LOCUS       sequence-63808-e         972 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  This plasmid separately encodes a human codon-optimized SpCas9 
            nickase, a tracrRNA and customizable crRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 972)
  AUTHORS   Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, 
            Jiang W, Marraffini LA, Zhang F
  TITLE     Multiplex Genome Engineering Using CRISPR/Cas Systems.
  JOURNAL   Science. 2013 Jan 3.
  PUBMED    23287718
REFERENCE   2  (bases 1 to 972)
  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: "Science. 
            2013 Jan 3."
COMMENT     Sequence Label: 
            pX334-U6-DR-BB-DR-Cbh-NLS-hSpCas9n(D10A)-NLS-H1-shorttracr-PGK-puro
FEATURES             Location/Qualifiers
     source          1..972
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    complement(75..108)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(184..205)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        complement(279..357)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(368..582)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(428..447)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     polyA_signal    596..803
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     792..809
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(846..866)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 cgcctcccct acccggtaga atttcgaggt cgagatccta agcttggctg gacgtaaact
       61 cctcttcaga cctaataact tcgtatagca tacattatac gaagttatat taagggttat
      121 tgaatatgat cggaattctt acttgtacag ctcgtccatg ccgagagtga tcccggcggc
      181 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc
      241 ggactgggtg ctcaggtagt ggttgtcggc gccaaaaagc accgactcgg tgccactttt
      301 tcaagttgat aacggactag ccttatttta acttgctatg ctgttttgaa tggttccaac
      361 tagatctgtg gtctcataca gaacttataa gattcccaaa tccaaagaca tttcacgttt
      421 atggtgattt cccagaacac atagcgacat gcaaatattg cagggcgcca ctcccctgtc
      481 cctcacagcc atcttcctgc cagggcgcac gcgcgctggg tgttcccgcc tagtgacact
      541 gggcccgcga ttccttggag cgggttgatg acgtcagcgt tcgaattgcg gccgctcccc
      601 agcatgcctg ctattctctt cccaatcctc ccccttgctg tcctgcccca ccccaccccc
      661 cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt attaggaaag
      721 gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga
      781 tggctggcaa ctagaaggca cagtcgaggc tgatcagcga gctctaggaa ttcttagctg
      841 gcctccacct ttctcttctt cttggggctg tcgcctccca gctgagacag gtcgatccgt
      901 gtctcgtaca ggccggtgat gctctggtgg atcagggtgg cgtccagcac ctctttggtg
      961 ctggtgtacc tc
//
