LOCUS       sequence-62314-e         966 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of customizable guide RNA (gRNA) under cotnrol of murine 
            U6 promoter, also contains a CMV-puro-t2A-mCherry expression 
            cassette for mammalian gene knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 966)
  AUTHORS   Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 
            WA
  TITLE     Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific 
            Control of Gene Expression.
  JOURNAL   Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
  PUBMED    23452860
REFERENCE   2  (bases 1 to 966)
  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: 
            "10.1016/j.cell.2013.02"; journalName: "Cell"; date: "2013-02-28- 
            28"; volume: "152"; issue: "5"; pages: "1173-83"
COMMENT     Sequence Label: pgRNA-humanized
FEATURES             Location/Qualifiers
     source          1..966
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     regulatory      115..124
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(131..184)
                     /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(194..790)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(274..294)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     771..790
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(824..923)
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     complement(853..873)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 cctcgccctc gccctcgatc tcgaactcgt ggccgttcac ggagccctcc atgtgcacct
       61 tgaagcgcat gaactccttg atgatggcca tgttatcctc ctcgcccttg ctcaccatgg
      121 tggcgaccgg tgggccagga ttctcctcga cgtcaccgca tgttagcaga cttcctctgc
      181 cctctcccga tccggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca
      241 cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg
      301 cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca
      361 cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga
      421 accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg
      481 ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg
      541 cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca
      601 ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct
      661 cgatgtggcg gtccgggtcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg
      721 cgagggtgcg tacggcccgg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt
      781 actcggtcat ggtgggctag cggatctgac ggttcactaa accagctctg cttatataga
      841 cctcccaccg tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt
      901 ttggaaagtc ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga
      961 gacttg
//
