LOCUS       sequence-410890-        8028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized increased fidelity 
            eSpCas9-plus (w/o U6-sgRNA). Cleaves both 20nt and 5'-extended 21nt 
            spacer containing sgRNAs with close to the same fidelity as 
            eSpCas9..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8028)
  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 8028)
  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: eSpCas9-plus
FEATURES             Location/Qualifiers
     source          1..8028
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          136..363
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      375..384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             384..449
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             456..476
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             501..4580
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             4581..4628
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4656..4673)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4662..4869
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4878..5018
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4878..5007
                     /label=AAV2 ITR
     rep_origin      5093..5548
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5180..5199)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5390..5411
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5565..5584)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5684..5706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5744..5762)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5830..5934
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5935..6003,6004..6795)
                     /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(6153..6172)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6966..7554
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7455..7474
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7626..7911
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggggggggg ggcggggggg ggsgrggcgg agaggtgcgg cggcagccaa tcagagcggc
       61 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
      121 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
      181 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
      241 gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg
      301 ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt
      361 caggttggac cggtgccacc atggactata aggaccacga cggagactac aaggatcatg
      421 atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta
      481 tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact
      541 ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc
      601 tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca
      661 gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac
      721 ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg
      781 acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc
      841 ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca
      901 tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct
      961 atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga
     1021 accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc
     1081 tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca
     1141 gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga
     1201 atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca
     1261 acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc
     1321 tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga
     1381 acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg
     1441 cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc
     1501 tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga
     1561 gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt
     1621 tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca
     1681 gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc
     1741 acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg
     1801 acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc
     1861 tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc
     1921 cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga
     1981 tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt
     2041 acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga
     2101 gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga
     2161 ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct
     2221 tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc
     2281 acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca
     2341 ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac
     2401 ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga
     2461 gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt
     2521 ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc
     2581 agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg
     2641 gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga
     2701 agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca
     2761 agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga
     2821 agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga
     2881 tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact
     2941 acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg
     3001 actacgatgt ggaccatatc gtgcctcaga gctttctggc cgacgactcc atcgacaaca
     3061 aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg
     3121 tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga
     3181 gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg
     3241 gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc
     3301 tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag
     3361 tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag
     3421 tgcgcgagat caacaactac caccacgccc acgacgcgta cctgaacgcc gtcgtgggaa
     3481 ccgccctgat caaaaagtac cctaagctgg gtggcaaggt gtacgatgta cggaagatga
     3541 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     3601 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aaggcgcctc
     3661 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     3721 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     3781 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
     3841 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     3901 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     3961 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     4021 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     4081 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     4141 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     4201 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     4261 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     4321 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
     4381 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     4441 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     4501 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     4561 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     4621 agaaaaagta agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca
     4681 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     4741 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     4801 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca
     4861 tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4921 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4981 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     5041 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5101 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5161 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5221 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5281 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5341 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5401 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5461 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5521 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5581 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5641 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5701 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5761 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5821 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5881 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5941 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6001 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6061 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6121 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6181 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6241 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6301 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6361 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6421 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6481 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6541 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6601 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     6661 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6721 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6781 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6841 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6901 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6961 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7021 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7081 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7141 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7201 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7261 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7321 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7381 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7441 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7501 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7561 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgctagag
     7621 gtacccgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     7681 ccattgacgt caatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     7741 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     7801 attgacgtca atgacggtaa atggcccgcc tggcattgtg cccagtacat gaccttatgg
     7861 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg
     7921 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat
     7981 ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggg
//
