LOCUS       sequence-163814-        1031 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  AAV backbone with a full length U6/TO promoter driving expression of
            a sgRNA. The gRNA can be inserted via Sapl sites. TracrRNA 
            compatible with SpCas9. gRNA transcription is doxycycline 
            dependent..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 1031)
  AUTHORS   de Solis CA, Ho A, Holehonnur R, Ploski JE
  TITLE     The Development of a Viral Mediated CRISPR/Cas9 System with 
            Doxycycline Dependent gRNA Expression for Inducible In vitro and In 
            vivo Genome Editing.
  JOURNAL   Front Mol Neurosci. 2016 Aug 18;9:70. doi: 10.3389/fnmol.2016.00070.
            eCollection 2016.
  PUBMED    27587996
REFERENCE   2  (bases 1 to 1031)
  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: "Front Mol 
            Neurosci."; date: "2016-08-18"; volume: "9:70. doi"; pages: " 
            10.3389/fnmol.2016.00070. eCollection 2016"
COMMENT     Sequence Label: 
            pJEP10-AAV-U6/TO-gRNA(Empty)-CMV-TetR-P2A-eGFP-KASH-pA
FEATURES             Location/Qualifiers
     source          1..1031
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             75..128
                     /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(129..131,132..842)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(129..131,132..842)
                     /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(171..192)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(432..451)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     779..800
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gccttgaatt gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtccgcgg
       61 ctagcggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
      121 ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtacccatc ctggtcgagc
      181 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      241 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      301 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
      361 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      421 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
      481 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
      541 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
      601 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
      661 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
      721 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
      781 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
      841 agtccggact cagatctcga gaggaggagg aggagacaga cagcaggatg ccccacctcg
      901 acagccccgg cagctcccag ccgagacgct ccttcctctc aagggtgatc agggcagcgc
      961 taccgttgca gctgcttctg ctgctgctgc tgctcctggc ctgcctgcta cctgcctctg
     1021 aagatgacta c
//
