LOCUS       sequence-194897-        7916 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized increased fidelity 
            HeFm1SpCas9 (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7916)
  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 7916)
  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: HeFm1SpCas9
FEATURES             Location/Qualifiers
     source          1..7916
                     /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          417..644
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      656..665
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             665..730
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             737..757
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(782..3319,3320..3322,3323..3784,3785..3787,3788..3955,
                     3956..3958,3959..4882)
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4883..4930
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4958..4975)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4964..5171
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5180..5320
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5180..5309
                     /label=AAV2 ITR
     rep_origin      5395..5850
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5482..5501)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5692..5713
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5867..5886)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5986..6008
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6046..6064)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6132..6236
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6237..6305,6306..7097)
                     /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(6455..6474)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7268..7856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7757..7776
                     /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 ggggggggag
      421 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      481 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      541 gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa
      601 tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac
      661 catggactat aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga
      721 tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc
      781 cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat
      841 caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca
      901 cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc
      961 cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta
     1021 tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct tccacagact
     1081 ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa
     1141 catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa
     1201 actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat
     1261 gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt
     1321 ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat
     1381 caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg
     1441 gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct
     1501 gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga
     1561 tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca
     1621 gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct
     1681 gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat
     1741 gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca
     1801 gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg
     1861 ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga
     1921 aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa
     1981 gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc
     2041 cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga
     2101 gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag
     2161 attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt
     2221 ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa
     2281 cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta
     2341 taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag
     2401 cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt
     2461 gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc
     2521 cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat
     2581 caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct
     2641 gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca
     2701 cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcgc
     2761 gctgagccgg aagctgatca acggcatccg cgacaagcag tccggcaaga caatcctgga
     2821 tttcctgaag tccgacggct tcgccaacag aaacttcatg gcgctgatcc acgacgacag
     2881 cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca
     2941 cgagcacatt gccaatctcg ccggcagccc cgccattaag aagggcatcc tccagacagt
     3001 gaaggtggtg gacgagctgg tgaaagtgat gggccggcac aagcccgaga acatcgtgat
     3061 cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagtc gcgagagaat
     3121 gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt
     3181 ggaaaacacc cagctccaga acgagaagct gtacctgtac tacctccaga atgggcggga
     3241 tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat
     3301 cgtgcctcag agctttctgg cggacgactc catcgacaac aaggtgctga ccagaagcga
     3361 caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa
     3421 ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac
     3481 caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct
     3541 ggtggaaacc cgggcgatca caaagcacgt ggcacagatc ctggactccc ggatgaacac
     3601 taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa
     3661 gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta
     3721 ccaccacgcc cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta
     3781 ccctgcgctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat
     3841 gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa
     3901 catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaaggcgcc
     3961 tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc
     4021 caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca
     4081 gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc
     4141 cagaaagaag gactgggatc ctaagaagta cggcggcttc gacagcccca ccgtggccta
     4201 ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaagctta agagtgtgaa
     4261 agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt
     4321 tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta
     4381 ctcgctcttc gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaattgca
     4441 gaagggaaac gaactggctc ttccctccaa atatgtgaac ttcctgtacc tggccagcca
     4501 ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca
     4561 gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat
     4621 cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc
     4681 catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc
     4741 tgccgccttc aagtactttg acaccaccat cgataggaag aggtacacgt cgaccaaaga
     4801 ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaaa cgcgtatcga
     4861 cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa
     4921 aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     4981 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5041 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     5101 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     5161 catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     5221 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     5281 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     5341 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     5401 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5461 ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5521 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5581 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     5641 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5701 tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga
     5761 ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5821 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5881 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5941 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6001 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     6061 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     6121 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6181 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6241 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6301 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6361 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6421 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6481 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6541 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6601 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6661 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6721 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6781 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6841 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6901 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg
     6961 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7021 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7081 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7141 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7201 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7261 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7321 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7381 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7441 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7501 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7561 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7621 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7681 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7741 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7801 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7861 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatg
//
