LOCUS       sequence-125195-        8175 bp    DNA     circular SYN 14-APR-2025
DEFINITION  SpCas9 N863A nickase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8175)
  AUTHORS   Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, 
            Ishitani R, Zhang F, Nureki O
  TITLE     Crystal structure of Cas9 in complex with guide RNA and target DNA.
  JOURNAL   Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. 
            Epub 2014 Feb 13.
  PUBMED    24529477
REFERENCE   2  (bases 1 to 8175)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2014.02.001"; journalName: "Cell"; date: 
            "2014-02-27- 27"; volume: "156"; issue: "5"; pages: "935-49"
COMMENT     Sequence Label: PX429 SpCas9 N863A nickase
FEATURES             Location/Qualifiers
     source          1..8175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        160..463
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        465..742
                     /label=chicken beta-actin promoter
     intron          743..971
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      983..992
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             995..1015
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1040..1063,1064..1066,1067..3622,3623..3625,
                     3626..5140)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5141..5188
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5216..5233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5222..5429
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5438..5578
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5438..5567
                     /label=AAV2 ITR
     rep_origin      5653..6108
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5740..5759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5950..5971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6125..6144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6244..6266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6304..6322)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6390..6494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6495..6563,6564..7355)
                     /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(6713..6732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7526..8114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8015..8034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggcctct agaggtaccc gttacataac ttacggtaaa
      181 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      241 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      301 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      361 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      421 tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac
      481 gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat
      541 tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg
      601 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
      661 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
      721 aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct
      781 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
      841 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
      901 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
      961 ctttttttca ggttggaccg gtgccaccat ggccccaaag aagaagcgga aggtcggtat
     1021 ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg gcaccaactc
     1081 tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct
     1141 gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag
     1201 cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg
     1261 gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga
     1321 cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg
     1381 gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat
     1441 ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta
     1501 tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa
     1561 ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct
     1621 gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag
     1681 actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa
     1741 tggcctgttc ggcaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa
     1801 cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct
     1861 ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa
     1921 cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc
     1981 ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct
     2041 gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag
     2101 caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt
     2161 catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag
     2221 agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca
     2281 cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga
     2341 caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct
     2401 ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc
     2461 ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat
     2521 gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta
     2581 cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag
     2641 aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac
     2701 caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt
     2761 cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca
     2821 cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat
     2881 tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg
     2941 gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag
     3001 atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg acaagcagtc
     3061 cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatgca
     3121 gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg
     3181 ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg ccattaagaa
     3241 gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa
     3301 gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa
     3361 gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat
     3421 cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt acctgtacta
     3481 cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga
     3541 ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca tcgacaacaa
     3601 ggtgctgacc agaagcgaca aggcccgggg caagagcgac aacgtgccct ccgaagaggt
     3661 cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag
     3721 aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg
     3781 cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg cacagatcct
     3841 ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt
     3901 gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt
     3961 gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg tcgtgggaac
     4021 cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg actacaaggt
     4081 gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa
     4141 gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg
     4201 cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga
     4261 taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt
     4321 gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa
     4381 cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga
     4441 cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa
     4501 gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga
     4561 gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat
     4621 catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc
     4681 ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt
     4741 cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa
     4801 acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga
     4861 gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa
     4921 caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct
     4981 gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag
     5041 gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct
     5101 gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa
     5161 aaaggccggc caggcaaaaa agaaaaagta agaattccta gagctcgctg atcagcctcg
     5221 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     5281 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     5341 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     5401 tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg
     5461 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     5521 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     5581 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     5641 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5701 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5761 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5821 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     5881 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5941 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt
     6001 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6061 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     6121 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     6181 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6241 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     6301 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6361 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6421 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     6481 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     6541 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     6601 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     6661 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6721 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6781 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6841 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6901 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6961 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7021 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7081 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7141 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7201 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7261 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7321 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7381 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     7441 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     7501 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     7561 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     7621 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     7681 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7741 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7801 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7861 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7921 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7981 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     8041 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     8101 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     8161 cttttgctca catgt
//
