LOCUS       sequence-161525-         918 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expresses GFP with an Csy4 target hairpin inserted immediately 
            following the start codon for Csy4-mediated regulation. Cloned into 
            an Adeno-Associated Virus backbone for packaging into AAV..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 918)
  AUTHORS   Borchardt EK, Vandoros LA, Huang M, Lackey PE, Marzluff WF, Asokan A
  TITLE     Controlling mRNA stability and translation with the CRISPR 
            endoribonuclease Csy4.
  JOURNAL   RNA. 2015 Nov;21(11):1921-30. doi: 10.1261/rna.051227.115. Epub 2015
            Sep 9.
  PUBMED    26354771
REFERENCE   2  (bases 1 to 918)
  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: 
            "10.1261/rna.051227.115"; journalName: "RNA"; date: "2015-11"; 
            volume: "21"; issue: "11"; pages: "1921-30"
COMMENT     Sequence Label: TR- ATG-HP-GFP
FEATURES             Location/Qualifiers
     source          1..918
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             complement(join(17..730,731..733))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(20..730,731..733))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(62..83)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     411..430
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     670..691
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(743..762)
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
ORIGIN
        1 gaagcggccg ctcgagttac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
       61 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg
      121 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
      181 tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
      241 agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
      301 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
      361 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
      421 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
      481 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
      541 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
      601 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
      661 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
      721 cgcccttgct cacatttctt agctgcctat acggcagtga actcatggtg gcgaccggtc
      781 caacctgaaa aaaagtgatt tcaggcaggt gctccaggta attaaacatt aataccccac
      841 caaccaacca tcccttaaac ccttacctct tgctcagcta attacagccc gtctagagcc
      901 gccggtcaca cgccagaa
//
