LOCUS       sequence-412365-        8076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized increased fidelity 
            evoSpCas9 (without U6-sgRNA coding sequence, with silent mutations).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8076)
  AUTHORS   Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E
  TITLE     Blackjack mutations improve the on-target activities of increased 
            fidelity variants of SpCas9 with 5'G-extended sgRNAs.
  JOURNAL   Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.
  PUBMED    32144253
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: "Nat 
            Commun."; date: "2020-03-6"; volume: "11(1):1223. doi"; pages: " 
            10.1038/s41467-020-15021-5"
COMMENT     Sequence Label: pX330-Flag-evoSpCas9 (without sgRNA; with sile...
FEATURES             Location/Qualifiers
     source          1..8076
                     /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"
     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             942..5042
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 variant with improved
                     specificity (Casini et al., 2018)"
                     /label=evoCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERVTNFDKNLPNEKVLPKHSLLNEYF
                     TVYNELTEVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGQLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     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(5118..5135)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5124..5331
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5340..5480
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5340..5469
                     /label=AAV2 ITR
     rep_origin      5555..6010
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5642..5661)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5852..5873
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6027..6046)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6146..6168
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6206..6224)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6292..6396
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6397..6465,6466..7257)
                     /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(6615..6634)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7428..8016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7917..7936
                     /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 sgsgsgcsgg gggggggggg gggggggggg gggggggggg gggsgaggcg 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 gcctggacat 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 gtgaccaact tcgataagaa cctgcccaac gagaaggtgc
     2461 tgcccaagca cagcctgctg aacgagtact tcaccgtgta taacgagctg accgaagtga
     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 gctggggcca 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 tggaccatat cgtgcctcag agctttctga
     3481 aggacgactc 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 cacgacgcgt
     3901 acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt
     3961 tcgtgtacgg cgactacaag gtgtacgacg tacggaagat gatcgccaag agcgagcagg
     4021 aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga
     4081 ccgagattac cctggccaac ggcgagatcc ggaagcggcc 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 taagaattcc
     5101 tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     5161 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     5221 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     5281 gcaggacagc aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca
     5341 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     5401 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     5461 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     5521 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
     5581 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
     5641 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5701 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5761 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5821 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5881 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
     5941 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     6001 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6061 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     6121 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     6181 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     6241 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     6301 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     6361 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     6421 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     6481 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     6541 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     6601 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6661 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6721 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6781 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6841 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6901 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6961 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     7021 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     7081 attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg
     7141 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     7201 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     7261 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     7321 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     7381 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     7441 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     7501 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     7561 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     7621 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7681 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7741 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7801 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7861 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7921 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7981 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     8041 cggttcctgg ccttttgctg gccttttgct cacatg
//
