LOCUS       sequence-141304-         993 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  CbAkt promoter driven Cas9-2A-GFP (from px458).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 993)
  AUTHORS   Lonowski LA, Narimatsu Y, Riaz A, Delay CE, Yang Z, Niola F, Duda K,
            Ober EA, Clausen H, Wandall HH, Hansen SH, Bennett EP, Frodin M
  TITLE     Genome editing using FACS enrichment of nuclease-expressing cells 
            and indel detection by amplicon analysis.
  JOURNAL   Nat Protoc. 2017 Mar;12(3):581-603. doi: 10.1038/nprot.2016.165. 
            Epub 2017 Feb 16.
  PUBMED    28207001
REFERENCE   2  (bases 1 to 993)
  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.2016.165"; journalName: "Nat Protoc"; date: 
            "2017-03"; volume: "12"; issue: "3"; pages: "581-603"
COMMENT     Sequence Label: CAS9PBKS
FEATURES             Location/Qualifiers
     source          1..993
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             37..84
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             100..153
                     /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             join(154..156,157..867)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(154..156,157..867)
                     /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(196..217)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(457..476)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     804..825
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(895..912)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc cacgaaaaag
       61 gccggccagg caaaaaagaa aaaggaattc ggcagtggag agggcagagg aagtctgcta
      121 acatgcggtg acgtcgagga gaatcctggc ccagtgagca agggcgagga gctgttcacc
      181 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      241 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      301 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      361 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      421 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      481 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      541 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      601 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
      661 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      721 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
      781 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
      841 actctcggca tggacgagct gtacaaggaa ttctaactag agctcgctga tcagcctcga
      901 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
      961 tggaaggtgc cactcccact gtcctttcct aat
//
