LOCUS       sequence-259607-        8019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-opt. increased fidelity 
            Blackjack-HeFSpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 
            5'-extended 21nt spacer containing sgRNAs with higher fidelity than 
            HeFSpCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8019)
  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 8019)
  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-HeFSpCas9
FEATURES             Location/Qualifiers
     source          1..8019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          127..354
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      366..375
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             375..440
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             447..467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(492..3029,3030..3032,3033..3494,3495..3497,3498..3644,
                     3645..3647,3648..4571)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             4572..4619
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4647..4664)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4653..4860
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4869..5009
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4869..4998
                     /label=AAV2 ITR
     rep_origin      5084..5539
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5171..5190)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5381..5402
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5556..5575)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5675..5697
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5735..5753)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5821..5925
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5926..5994,5995..6786)
                     /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(6144..6163)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6957..7545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7446..7465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7617..7902
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
       61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      241 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt
      301 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga
      361 ccggtgccac catggactat aaggaccacg acggagacta caaggatcat gatattgatt
      421 acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag
      481 tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
      541 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
      601 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
      661 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
      721 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
      781 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
      841 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
      901 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
      961 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     1021 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     1081 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     1141 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     1201 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     1261 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     1321 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     1381 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     1441 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     1501 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     1561 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     1621 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     1681 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     1741 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     1801 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     1861 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     1921 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccgcct
     1981 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     2041 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     2101 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     2161 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     2221 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     2281 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     2341 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     2401 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     2461 gctggggcgc gctgagccgg aagctgatca acggcatccg cgacaagcag tccggcaaga
     2521 caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg gcgctgatcc
     2581 acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     2641 atagcctgca cgagcacatt gccaatctcg ccggcagccc cgccattaag aagggcatcc
     2701 tccagacagt gaaggtggtg gacgagctgg tgaaagtgat gggccggcac aagcccgaga
     2761 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagtc
     2821 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     2881 aacaccccgt ggaaaacacc cagctccaga acgagaagct gtacctgtac tacctccaga
     2941 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     3001 tggaccatat cgtgcctcag agctttctgg cggacgactc catcgacaac aaggtgctga
     3061 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     3121 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     3181 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     3241 agagacagct ggtggaaacc cgggcgatca caaagcacgt ggcacagatc ctggactccc
     3301 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     3361 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     3421 tcaacaacta ccaccacgcc cacgacgcgt acctgaacgc cgtcgtggga accgccctga
     3481 tcaaaaagta ccctgcgctg ggtggcaagg tgtacgatgt acggaagatg atcgccaaga
     3541 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     3601 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaaggcgcct ctgatcgaga
     3661 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     3721 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     3781 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     3841 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     3901 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     3961 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     4021 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     4081 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     4141 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     4201 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     4261 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     4321 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     4381 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     4441 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     4501 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     4561 tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt
     4621 aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     4681 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     4741 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     4801 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     4861 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4921 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4981 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     5041 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     5101 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     5161 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     5221 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5281 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5341 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5401 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5461 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5521 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5581 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5641 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5701 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5761 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5821 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5881 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5941 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     6001 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     6061 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6121 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6181 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6241 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6301 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6361 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6421 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6481 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6541 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6601 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     6661 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6721 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6781 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6841 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6901 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6961 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7021 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7081 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     7141 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7201 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7261 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7321 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7381 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7441 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7501 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7561 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgctaga ggtacccgtt
     7621 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     7681 tcaatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     7741 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     7801 aatgacggta aatggcccgc ctggcattgt gcccagtaca tgaccttatg ggactttcct
     7861 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     7921 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     7981 ttttaattat tttgtgcagc gatgggggcg ggggggggg
//
