LOCUS       sequence-242654-        8077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized increased fidelity 
            HeFSpCas9 (without U6-sgRNA coding sequence, with silent mutations).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8077)
  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 8077)
  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: pX330-Flag-HeFSpCas9 (without sgRNA; with sile...
FEATURES             Location/Qualifiers
     source          1..8077
                     /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..4116,
                     4117..4119,4120..5043)
                     /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
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5044..5091
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5119..5136)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5125..5332
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5341..5481
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5341..5470
                     /label=AAV2 ITR
     rep_origin      5556..6011
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5643..5662)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5853..5874
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6028..6047)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6147..6169
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6207..6225)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6293..6397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6398..6466,6467..7258)
                     /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(6616..6635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7429..8017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7918..7937
                     /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 ggaaagcgag
     3961 ttcgtgtacg gcgactacaa ggtgtacgac gtacggaaga tgatcgccaa gagcgagcag
     4021 gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag
     4081 accgagatta ccctggccaa cggcgagatc cggaaggcgc ctctgatcga gacaaacggc
     4141 gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg
     4201 agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa
     4261 gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac
     4321 cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc
     4381 aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc
     4441 atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac
     4501 aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa
     4561 aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc
     4621 ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc
     4681 tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac
     4741 gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg
     4801 gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag
     4861 aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt
     4921 gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg
     4981 atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc
     5041 gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gtaagaattc
     5101 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     5161 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     5221 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     5281 ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc
     5341 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     5401 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     5461 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     5521 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     5581 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5641 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5701 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5761 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5821 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5881 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     5941 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     6001 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6061 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6121 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6181 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     6241 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     6301 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6361 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6421 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6481 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6541 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6601 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6661 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6721 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6781 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6841 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6901 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6961 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     7021 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7081 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg
     7141 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7201 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7261 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7321 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7381 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7441 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7501 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7561 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7621 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7681 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7741 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7801 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7861 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7921 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7981 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     8041 acggttcctg gccttttgct ggccttttgc tcacatg
//
