LOCUS       sequence-197615-        8073 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized dead/inactive increased
            fidelity eSpCas9 (1.1) (without U6-sgRNA coding sequence).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8073)
  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 8073)
  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-deSpCas9
FEATURES             Location/Qualifiers
     source          1..8073
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..804
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      816..825
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             825..890
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             897..917
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(942..3479,3480..3482,3483..3944,3945..3947,3948..4115,
                     4116..4118,4119..5042)
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5043..5090
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5115..5132)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5121..5328
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5337..5477
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5337..5466
                     /label=AAV2 ITR
     rep_origin      5552..6007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5639..5658)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5849..5870
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6024..6043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6143..6165
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6203..6221)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6289..6393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6394..6462,6463..7254)
                     /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(6612..6631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7425..8013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7914..7933
                     /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 gggcggggcg gggcgaggcg gagaggtgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      781 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg
      841 acggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa
      901 agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg
      961 ggctagccat cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc
     1021 ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga
     1081 tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg
     1141 ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca
     1201 acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg
     1261 aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct
     1321 accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca
     1381 aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact
     1441 tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc
     1501 tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg
     1561 ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc
     1621 agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc
     1681 tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca
     1741 aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg
     1801 acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag
     1861 tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc
     1921 accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca
     1981 aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca
     2041 gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg
     2101 aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg
     2161 gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag
     2221 atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca
     2281 tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa
     2341 agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg
     2401 cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2461 tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga
     2521 aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca
     2581 tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact
     2641 acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca
     2701 acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg
     2761 acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg
     2821 acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga
     2881 tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca
     2941 acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     3001 tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca
     3061 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg
     3121 ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg
     3181 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3241 agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca
     3301 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga
     3361 acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac
     3421 tggacatcaa ccggctgtcc gactacgatg tggacgccat cgtgcctcag agctttctgg
     3481 ccgacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg
     3541 acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga
     3601 acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc
     3661 tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca
     3721 caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca
     3781 agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga
     3841 aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct
     3901 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctgcgctg gaaagcgagt
     3961 tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg
     4021 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4081 ccgagattac cctggccaac ggcgagatcc ggaaggcgcc tctgatcgag acaaacggcg
     4141 aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga
     4201 gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag
     4261 agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc
     4321 ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca
     4381 aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca
     4441 tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca
     4501 aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa
     4561 acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc
     4621 tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct
     4681 cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg
     4741 agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg
     4801 acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga
     4861 atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg
     4921 acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga
     4981 tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg
     5041 acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taattcctag
     5101 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5161 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5221 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5281 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga
     5341 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     5401 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     5461 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5521 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     5581 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     5641 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5701 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5761 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5821 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5881 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     5941 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     6001 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     6061 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     6121 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     6181 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     6241 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     6301 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6361 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6421 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6481 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6541 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6601 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6661 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6721 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6781 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6841 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6901 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6961 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     7021 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     7081 gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca
     7141 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     7201 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     7261 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7321 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7381 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7441 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7501 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7561 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7621 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7681 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7741 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7801 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7861 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7921 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7981 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     8041 ttcctggcct tttgctggcc ttttgctcac atg
//
