LOCUS       sequence-408748-        8055 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression plasmid for human codon-optimized increased-fidelity 
            (i.e. high-fidelity) SpCas9 variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8055)
  AUTHORS   Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz 
            SL, Welker A, Vegi VL, Huszar K, Welker E
  TITLE     A cleavage rule for selection of increased-fidelity SpCas9 variants 
            with high efficiency and no detectable off-targets.
  JOURNAL   Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5.
  PUBMED    37717069
REFERENCE   2  (bases 1 to 8055)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2023-09-16"; volume: "14(1):5746. doi"; pages: " 
            10.1038/s41467-023-41393-5"
COMMENT     Sequence Label: B-HeFSpCas9-A661R
FEATURES             Location/Qualifiers
     source          1..8055
                     /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..4094,
                     4095..4097,4098..5021)
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             5022..5069
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5097..5114)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5103..5310
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5319..5459
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5319..5448
                     /label=AAV2 ITR
     rep_origin      5534..5989
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5621..5640)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5831..5852
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6006..6025)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6125..6147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6185..6203)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6271..6375
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6376..6444,6445..7236)
                     /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(6594..6613)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7407..7995
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7896..7915
                     /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 cgcgcgccgg ggggggcggg gcggggcgrg 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 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 atgaccgcct 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 cgacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct
     3001 tcgccaacag aaacttcatg gcgctgatcc acgacgacag cctgaccttt aaagaggaca
     3061 tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctcg
     3121 ccggcagccc cgccattaag aagggcatcc tccagacagt gaaggtggtg gacgagctgg
     3181 tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc
     3241 agaccaccca gaagggacag aagaacagtc gcgagagaat gaagcggatc gaagagggca
     3301 tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctccaga
     3361 acgagaagct gtacctgtac tacctccaga atgggcggga tatgtacgtg gaccaggaac
     3421 tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctgg
     3481 cggacgactc 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 cgggcgatca
     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 ccctgcgctg ggtggcaagg
     3961 tgtacgatgt acggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca
     4021 agtacttctt ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg
     4081 gcgagatccg gaaggcgcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg
     4141 ataagggccg ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg
     4201 tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga
     4261 acagcgataa gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg
     4321 acagccccac cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca
     4381 agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg
     4441 agaagaatcc catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga
     4501 tcatcaagct gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg
     4561 cctctgccgg cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact
     4621 tcctgtacct ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga
     4681 aacagctgtt tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg
     4741 agttctccaa gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca
     4801 acaagcaccg ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc
     4861 tgaccaatct gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga
     4921 ggtacaccag caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc
     4981 tgtacgagac acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga
     5041 aaaaggccgg ccaggcaaaa aagaaaaagt aagaattcct agagctcgct gatcagcctc
     5101 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     5161 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     5221 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     5281 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt
     5341 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg
     5401 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg
     5461 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     5521 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     5581 cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct
     5641 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     5701 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     5761 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     5821 ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat
     5881 tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt
     5941 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     6001 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     6061 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     6121 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     6181 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     6241 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     6301 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     6361 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     6421 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     6481 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     6541 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     6601 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6661 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6721 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6781 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6841 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6901 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6961 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     7021 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     7081 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     7141 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     7201 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     7261 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     7321 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7381 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     7441 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     7501 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     7561 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     7621 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7681 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7741 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7801 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7861 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7921 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7981 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     8041 ccttttgctc acatg
//
