LOCUS       sequence-252850-        8056 bp    DNA     circular SYN 15-APR-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 8056)
  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 8056)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..8056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..805
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      817..826
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             826..891
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             898..918
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(943..3480,3481..3483,3484..3945,3946..3948,3949..4095,
                     4096..4098,4099..5022)
                     /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             5023..5070
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5098..5115)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5104..5311
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5320..5460
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5320..5449
                     /label=AAV2 ITR
     rep_origin      5535..5990
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5622..5641)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5832..5853
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6007..6026)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6126..6148
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6186..6204)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6272..6376
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6377..6445,6446..7237)
                     /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(6595..6614)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7408..7996
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7897..7916
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg
      601 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
      661 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa
      721 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca
      781 cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta taaggaccac
      841 gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gatggcccca
      901 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtacagcatc
      961 ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg
     1021 cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg
     1081 atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc
     1141 gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc
     1201 aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg
     1261 gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc
     1321 taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac
     1381 aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac
     1441 ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag
     1501 ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac
     1561 gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc
     1621 cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc
     1681 ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc
     1741 aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc
     1801 gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga
     1861 gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag
     1921 caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac
     1981 aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc
     2041 agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag
     2101 gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac
     2161 ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa
     2221 gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc
     2281 atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga
     2341 aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc
     2401 gcccagagct tcatcgagcg gatgaccgcc ttcgataaga acctgcccaa cgagaaggtg
     2461 ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg
     2521 aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc
     2581 atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac
     2641 tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc
     2701 aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg
     2761 gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag
     2821 gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg
     2881 atgaagcagc tgaagcggcg gagatacacc ggctggggcg cgctgagccg gaagctgatc
     2941 aacggcatcc gcgacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc
     3001 ttcgccaaca gaaacttcat ggcgctgatc cacgacgaca gcctgacctt taaagaggac
     3061 atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctc
     3121 gccggcagcc ccgccattaa gaagggcatc ctccagacag tgaaggtggt ggacgagctg
     3181 gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac
     3241 cagaccaccc agaagggaca gaagaacagt cgcgagagaa tgaagcggat cgaagagggc
     3301 atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctccag
     3361 aacgagaagc tgtacctgta ctacctccag aatgggcggg atatgtacgt ggaccaggaa
     3421 ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg
     3481 gcggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg gggcaagagc
     3541 gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg
     3601 aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc
     3661 ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac ccgggcgatc
     3721 acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac
     3781 aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg
     3841 aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcg
     3901 tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctgcgct gggtggcaag
     3961 gtgtacgatg tacggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc
     4021 aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac
     4081 ggcgagatcc ggaaggcgcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg
     4141 gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc
     4201 gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg
     4261 aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc
     4321 gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc
     4381 aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc
     4441 gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg
     4501 atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg
     4561 gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac
     4621 ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag
     4681 aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc
     4741 gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac
     4801 aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc
     4861 ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag
     4921 aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc
     4981 ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg
     5041 aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct
     5101 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     5161 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5221 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5281 attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
     5341 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     5401 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     5461 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5521 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5581 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5641 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5701 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5761 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5821 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     5881 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5941 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     6001 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     6061 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6121 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     6181 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     6241 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6301 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6361 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6421 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6481 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6541 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6601 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6661 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6721 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6781 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6841 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6901 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6961 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     7021 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     7081 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7141 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7201 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7261 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7321 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7381 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7441 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7501 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     7561 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7621 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7681 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7741 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7801 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7861 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7921 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7981 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     8041 gccttttgct cacatg
//
