LOCUS       sequence-152065-        5957 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses GFP from the CBA promoter. Includes a Csy4 target hairpin 
            in the 5'UTR for Csy4-mediated regulation. Cloned into an 
            Adeno-Associated Virus backbone for packaging into AAV..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5957)
  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 5957)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-5'UTR-HP-GFP
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    16..137
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(27..46)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     81..100
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      488..943
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(575..594)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     785..806
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1010..1029)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1129..1151
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1189..1207)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1275..1379
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1380..1448,1449..2240)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1598..1617)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2411..2999
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2900..2919
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3434..3813
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3815..4092
                     /label=chicken beta-actin promoter
     primer_bind     5035..5057
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     protein_bind    5173..5192
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     regulatory      5219..5228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5225..5227,5228..5230,5231..5944)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5225..5227,5228..5230,5231..5941)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5270..5291)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5531..5550)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5878..5899
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt
       61 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa
      121 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg
      181 agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg
      241 atctaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
      301 gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
      361 gagcgagcgc gcagagaggg agtggccaac cccccccccc ccccccctgc agcctggcgt
      421 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgtagcct gaatggcgaa
      481 tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
      541 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      601 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      661 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      721 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      781 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      841 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      901 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcctgatg cgctattttc
      961 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     1021 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
     1081 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     1141 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
     1201 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1261 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatactt tcaaatatgt
     1321 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1381 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1441 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1501 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1561 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1621 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1681 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1741 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1801 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1861 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1921 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1981 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2041 cggcccttcc ggctggctgg tttattgcgg ataaatctgg agccggtgag cgtgggtctc
     2101 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2161 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2221 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2281 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2341 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2401 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2461 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2521 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2581 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2641 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2701 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2761 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc
     2821 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2881 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2941 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3001 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3061 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3121 ataccgctgg gggggggggg gggggttggc cactccctct ctgcgcgctc gctcgctcac
     3181 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3241 cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct agatcttcaa
     3301 tattggccat tagccatatt attcattggt tatatagcat aaatcaatat tggatattgg
     3361 ccattgcata cgttgtatct atatcataat atgtacattt atattggctc atgtccaata
     3421 tgaccgccat gttggcattg attattgact agttattaat agtaatcaat tacggggtca
     3481 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     3541 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     3601 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     3661 ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt
     3721 aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag
     3781 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
     3841 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
     3901 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
     3961 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
     4021 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
     4081 gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct ccgccgccgc
     4141 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
     4201 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
     4261 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg gggagcggct
     4321 cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggcccg cgctgcccgg
     4381 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
     4441 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
     4501 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtatg ggcgcggcgg tcgggctgta
     4561 acccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc
     4621 tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg
     4681 tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc
     4741 cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat
     4801 cgtgcgagag ggcgcaggga cttactttgt cccaaatctg tgcggagccg aaatctggga
     4861 ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg
     4921 aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc
     4981 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
     5041 gcttctggcg tgtgaccggc ggctctagac gggctgtaat tagctgagca agaggtaagg
     5101 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
     5161 aatcactttt tagttcactg ccgtataggc agctaagaaa tttcaggttg gaccggtcgc
     5221 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     5281 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     5341 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     5401 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     5461 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     5521 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     5581 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     5641 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     5701 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     5761 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     5821 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     5881 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     5941 gtaactcgag cggccgc
//
