LOCUS       sequence-78360-e        1126 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Cas9n (D10A nickase mutant) from S. pyogenes with 2A-EGFP, and 
            cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1126)
  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 1126)
  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-GFP (PX461)
FEATURES             Location/Qualifiers
     source          1..1126
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(join(13..723,724..726))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(13..723,724..726))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(55..76)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     404..423
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     663..684
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(727..780)
                     /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(796..843)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
ORIGIN
        1 tagttagaat tccttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa
       61 ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt
      121 gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg
      181 gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac
      241 cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta
      301 ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat
      361 gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc
      421 gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa
      481 gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt
      541 ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt
      601 cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt
      661 tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt
      721 gctcactggg ccaggattct cctcgacgtc accgcatgtt agcagacttc ctctgccctc
      781 tccactgccg aattcctttt tcttttttgc ctggccggcc tttttcgtgg ccgccggcct
      841 tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg tacaggccgg tgatgctctg
      901 gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg tacctcttcc ggtcgatggt
      961 ggtgtcaaag tacttgaagg cggcaggggc tcccagattg gtcagggtaa acaggtggat
     1021 gatattctcg gcctgctctc tgatgggctt atcccggtgc ttgttgtagg cggacagcac
     1081 tttgtccaga ttagcgtcgg ccaggatcac tctcttggag aactcg
//
