LOCUS       sequence-152070-        5957 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses GFP from the CBA promoter. Includes a Csy4 target hairpin 
            in the 3'UTR. 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-GFP-3'UTR-HP
FEATURES             Location/Qualifiers
     source          1..5957
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      120..129
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(126..128,129..131,132..845)
                     /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(126..128,129..131,132..842)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     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"
     protein_bind    876..895
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     polyA_signal    904..1025
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(915..934)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     969..988
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      1376..1831
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1463..1482)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1673..1694
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1898..1917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2017..2039
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2077..2095)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2163..2267
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2268..2336,2337..3128)
                     /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(2486..2505)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3299..3887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3788..3807
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4322..4701
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4703..4980
                     /label=chicken beta-actin promoter
     primer_bind     5923..5945
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
ORIGIN
        1 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat
       61 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtcg
      121 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 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 agtaactcga gcggccgctt cgagcagaca tgatagttca ctgccgtata ggcagctaag
      901 aaataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
      961 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     1021 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag atgtgggagg
     1081 ttttttaaag caagtaaaac ctctacaaat gtggtaaaat cgataaggat ctaggaaccc
     1141 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgcccgg
     1201 gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
     1261 agagagggag tggccaaccc cccccccccc ccccctgcag cctggcgtaa tagcgaagag
     1321 gcccgcaccg atcgcccttc ccaacagttg cgtagcctga atggcgaatg gcgcgacgcg
     1381 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1441 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1501 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1561 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1621 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1681 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1741 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1801 aattttaaca aaatattaac gtttacaatt tcctgatgcg ctattttctc cttacgcatc
     1861 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     1921 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     1981 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     2041 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     2101 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     2161 tgcgcggaac ccctatttgt ttatttttct aaatactttc aaatatgtat ccgctcatga
     2221 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     2281 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     2341 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     2401 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     2461 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     2521 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2581 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2641 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2701 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2761 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     2821 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2881 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2941 ctggctggtt tattgcggat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     3001 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     3061 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     3121 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     3181 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3241 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3301 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3361 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3421 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3481 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3541 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3601 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3661 acaccgaact gagataccta cagcgtgagc attgagaaag cgccacgctt cccgaaggga
     3721 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3781 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3841 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3901 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3961 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctgggg
     4021 gggggggggg gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     4081 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     4141 cagagaggga gtggccaact ccatcactag gggttcctag atcttcaata ttggccatta
     4201 gccatattat tcattggtta tatagcataa atcaatattg gatattggcc attgcatacg
     4261 ttgtatctat atcataatat gtacatttat attggctcat gtccaatatg accgccatgt
     4321 tggcattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     4381 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4441 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4501 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4561 caagtgtatc atatgccaag tccgccccct attgacgtca atgacggtaa atggcccgcc
     4621 tggcattatg cccagtacat gaccttacgg gactttccta cttggcagta catctacgta
     4681 ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc
     4741 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg
     4801 atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
     4861 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
     4921 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     4981 ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
     5041 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
     5101 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
     5161 aagccttgag gggctccggg agggcccttt gtgcgggggg gagcggctcg gggggtgcgt
     5221 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg
     5281 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     5341 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     5401 gtgcgtgggg gggtgagcag ggggtatggg cgcggcggtc gggctgtaac ccccccctgc
     5461 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     5521 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     5581 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     5641 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     5701 cgcagggact tactttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     5761 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     5821 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     5881 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     5941 tgaccggcgg ctctaga
//
