LOCUS       sequence-410968-        8087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-opt. increased fidelity 
            Blackjack-HypaSpCas9 (w/o U6-sgRNA). Cleaves both 20nt and 
            5'-extended 21nt spacer containing sgRNAs with higher fidelity than 
            HypaSpCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8087)
  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 8087)
  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-HypaSpCas9
FEATURES             Location/Qualifiers
     source          1..8087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        195..480
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     rep_origin      complement(552..1140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(632..651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1311..2102,2103..2171))
                     /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     1934..1953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2172..2276)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2344..2362
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2400..2422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2522..2541
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2558..3013)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2695..2716)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2907..2926
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3088..3228
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3099..3228
                     /label=AAV2 ITR
     polyA_signal    3237..3444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3433..3450
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3478..3525)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3526..7605)
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRAFAA
                     LIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             complement(7630..7650)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7657..7722)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      7722..7731
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          7743..7970
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
ORIGIN
        1 cgccccccyc acccccgccc cscccscgcc ccccccccmc cccncccccc cccccccccc
       61 csacwggsnn nnnccccccc cccccccgcc cccatcgctg cacaaaataa ttaaaaaata
      121 aataaataca aaattggggg tggggagggg ggggagatgg ggagagtgaa gcagaacgtg
      181 gggctcacct cgaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
      241 aagtcccata aggtcatgta ctgggcacaa tgccaggcgg gccatttacc gtcattgacg
      301 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
      361 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tattgacgtc
      421 aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg
      481 ggtacctcta gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      541 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      601 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      661 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      721 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
      781 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
      841 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
      901 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      961 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1021 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1081 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1141 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1201 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1261 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1321 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     1381 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     1441 atgataccgc ggcttccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     1501 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     1561 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     1621 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     1681 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     1741 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     1801 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     1861 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     1921 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     1981 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2041 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2101 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2161 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2221 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2281 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     2341 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     2401 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     2461 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     2521 gcggcatcag agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg
     2581 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc
     2641 ggcaaaatcc cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt
     2701 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2761 tatcagggcg atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg
     2821 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     2881 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg
     2941 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3001 ctacagggcg cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg
     3061 taaggagaaa ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg
     3121 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     3181 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgctccc
     3241 cagcatgcct gctattctct tcccaatcct cccccttgct gtcctgcccc accccacccc
     3301 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     3361 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     3421 atggctggca actagaaggc acagtcgagg ctgatcagcg agctctagga attcttactt
