LOCUS       sequence-194896-        8018 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized high-fidelity 
            SpCas9-HF1, px330-like backbone (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 8018)
  AUTHORS   Kulcsar PI, Talas A, Huszar K, Ligeti Z, Toth E, Weinhardt N, Fodor 
            E, Welker E
  TITLE     Crossing enhanced and high fidelity SpCas9 nucleases to optimize 
            specificity and cleavage.
  JOURNAL   Genome Biol. 2017 Oct 6;18(1):190. doi: 10.1186/s13059-017-1318-8.
  PUBMED    28985763
REFERENCE   2  (bases 1 to 8018)
  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: "Genome 
            Biol."; date: "2017-10-6"; volume: "18(1):190. doi"; pages: " 
            10.1186/s13059-017-1318-8"
COMMENT     Sequence Label: pX330-Flag-SpCas9-HF1  (without sgRNA)
FEATURES             Location/Qualifiers
     source          1..8018
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          519..746
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      758..767
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             767..832
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             839..859
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(884..2368,2369..2371,2372..2860,2861..2863,2864..2962,
                     2963..2965,2966..3655,3656..3658,3659..4984)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4985..5032
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5060..5077)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5066..5273
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5282..5422
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5282..5411
                     /label=AAV2 ITR
     rep_origin      5497..5952
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5584..5603)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5794..5815
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5969..5988)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6088..6110
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6148..6166)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6234..6338
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6339..6407,6408..7199)
                     /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(6557..6576)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7370..7958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7859..7878
                     /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 gggagaggtg
      421 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
      481 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
      541 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
      601 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca
      661 agaggtaagg gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc
      721 acctgcctga aatcactttt tttcaggttg gaccggtgcc accatggact ataaggacca
      781 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
      841 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
      901 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
      961 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     1021 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     1081 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     1141 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     1201 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     1261 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     1321 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     1381 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     1441 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     1501 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     1561 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     1621 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     1681 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     1741 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     1801 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     1861 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     1921 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     1981 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     2041 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     2101 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     2161 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     2221 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgacaag
     2281 aaagtccgaa gaaacgatta ctccctggaa ttttgaggaa gttgtcgata aaggtgcgtc
     2341 agctcaatcg ttcatcgaga ggatgaccgc ctttgacaag aatttaccga acgaaaaagt
     2401 attgcctaag cacagtttac tttacgagta tttcacagtg tacaatgaac tcacgaaagt
     2461 taagtatgtc actgagggca tgcgtaaacc cgcctttcta agcggagaac agaagaaagc
     2521 aatagtagat ctgttattca agaccaaccg caaagtgaca gttaagcaat tgaaagagga
     2581 ctactttaag aaaattgaat gcttcgattc tgtcgagatc tccggggtag aagatcgatt
     2641 taatgcgtca cttggtacgt atcatgacct cctaaagata attaaagata aggacttcct
     2701 ggataacgaa gagaatgaag atatcttaga agatatagtg ttgactctta ccctctttga
     2761 agatcgggaa atgattgagg aaagactaaa aacatacgct cacctgttcg acgataaggt
     2821 tatgaaacag ttaaagaggc gtcgctatac gggctgggga gccttgtcgc ggaaacttat
     2881 caacgggata agagacaagc aaagtggtaa aactattctc gattttctaa agagcgacgg
     2941 cttcgccaat aggaacttta tggccctgat ccatgatgac tctttaacct tcaaagagga
     3001 tatacaaaag gcacaggttt ccggacaagg ggactcattg cacgaacata ttgcgaatct
