LOCUS       sequence-410886-        8191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-opt. increased fidelity 
            Blackjack-Hypa2SpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 
            5'-extended 21nt spacer containing sgRNAs with higher fidelity than 
            Hypa2SpCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8191)
  AUTHORS   Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz 
            SL, Welker A, Vegi VL, Huszar K, Welker E
  TITLE     A cleavage rule for selection of increased-fidelity SpCas9 variants 
            with high efficiency and no detectable off-targets.
  JOURNAL   Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5.
  PUBMED    37717069
REFERENCE   2  (bases 1 to 8191)
  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: "2023-09-16"; volume: "14(1):5746. doi"; pages: " 
            10.1038/s41467-023-41393-5"
COMMENT     Sequence Label: B-HypaR-SpCas9
FEATURES             Location/Qualifiers
     source          1..8191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          208..435
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      447..456
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             456..521
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             528..548
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             573..4652
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRAFAA
                     LIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             4653..4700
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4728..4745)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4734..4941
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4950..5090
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4950..5079
                     /label=AAV2 ITR
     rep_origin      5165..5620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5252..5271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5462..5483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5637..5656)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5756..5778
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5816..5834)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5902..6006
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6007..6075,6076..6867)
                     /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(6225..6244)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7038..7626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7527..7546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7698..7983
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 ggbsgdgggg gkggkknggg gggggggggg gggggggggg gggggggggg gsgggggvgg
       61 ggsggggsgg gggggggsgg ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc
      121 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc
      181 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc
      241 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      301 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa gaggtaaggg
      361 tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca cctgcctgaa
      421 atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac gacggagact
      481 acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca aagaagaagc
      541 ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc ggcctggaca
      601 tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga
      661 aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc
      721 tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc gccagaagaa
      781 gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg
      841 ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata
      901 agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga
      961 agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc
     1021 tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg
     1081 agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga
     1141 cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca
     1201 tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg
     1261 gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca
     1321 acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct
     1381 acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc
     1441 tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg
     1501 agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg
     1561 acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt
     1621 tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag
     1681 agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg
     1741 tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc
     1801 cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc
     1861 cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact
     1921 acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg
     1981 aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct
     2041 tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc
     2101 acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga
     2161 ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc
     2221 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga
     2281 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc
     2341 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg
     2401 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga
     2461 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc
     2521 tgaagcggcg gagatacacc ggctggggcg cgctgagccg gaagctgatc aacggcatcc
     2581 gcgacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca
     2641 gagcctttgc agccctgatc gctgacgaca gcctgacctt taaagaggac atccagaaag
     2701 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc
     2761 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga
     2821 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc
     2881 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc
     2941 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc
     3001 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca
     3061 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact
     3121 ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc gacaacgtgc
     3181 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc
     3241 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac
     3301 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg
     3361 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc
     3421 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc
     3481 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcg tacctgaacg
     3541 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct gggtggcaag gtgtacgatg
     3601 tacggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     3661 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     3721 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     3781 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     3841 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     3901 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     3961 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     4021 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     4081 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     4141 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     4201 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     4261 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     4321 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     4381 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     4441 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     4501 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     4561 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     4621 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
     4681 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     4741 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4801 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4861 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4921 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4981 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     5041 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     5101 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     5161 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5221 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     5281 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5341 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     5401 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5461 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     5521 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     5581 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5641 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5701 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5761 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5821 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5881 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5941 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     6001 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6061 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6121 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6181 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6241 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6301 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6361 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6421 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6481 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6541 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6601 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6661 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6721 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6781 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6841 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6901 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6961 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     7021 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7081 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7141 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7201 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7261 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7321 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7381 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7441 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7501 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7561 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7621 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7681 cacatgctag aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     7741 aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa
     7801 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     7861 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac
     7921 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     7981 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc
     8041 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg
     8101 ggggggbgcg cgccaggsgg gggggggggg gggggggggg gggggggggg gggggggggg
     8161 gggggggggg gggggggggg gggggggggs g
//
