LOCUS       sequence-161521-        1029 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 1029)
  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 1029)
  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..1029
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     protein_bind    128..147
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     CDS             join(157..159,160..873)
                     /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             join(157..159,160..870)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(199..220)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(460..479)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     807..828
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    902..1023
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(913..932)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     967..986
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 ggctgtaatt agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta
       61 atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtcgcc
      121 accatgagtt cactgccgta taggcagcta agaaatgtga gcaagggcga ggagctgttc
      181 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      241 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
      301 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
      361 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      421 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      481 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      541 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      601 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
      661 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
      721 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
      781 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
      841 atcactctcg gcatggacga gctgtacaag taactcgagc ggccgcttcg agcagacatg
      901 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
      961 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     1021 gttaacaac
//
