LOCUS       sequence-412375-        8046 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 8046)
  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 8046)
  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..8046
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          133..360
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      372..381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             381..446
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             453..473
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(498..3035,3036..3038,3039..3500,3501..3503,3504..3671,
                     3672..3674,3675..4598)
                     /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             4599..4646
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4674..4691)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4680..4887
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4896..5036
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4896..5025
                     /label=AAV2 ITR
     rep_origin      5111..5566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5198..5217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5408..5429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5583..5602)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5702..5724
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5762..5780)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5848..5952
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5953..6021,6022..6813)
                     /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(6171..6190)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6984..7572
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7473..7492
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7644..7929
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggggggggs ggggsggggs grggsggrgr ggtgcggcgg cagccaatca gagcggcgcg
       61 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      121 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      181 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      241 ggcccttctc ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg
      301 gttggtgggg tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag
      361 gttggaccgg tgccaccatg gactataagg accacgacgg agactacaag gatcatgata
      421 ttgattacaa agacgatgac gataagatgg ccccaaagaa gaagcggaag gtcggtatcc
      481 acggagtccc agcagccgac aagaagtaca gcatcggcct ggacatcggc accaactctg
      541 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
      601 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
      661 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
      721 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
      781 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
      841 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
      901 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
      961 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     1021 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     1081 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     1141 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     1201 gcctgttcgg aaacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     1261 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     1321 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     1381 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     1441 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     1501 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     1561 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     1621 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     1681 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     1741 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     1801 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     1861 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     1921 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     1981 ccgccttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     2041 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     2101 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     2161 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     2221 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     2281 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     2341 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     2401 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     2461 acaccggctg gggcgcgctg agccggaagc tgatcaacgg catccgcgac aagcagtccg
     2521 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatggcgc
     2581 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     2641 agggcgatag cctgcacgag cacattgcca atctcgccgg cagccccgcc attaagaagg
     2701 gcatcctcca gacagtgaag gtggtggacg agctggtgaa agtgatgggc cggcacaagc
     2761 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     2821 acagtcgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     2881 tgaaagaaca ccccgtggaa aacacccagc tccagaacga gaagctgtac ctgtactacc
     2941 tccagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     3001 acgatgtgga ccatatcgtg cctcagagct ttctggcgga cgactccatc gacaacaagg
     3061 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     3121 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     3181 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     3241 tcatcaagag acagctggtg gaaacccggg cgatcacaaa gcacgtggca cagatcctgg
     3301 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     3361 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     3421 gcgagatcaa caactaccac cacgcccacg acgcgtacct gaacgccgtc gtgggaaccg
     3481 ccctgatcaa aaagtaccct gcgctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     3541 acgacgtacg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     3601 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     3661 agatccggaa ggcgcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     3721 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     3781 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     3841 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     3901 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     3961 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     4021 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     4081 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     4141 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     4201 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     4261 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     4321 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     4381 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     4441 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     4501 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     4561 acgagacacg gatcgacctg tctcagctgg gaggcgacaa aaggccggcg gccacgaaaa
     4621 aggccggcca ggcaaaaaag aaaaagtaag aattcctaga gctcgctgat cagcctcgac
     4681 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     4741 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     4801 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     4861 ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga tggagttggc
     4921 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4981 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     5041 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     5101 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5161 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     5221 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5281 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5341 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5401 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     5461 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     5521 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5581 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5641 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5701 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     5761 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     5821 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5881 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5941 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6001 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6061 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6121 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     6181 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     6241 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     6301 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     6361 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     6421 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     6481 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     6541 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     6601 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     6661 gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6721 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6781 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6841 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6901 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6961 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     7021 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     7081 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     7141 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     7201 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7261 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7321 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7381 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7441 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7501 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7561 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7621 tttgctcaca tgctagaggt acccgttaca taacttacgg taaatggccc gcctggctga
     7681 ccgcccaacg acccccgccc attgacgtca atagtaacgc caatagggac tttccattga
     7741 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     7801 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattgtgcc
     7861 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     7921 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
     7981 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
     8041 gggggg
//