     3061 tgctggttcg ccagccatca aaaagggcat actccagaca gtcaaagtag tggatgagct
     3121 agttaaggtc atgggacgtc acaaaccgga aaacattgta atcgagatgg cacgcgaaaa
     3181 tcaaacgact cagaaggggc aaaaaaacag tcgagagcgg atgaagagaa tagaagaggg
     3241 tattaaagaa ctgggcagcc agatcttaaa ggagcatcct gtggaaaata cccaattgca
     3301 gaacgagaaa ctttacctct attacctaca aaatggaagg gacatgtatg ttgatcagga
     3361 actggacata aaccgtttat ctgattacga cgtcgatcac attgtacccc aatccttttt
     3421 gaaggacgat tcaatcgaca ataaagtgct tacacgctcg gataagaacc gagggaaaag
     3481 tgacaatgtt ccaagcgagg aagtcgtaaa gaaaatgaag aactattggc ggcagctcct
     3541 aaatgcgaaa ctgataacgc aaagaaagtt cgataactta actaaagctg agaggggtgg
     3601 cttgtctgaa cttgacaagg ccggatttat taaacgtcag ctcgtggaaa cccgcgccat
     3661 cacaaagcat gttgcgcaga tactagattc ccgaatgaat acgaaatacg acgagaacga
     3721 taagctgatt cgggaagtca aagtaatcac tttaaagtca aaattggtgt cggacttcag
     3781 aaaggatttt caattctata aagttaggga gataaataac taccaccatg cgcacgacgc
     3841 ttatcttaat gccgtcgtag ggaccgcact cattaagaaa tacccgaagc tagaaagtga
     3901 gtttgtgtat ggtgattaca aagtttatga cgtccgtaag atgatcgcga aaagcgaaca
     3961 ggagataggc aaggctacag ccaaatactt cttttattct aacattatga atttctttaa
     4021 gacggaaatc actctggcaa acggagagat acgcaaacga cctttaattg aaaccaatgg
     4081 ggagacaggt gaaatcgtat gggataaggg ccgggacttc gcgacggtga gaaaagtttt
     4141 gtccatgccc caagtcaaca tagtaaagaa aactgaggtg cagaccggag ggttttcaaa
     4201 ggaatcgatt cttccaaaaa ggaatagtga taagctcatc gctcgtaaaa aggactggga
     4261 cccgaaaaag tacggtggct tcgatagccc tacagttgcc tattctgtcc tagtagtggc
     4321 aaaagttgag aagggaaaat ccaagaaact gaagtcagtc aaagaattat tggggataac
     4381 gattatggag cgctcgtctt ttgaaaagaa ccccatcgac ttccttgagg cgaaaggtta
     4441 caaggaagta aaaaaggatc tcataattaa actaccaaag tatagtctgt ttgagttaga
     4501 aaatggccga aaacggatgt tggctagcgc cggagagctt caaaagggga acgaactcgc
     4561 actaccgtct aaatacgtga atttcctgta tttagcgtcc cattacgaga agttgaaagg
     4621 ttcacctgaa gataacgaac agaagcaact ttttgttgag cagcacaaac attatctcga
     4681 cgaaatcata gagcaaattt cggaattcag taagagagtc atcctagctg atgccaatct
     4741 ggacaaagta ttaagcgcat acaacaagca cagggataaa cccatacgtg agcaggcgga
     4801 aaatattatc catttgttta ctcttaccaa cctcggcgct ccagccgcat tcaagtattt
     4861 tgacacaacg atagatcgca aacgatacac ttctaccaag gaggtgctag acgcgacact
     4921 gattcaccaa tccatcacgg gattatatga aactcggata gatttgtcac agcttggggg
     4981 tgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt
     5041 cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     5101 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     5161 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     5221 gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagcggccg
     5281 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5341 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5401 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5461 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5521 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5581 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5641 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5701 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5761 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5821 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     5881 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5941 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6001 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6061 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6121 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6181 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6241 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6301 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6361 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6421 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6481 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6541 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6601 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6661 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6721 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6781 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6841 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     6901 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     6961 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7021 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7081 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7141 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7201 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7261 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7321 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7381 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7441 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7501 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7561 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7621 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7681 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7741 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7801 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7861 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     7921 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     7981 tacggttcct ggccttttgc tggccttttg ctcacatg
//
