LOCUS       sequence-106837-        1035 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 1035)
  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 1035)
  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..1035
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(7..606)
                     /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(90..110)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     587..606
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(607..660)
                     /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             complement(676..723)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 gaattctcag gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt cgggcacctc
       61 gacgtcggcg gtgacggtga agccgagccg ctcgtagaag gggaggttgc ggggcgcgga
      121 ggtctccagg aaggcgggca ccccggcgcg ctcggccgcc tccactccgg ggagcacgac
      181 ggcgctgccc agacccttgc cctggtggtc gggcgagact ccgacggtgg ccaggaacca
      241 cgcgggctcc ttgggccggt gcggcgccag gaggccttcc atctgttgct gcgcggccag
      301 ccgggaaccg ctcaactcgg ccatgcgcgg gccgatctcg gcgaacaccg cccccgcttc
      361 gacgctctcc ggcgtggtcc agaccgccac cgcggcgccg tcgtccgcga cccacacctt
      421 gccgatgtcg agcccgacgc gcgtgaggaa gagttcttgc agctcggtga cccgctcgat
      481 gtggcggtcc ggatcgacgg tgtggcgcgt ggcggggtag tcggcgaacg cggcggcgag
      541 ggtgcgtacg gccctgggga cgtcgtcgcg ggtggcgagg cgcaccgtgg gcttgtactc
      601 ggtcattggg ccaggattct cctcgacgtc accgcatgtt agcagacttc ctctgccctc
      661 tccactgccg aattcctttt tcttttttgc ctggccggcc tttttcgtgg ccgccggcct
      721 tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg tacaggccgg tgatgctctg
      781 gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg tacctcttcc ggtcgatggt
      841 ggtgtcaaag tacttgaagg cggcaggggc tcccagattg gtcagggtaa acaggtggat
      901 gatattctcg gcctgctctc tgatgggctt atcccggtgc ttgttgtagg cggacagcac
      961 tttgtccaga ttagcgtcgg ccaggatcac tctcttggag aactcgctga tctgctcgat
     1021 gatctcgtcc aggta
//
