgtcggtga tcacggccca gcccacagag ttggtgccga
     7501 tgtccaggcc gatgctgtac ttcttgtcgg ctgctgggac tccgtggata ccgaccttcc
     7561 gcttcttctt tggggccatc ttatcgtcat cgtctttgta atcaatatca tgatccttgt
     7621 agtctccgtc gtggtcctta tagtccatgg tggcaccggt ccaacctgaa aaaaagtgat
     7681 ttcaggcagg tgctccaggt aattaaacat taatacccca ccaaccaacc atcccttaaa
     7741 cccttacctc ttgctcagct aattacagcc cggaggagaa gggccgtccc gcccgctcac
     7801 ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg cggcggagcg
     7861 gggcacgggg cgaaggcagc gcgcagcgac tcccgcccgc cgcgcgcttc gctttttata
     7921 gggccgccgc cgccgccgcc tcgccataaa aggaaacttt cggagcgcgc cgctctgatt
     7981 ggctgccgcc gcacctctcc gcctcgcccc g
//
