LOCUS       sequence-145604-         660 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  All-in-One plasmid encoding dual U6 promoter-driven sgRNAs and 
            Cas9-D10A nickase linked via 2A peptide with puromycin resistant 
            marker to enhance efficient and accurate genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 660)
  AUTHORS   Chiang TW, le Sage C, Larrieu D, Demir M, Jackson SP
  TITLE     CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening 
            to enhance genome editing.
  JOURNAL   Sci Rep. 2016 Apr 15;6:24356. doi: 10.1038/srep24356.
  PUBMED    27079678
REFERENCE   2  (bases 1 to 660)
  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"; journalName: "Sci Rep."; 
            date: "2016-04-15"; volume: "6:24356. doi"; pages: " 
            10.1038/srep24356"
COMMENT     Sequence Label: AIO-Puro
FEATURES             Location/Qualifiers
     source          1..660
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        complement(79..327)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(307..327)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_RNA        complement(452..527)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 cagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa actgagaccc
       61 tctcatcgat ggtctcccgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc
      121 gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac
      181 tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt
      241 gtttacagtc aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata
      301 tgaaggaatc atgggaaata ggccctcttc ctgcccgacc tttcccttta gtgagggtta
      361 atctagagcc atttgtctgc agaattggcg cacgcgctaa aaacggacta gccttatttt
      421 aacttgctat ttctagctct aaaacaaaaa agcaccgact cggtgccact ttttcaagtt
      481 gataacggac tagccttatt ttaacttgct atttctagct ctaaaacagg tcttcgcatg
      541 gatccgaaga ccccggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
      601 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
//
