LOCUS       sequence-197504-        8076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized high-fidelity eSpCas9 
            (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8076)
  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 8076)
  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-eSpCas9 (without sgRNA)
FEATURES             Location/Qualifiers
     source          1..8076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        288..565
                     /label=chicken beta-actin promoter
     intron          566..793
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      805..814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             814..879
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             886..906
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(931..3468,3469..3471,3472..3933,3934..3936,3937..4104,
                     4105..4107,4108..5031)
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5032..5079
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5107..5124)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5113..5320
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5329..5469
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5329..5458
                     /label=AAV2 ITR
     rep_origin      5544..5999
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5631..5650)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5841..5862
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6016..6035)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6135..6157
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6195..6213)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6281..6385
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6386..6454,6455..7246)
                     /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(6604..6623)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7417..8005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7906..7925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      181 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
      241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      301 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      361 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
      421 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
      481 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      541 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
      601 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
      661 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
      721 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
      781 cacttttttt caggttggac cggtgccacc atggactata aggaccacga cggagactac
      841 aaggatcatg atattgatta caaagacgat gacgataaga tggccccaaa gaagaagcgg
      901 aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg cctggacatc
      961 ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa
     1021 ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat cggagccctg
     1081 ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc cagaagaaga
     1141 tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa cgagatggcc
     1201 aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga agaggataag
     1261 aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta ccacgagaag
     1321 taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg
     1381 cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt cctgatcgag
     1441 ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct ggtgcagacc
     1501 tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc caaggccatc
     1561 ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca gctgcccggc
     1621 gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct gacccccaac
     1681 ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa ggacacctac
     1741 gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga cctgtttctg
     1801 gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt gaacaccgag
     1861 atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca ccaccaggac
     1921 ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa agagattttc
     1981 ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag ccaggaagag
     2041 ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga actgctcgtg
     2101 aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg cagcatcccc
     2161 caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga tttttaccca
     2221 ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat cccctactac
     2281 gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa
     2341 accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc
     2401 atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct gcccaagcac
     2461 agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa atacgtgacc
     2521 gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat cgtggacctg
     2581 ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta cttcaagaaa
     2641 atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg
     2701 ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga caatgaggaa
     2761 aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga cagagagatg
     2821 atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat gaagcagctg
     2881 aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa cggcatccgg
     2941 gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt cgccaacaga
     3001 aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat ccagaaagcc
     3061 caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc cggcagcccc
     3121 gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg
     3181 ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca gaccacccag
     3241 aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat caaagagctg
     3301 ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa cgagaagctg
     3361 tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact ggacatcaac
     3421 cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctggc cgacgactcc
     3481 atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga caacgtgccc
     3541 tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa cgccaagctg
     3601 attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct gagcgaactg
     3661 gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac aaagcacgtg
     3721 gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa gctgatccgg
     3781 gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa ggatttccag
     3841 ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta cctgaacgcc
     3901 gtcgtgggaa ccgccctgat caaaaagtac cctgcgctgg aaagcgagtt cgtgtacggc
     3961 gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga aatcggcaag
     4021 gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc
     4081 ctggccaacg gcgagatccg gaaggcgcct ctgatcgaga caaacggcga aaccggggag
     4141 atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa
     4201 gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg
     4261 cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac
     4321 ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag
     4381 ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga
     4441 agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa
     4501 aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag
     4561 agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa
     4621 tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat
     4681 aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag
     4741 cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg
     4801 tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac
     4861 ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc
     4921 gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc
     4981 atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg
     5041 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct
     5101 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     5161 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     5221 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     5281 agggggagga ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag
     5341 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     5401 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     5461 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     5521 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5581 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     5641 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5701 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5761 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5821 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5881 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5941 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     6001 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     6061 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     6121 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     6181 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     6241 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6301 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6361 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6421 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6481 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     6541 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     6601 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6661 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6721 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6781 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6841 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6901 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6961 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     7021 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     7081 atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa
     7141 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     7201 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     7261 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     7321 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7381 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7441 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7501 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7561 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7621 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7681 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7741 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7801 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7861 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7921 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7981 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     8041 cttttgctgg ccttttgctc acatgctaga ggtacc
//