     3481 tttctttttt gcctggccgg cctttttcgt ggccgccggc cttttgtcgc ctcccagctg
     3541 agacaggtcg atccgtgtct cgtacaggcc ggtgatgctc tggtggatca gggtggcgtc
     3601 cagcacctct ttggtgctgg tgtacctctt ccggtcgatg gtggtgtcaa agtacttgaa
     3661 ggcggcaggg gctcccagat tggtcagggt aaacaggtgg atgatattct cggcctgctc
     3721 tctgatgggc ttatcccggt gcttgttgta ggcggacagc actttgtcca gattagcgtc
     3781 ggccaggatc actctcttgg agaactcgct gatctgctcg atgatctcgt ccaggtagtg
     3841 cttgtgctgt tccacaaaca gctgtttctg ctcattatcc tcgggggagc ccttcagctt
     3901 ctcatagtgg ctggccaggt acaggaagtt cacatatttg gagggcaggg ccagttcgtt
     3961 tcccttctgc agttcgccgg cagaggccag cattctcttc cggccgtttt ccagctcgaa
     4021 cagggagtac ttaggcagct tgatgatcag gtcctttttc acttctttgt agcccttggc
     4081 ttccagaaag tcgatgggat tcttctcgaa gctgcttctt tccatgatgg tgatccccag
     4141 cagctctttc acactcttca gtttcttgga cttgcccttt tccactttgg ccaccaccag
     4201 cacagaatag gccacggtgg ggctgtcgaa gccgccgtac ttcttagggt cccagtcctt
     4261 ctttctggcg atcagcttat cgctgttcct cttgggcagg atagactctt tgctgaagcc
     4321 gcctgtctgc acctcggtct ttttcacgat attcacttgg ggcatgctca gcactttccg
     4381 cacggtggca aaatcccggc ccttatccca cacgatctcc ccggtttcgc cgtttgtctc
     4441 gatcagaggc cgcttccgga tctcgccgtt ggccagggta atctcggtct tgaaaaagtt
     4501 catgatgttg ctgtagaaga agtacttggc ggtagccttg ccgatttcct gctcgctctt
     4561 ggcgatcatc ttccgtacat cgtacacctt gccacccagc ttagggtact ttttgatcag
     4621 ggcggttccc acgacggcgt tcaggtacgc gtcgtgggcg tggtggtagt tgttgatctc
     4681 gcgcactttg taaaactgga aatccttccg gaaatcggac accagcttgg acttcagggt
     4741 gatcactttc acttcccgga tcagcttgtc attctcgtcg tacttagtgt tcatccggga
     4801 gtccaggatc tgtgccacgt gctttgtgat ctgccgggtt tccaccagct gtctcttgat
     4861 gaagccggcc ttatccagtt cgctcaggcc gcctctctcg gccttggtca gattgtcgaa
     4921 ctttctctgg gtaatcagct tggcgttcag cagctgccgc cagtagttct tcatcttctt
     4981 cacgacctct tcggagggca cgttgtcgct cttgccccgg ttcttgtcgc ttctggtcag
     5041 caccttgttg tcgatggagt cgtccttcag aaagctctga ggcacgatat ggtccacatc
     5101 gtagtcggac agccggttga tgtccagttc ctggtccacg tacatatccc gcccattctg
     5161 caggtagtac aggtacagct tctcgttctg cagctgggtg ttttccacgg ggtgttcttt
     5221 caggatctgg ctgcccagct ctttgatgcc ctcttcgatc cgcttcattc tctcgcggct
     5281 gttcttctgt cccttctggg tggtctggtt ctctctggcc atttcgatca cgatgttctc
     5341 gggcttgtgc cggcccatca ctttcacgag ctcgtccacc accttcactg tctgcaggat
     5401 gcccttctta atggcggggc tgccggccag attggcaatg tgctcgtgca ggctatcgcc
     5461 ctggccggac acctgggctt tctggatgtc ctctttaaag gtcaggctgt cgtcagcgat
     5521 cagggctgca aaggctctgt tggcgaagcc gtcggacttc aggaaatcca ggattgtctt
     5581 gccggactgc ttgtcgcgga tgccgttgat cagcttccgg ctcagcctgc cccagccggt
     5641 gtatctccgc cgcttcagct gcttcatcac tttgtcgtcg aacaggtggg cataggtttt
     5701 cagccgttcc tcgatcatct ctctgtcctc aaacagtgtc agggtcagca cgatatcttc
     5761 cagaatgtcc tcgttttcct cattgtccag gaagtccttg tccttgataa ttttcagcag
     5821 atcgtggtat gtgcccaggg aggcgttgaa ccgatcttcc acgccggaga tttccacgga
     5881 gtcgaagcac tcgattttct tgaagtagtc ctctttcagc tgcttcacgg tcactttccg
     5941 gttggtcttg aacagcaggt ccacgatggc ctttttctgc tcgccgctca ggaaggcggg
     6001 ctttctcatt ccctcggtca cgtatttcac tttggtcagc tcgttataca cggtgaagta
     6061 ctcgtacagc aggctgtgct tgggcagcac cttctcgttg ggcaggttct tatcgaagtt
     6121 ggtcatccgc tcgatgaagc tctgggcgga agcgcccttg tccaccactt cctcgaagtt
     6181 ccagggggtg atggtttcct cgctctttct ggtcatccag gcgaatctgc tgtttcccct
     6241 ggccagaggg cccacgtagt aggggatgcg gaaggtcagg atcttctcga tcttttcccg
     6301 gttgtccttc aggaatgggt aaaaatcttc ctgccgccgc agaatggcgt gcagctctcc
     6361 caggtggatc tggtggggga tgctgccgtt gtcgaaggtc cgctgcttcc gcagcaggtc
     6421 ctctctgttc agcttcacga gcagttcctc ggtgccgtcc atcttttcca ggatgggctt
     6481 gatgaacttg tagaactctt cctggctggc tccgccgtca atgtagccgg cgtagccgtt
     6541 cttgctctgg tcgaagaaaa tctctttgta cttctcaggc agctgctgcc gcacgagagc
     6601 tttcagcagg gtcaggtcct ggtggtgctc gtcgtatctc ttgatcatag aggcgctcag
     6661 gggggccttg gtgatctcgg tgttcactct caggatgtcg ctcagcagga tggcgtcgga
     6721 caggttcttg gcggccagaa acaggtcggc gtactggtcg ccgatctggg ccagcaggtt
     6781 gtccaggtcg tcgtcgtagg tgtccttgct cagctgcagt ttggcatcct cggccaggtc
     6841 gaagttgctc ttgaagttgg gggtcaggcc caggctcagg gcaatcaggt ttccgaacag
     6901 gccattcttc ttctcgccgg gcagctgggc gatcagattt tccagccgtc tgctcttgct
     6961 cagtctggca gacaggatgg ccttggcgtc cacgccgctg gcgttgatgg ggttttcctc
     7021 gaacagctgg ttgtaggtct gcaccagctg gatgaacagc ttgtccacgt cgctgttgtc
     7081 ggggttcagg tcgccctcga tcaggaagtg gccccggaac ttgatcatgt gggccagggc
     7141 cagatagatc agccgcaggt cggccttgtc ggtgctgtcc accagtttct ttctcaggtg
     7201 gtagatggtg gggtacttct cgtggtaggc cacctcgtcc acgatgttgc cgaagatggg
     7261 gtgccgctcg tgcttcttat cctcttccac caggaaggac tcttccagtc tgtggaagaa
     7321 gctgtcgtcc accttggcca tctcgttgct gaagatctct tgcagatagc agatccggtt
     7381 cttccgtctg gtgtatcttc ttctggcggt tctcttcagc cgggtggcct cggctgtttc
     7441 gccgctgtcg aacagcaggg ctccgatcag gttcttcttg atgctgtgcc ggtcggtgtt
     7501 gcccagcacc ttgaatttct tgctgggcac cttgtactcg tcggtgatca cggcccagcc
     7561 cacagagttg gtgccgatgt ccaggccgat gctgtacttc ttgtcggctg ctgggactcc
     7621 gtggataccg accttccgct tcttctttgg ggccatctta tcgtcatcgt ctttgtaatc
     7681 aatatcatga tccttgtagt ctccgtcgtg gtccttatag tccatggtgg caccggtcca
     7741 acctgaaaaa aagtgatttc aggcaggtgc tccaggtaat taaacattaa taccccacca
     7801 accaaccatc ccttaaaccc ttacctcttg ctcagctaat tacagcccgg aggagaaggg
     7861 ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc agagccgggg cgggcggcgc
     7921 gaggcggcgg cggagcgggg cacggggcga aggcagcgcg cagcgactcc cgcccgccgc
     7981 gcgcttcgct ttttataggg ccgccgccgc cgccgcctcg ccataaaagg aaactttcgg
     8041 agcgcgccgc tctgattggc tgccgccgca cctctccgcc tcgcccc
//
