LOCUS       sequence-194895-        7940 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized dead/inactive increased
            fidelity HeFSpCas9 (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7940)
  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 7940)
  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-dHeFSpCas9
FEATURES             Location/Qualifiers
     source          1..7940
                     /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"
     regulatory      683..692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             692..757
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             764..784
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(809..3346,3347..3349,3350..3811,3812..3814,3815..3982,
                     3983..3985,3986..4909)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4910..4957
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4982..4999)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4988..5195
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5204..5344
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5204..5333
                     /label=AAV2 ITR
     rep_origin      5419..5874
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5506..5525)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5716..5737
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5891..5910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6010..6032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6070..6088)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6156..6260
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6261..6329,6330..7121)
                     /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(6479..6498)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7292..7880
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7781..7800
                     /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 gggcggggtg ccttcgcccc gtgccccgct
      481 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      541 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
      601 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
      661 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
      721 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
      781 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcgggc tagccatcgg
      841 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
      901 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
      961 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1021 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1081 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1141 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1201 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1261 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1321 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1381 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1441 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     1501 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     1561 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     1621 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     1681 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     1741 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     1801 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     1861 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     1921 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     1981 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2041 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2101 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2161 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2221 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2281 cgagcggatg accgccttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2341 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2401 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     2461 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     2521 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     2581 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     2641 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     2701 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     2761 gcggcggaga tacaccggct ggggcgcgct gagccggaag ctgatcaacg gcatccgcga
     2821 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     2881 cttcatggcg ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     2941 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctcgccg gcagccccgc
     3001 cattaagaag ggcatcctcc agacagtgaa ggtggtggac gagctggtga aagtgatggg
     3061 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     3121 gggacagaag aacagtcgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     3181 cagccagatc ctgaaagaac accccgtgga aaacacccag ctccagaacg agaagctgta
     3241 cctgtactac ctccagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3301 gctgtccgac tacgatgtgg acgccatcgt gcctcagagc tttctggcgg acgactccat
     3361 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     3421 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     3481 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     3541 taaggccggc ttcatcaaga gacagctggt ggaaacccgg gcgatcacaa agcacgtggc
     3601 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     3661 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     3721 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     3781 cgtgggaacc gccctgatca aaaagtaccc tgcgctggaa agcgagttcg tgtacggcga
     3841 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     3901 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     3961 ggccaacggc gagatccgga aggcgcctct gatcgagaca aacggcgaaa ccggggagat
     4021 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     4081 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     4141 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggatccta agaagtacgg
     4201 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     4261 caagtccaag aagcttaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     4321 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     4381 ggacctgatc atcaagctgc ctaagtactc gctcttcgag ctggaaaacg gccggaagag
     4441 aatgctggcc tctgccggcg aattgcagaa gggaaacgaa ctggctcttc cctccaaata
     4501 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     4561 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     4621 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     4681 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     4741 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     4801 taggaagagg tacacgtcga ccaaagaggt gctggacgcc accctgatcc accagagcat
     4861 caccggcctg tacgaaacgc gtatcgacct gtctcagctg ggaggcgaca aaaggccggc
     4921 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa ttcctagagc tcgctgatca
     4981 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     5041 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     5101 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     5161 gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc cctagtgatg
     5221 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     5281 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     5341 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5401 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5461 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     5521 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5581 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5641 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5701 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     5761 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     5821 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5881 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5941 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     6001 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     6061 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     6121 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6181 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6241 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6301 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6361 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6421 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6481 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6541 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6601 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6661 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6721 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6781 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6841 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6901 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6961 gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat ggtaagccct
     7021 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7081 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7141 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7201 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7261 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7321 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7381 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7441 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7501 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7561 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7621 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7681 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7741 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7801 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7861 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7921 gctggccttt tgctcacatg
//
