LOCUS       sequence-410972-        8240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized increased fidelity 
            eSpCas9 (without U6-sgRNA coding sequence, with silent mutations).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8240)
  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 8240)
  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-eSpCas9 (without sgRNA; with silent...
FEATURES             Location/Qualifiers
     source          1..8240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          152..379
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      391..400
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             400..465
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             472..492
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(517..3054,3055..3057,3058..3519,3520..3522,3523..3690,
                     3691..3693,3694..4617)
                     /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             4618..4665
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4693..4710)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4699..4906
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4915..5055
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4915..5044
                     /label=AAV2 ITR
     rep_origin      5130..5585
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5217..5236)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5427..5448
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5602..5621)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5721..5743
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5781..5799)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5867..5971
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5972..6040,6041..6832)
                     /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(6190..6209)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7003..7591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7492..7511
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7663..7948
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gcsgrgsggg gsgggggggg gsgrggggcg gggcggggcg aggcggagag gtgcggcggc
       61 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
      121 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
      181 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
      241 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga gcaagaggta
      301 agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg agcacctgcc
      361 tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga ccacgacgga
      421 gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc cccaaagaag
      481 aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag catcggcctg
      541 gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc
      601 aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa cctgatcgga
      661 gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag aaccgccaga
      721 agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt cagcaacgag
      781 atggccaagg tggacgacag cttcttccac agactggaag agtccttcct ggtggaagag
      841 gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt ggcctaccac
      901 gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac cgacaaggcc
      961 gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg ccacttcctg
     1021 atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat ccagctggtg
     1081 cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag
     1141 gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat cgcccagctg
     1201 cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct gggcctgacc
     1261 cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct gagcaaggac
     1321 acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta cgccgacctg
     1381 tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct gagagtgaac
     1441 accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga cgagcaccac
     1501 caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa gtacaaagag
     1561 attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg agccagccag
     1621 gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac cgaggaactg
     1681 ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga caacggcagc
     1741 atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca ggaagatttt
     1801 tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc
     1861 tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac cagaaagagc
     1921 gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc ttccgcccag
     1981 agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa ggtgctgccc
     2041 aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa agtgaaatac
     2101 gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa ggccatcgtg
     2161 gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga ggactacttc
     2221 aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg gttcaacgcc
     2281 tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt cctggacaat
     2341 gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt tgaggacaga
     2401 gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa agtgatgaag
     2461 cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct gatcaacggc
     2521 atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga cggcttcgcc
     2581 aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga ggacatccag
     2641 aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa tctggccggc
     2701 agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga gctcgtgaaa
     2761 gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga gaaccagacc
     2821 acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa
     2881 gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct gcagaacgag
     2941 aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca ggaactggac
     3001 atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt tctggccgac
     3061 gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa gagcgacaac
     3121 gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct gctgaacgcc
     3181 aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg cggcctgagc
     3241 gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca gatcacaaag
     3301 cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa tgacaagctg
     3361 atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt ccggaaggat
     3421 ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga cgcgtacctg
     3481 aacgccgtcg tgggaaccgc cctgatcaaa aagtaccctg cgctggaaag cgagttcgtg
     3541 tacggcgact acaaggtgta cgacgtacgg aagatgatcg ccaagagcga gcaggaaatc
     3601 ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt caagaccgag
     3661 attaccctgg ccaacggcga gatccggaag gcgcctctga tcgagacaaa cggcgaaacc
     3721 ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt gctgagcatg
     3781 ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag caaagagtct
     3841 atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg ggaccctaag
     3901 aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt ggccaaagtg
     3961 gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat caccatcatg
     4021 gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg ctacaaagaa
     4081 gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct ggaaaacggc
     4141 cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc
     4201 tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa gggctccccc
     4261 gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct ggacgagatc
     4321 atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa tctggacaaa
     4381 gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc cgagaatatc
     4441 atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta ctttgacacc
     4501 accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac cctgatccac
     4561 cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg aggcgacaaa
     4621 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagtaaga attcctagag
     4681 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     4741 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     4801 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     4861 acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg ccgcaggaac
     4921 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     4981 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5041 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     5101 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     5161 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     5221 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5281 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5341 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5401 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5461 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     5521 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     5581 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     5641 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5701 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5761 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     5821 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     5881 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     5941 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6001 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6061 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6121 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6181 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6241 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6301 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     6361 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     6421 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     6481 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     6541 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     6601 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     6661 tgataaatct ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga
     6721 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     6781 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     6841 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     6901 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6961 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     7021 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     7081 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     7141 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     7201 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     7261 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     7321 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     7381 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     7441 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     7501 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7561 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7621 cctggccttt tgctggcctt ttgctcacat gctagaggta cccgttacat aacttacggt
     7681 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc
     7741 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     7801 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     7861 gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     7921 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
     7981 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
     8041 tgcagcgatg ggggcggggg gggggggggg gggsngcccg gsgggggggg gggggggggg
     8101 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     8161 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     8221 gggggggggg gggggggggs
//
