LOCUS       sequence-64847-e        1045 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 1045)
  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 1045)
  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..1045
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     primer_bind     2..25
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    complement(121..154)
                     /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(230..251)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_RNA        complement(325..403)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(414..628)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(474..493)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     polyA_signal    642..849
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     838..855
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(892..912)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 ccagcggggc tgctaaagcg catgctccag actgccttgg gaaaagcgcc tcccctaccc
       61 ggtagaattt cgaggtcgag atcctaagct tggctggacg taaactcctc ttcagaccta
      121 ataacttcgt atagcataca ttatacgaag ttatattaag ggttattgaa tatgatcgga
      181 attcttactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
      241 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca
      301 ggtagtggtt gtcggcgcca aaaagcaccg actcggtgcc actttttcaa gttgataacg
      361 gactagcctt attttaactt gctatgctgt tttgaatggt tccaactaga tctgtggtct
      421 catacagaac ttataagatt cccaaatcca aagacatttc acgtttatgg tgatttccca
      481 gaacacatag cgacatgcaa atattgcagg gcgccactcc cctgtccctc acagccatct
      541 tcctgccagg gcgcacgcgc gctgggtgtt cccgcctagt gacactgggc ccgcgattcc
      601 ttggagcggg ttgatgacgt cagcgttcga attgcggccg ctccccagca tgcctgctat
      661 tctcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
      721 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
      781 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
      841 aaggcacagt cgaggctgat cagcgagctc taggaattct tagctggcct ccacctttct
      901 cttcttcttg gggctgtcgc ctcccagctg agacaggtcg atccgtgtct cgtacaggcc
      961 ggtgatgctc tggtggatca gggtggcgtc cagcacctct ttggtgctgg tgtacctctt
     1021 ccggtcgatg gtggtgtcaa agtac
//
