LOCUS       sequence-106365-        5990 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cas9n (D10A nickase mutant) from S. pyogenes with 2A-Puro, and 
            cloning backbone for sgRNA (V2.0).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5990)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 5990)
  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: 
            "10.1038/nprot.2013.143"; journalName: "Nat Protoc"; date: 
            "2013-11"; volume: "8"; issue: "11"; pages: "2281-308"
COMMENT     Sequence Label: pSpCas9n(BB)-2A-Puro (PX462) V2.0
FEATURES             Location/Qualifiers
     source          1..5990
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     enhancer        1..286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        288..565
                     /label=chicken beta-actin promoter
     intron          566..794
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      806..815
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             815..880
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             887..907
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(932..955,956..958,959..5032)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             932..5032
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5033..5080
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5096..5149
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5150..5749
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(5150..5169)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5646..5666
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5777..5794)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5783..5990
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      181 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
      241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      301 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      361 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
      421 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
      481 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      541 taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc
      601 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
      661 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt
      721 ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa
      781 tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta
      841 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
      901 gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggccat
      961 cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa
     1021 attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct
     1081 gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag
     1141 atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc
     1201 caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa
     1261 gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa
     1321 gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct
     1381 gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga
     1441 gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac
     1501 ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat
     1561 cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg
     1621 cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa
     1681 cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta
     1741 cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct
     1801 ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga
     1861 gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga
     1921 cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt
     1981 cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga
     2041 gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt
     2101 gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc
     2161 ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc
     2221 attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta
     2281 cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga
     2341 aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt
     2401 catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca
     2461 cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac
     2521 cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct
     2581 gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa
     2641 aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct
     2701 gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga
     2761 aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat
     2821 gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct
     2881 gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg
     2941 ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag
     3001 aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc
     3061 ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc
     3121 cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat
     3181 gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca
     3241 gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct
     3301 gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct
     3361 gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa
     3421 ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc
     3481 catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc
     3541 ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct
     3601 gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact
     3661 ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt
     3721 ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg
     3781 ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca
     3841 gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc
     3901 cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg
     3961 cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa
     4021 ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac
     4081 cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga
     4141 gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca
     4201 agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct
     4261 gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta
     4321 cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa
     4381 gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag
     4441 aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa
     4501 aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa
     4561 gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa
     4621 atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga
     4681 taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga
     4741 gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct
     4801 gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca
     4861 cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat
     4921 cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag
     4981 catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc
     5041 ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag gaattcggca gtggagaggg
     5101 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccaa tgaccgagta
     5161 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     5221 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     5281 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     5341 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     5401 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     5461 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     5521 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     5581 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     5641 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     5701 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag aattctaact
     5761 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     5821 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     5881 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     5941 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
//
