LOCUS       sequence-97158-e        7845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Truncated form of SpCas9 without REC2 domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7845)
  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 7845)
  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"; 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: PX543 SpCas9(del175-307)
FEATURES             Location/Qualifiers
     source          1..7845
                     /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             992..1057
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1064..1084
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2312..4810
                     /codon_start=1
                     /product="C-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(C)
                     /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI
                     SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY
                     AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH
                     DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE
                     NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL
                     QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV
                     VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI
                     LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV
                     GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL
                     ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL
                     PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME
                     RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP
                     SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD
                     KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL
                     IHQSITGLYETRIDLSQLGGD"
     CDS             4811..4858
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4886..4903)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4892..5099
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5108..5248
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5108..5237
                     /label=AAV2 ITR
     rep_origin      5323..5778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5410..5429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5620..5641
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5795..5814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5914..5936
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5974..5992)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6060..6164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6165..6233,6234..7025)
                     /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(6383..6402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7196..7784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7685..7704
                     /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 ggactataag gaccacgacg gagactacaa
     1021 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1081 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1141 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1201 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1261 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1321 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1381 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1441 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1501 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1561 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1621 cgacctggtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     1681 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     1741 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     1801 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     1861 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     1921 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     1981 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     2041 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     2101 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     2161 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     2221 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     2281 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     2341 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     2401 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     2461 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     2521 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     2581 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     2641 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     2701 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     2761 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     2821 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     2881 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     2941 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     3001 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     3061 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     3121 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     3181 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg tgcctcagag
     3241 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3301 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     3361 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     3421 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     3481 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     3541 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     3601 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     3661 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     3721 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     3781 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     3841 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     3901 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     3961 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     4021 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     4081 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     4141 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     4201 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     4261 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4321 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     4381 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     4441 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     4501 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     4561 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     4621 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     4681 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     4741 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     4801 gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaagta
     4861 agaattccta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     4921 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4981 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5041 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     5101 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     5161 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     5221 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     5281 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5341 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5401 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5461 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5521 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5581 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5641 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     5701 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5761 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5821 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5881 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5941 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6001 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6061 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6121 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6181 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6241 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6301 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6361 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6421 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6481 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6541 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6601 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6661 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6721 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6781 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6841 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     6901 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6961 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7021 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7081 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7141 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7201 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7261 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7321 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     7381 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7441 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7501 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7561 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7621 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7681 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7741 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7801 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
