LOCUS       sequence-376673-        5939 bp    DNA     circular SYN 15-DEC-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 5939)
  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 5939)
  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-GFP-3'UTR-HP
FEATURES             Location/Qualifiers
     source          1..5939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        94..473
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        475..752
                     /label=chicken beta-actin promoter
     primer_bind     1693..1715
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1847..1856
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1853..1855,1856..1858,1859..2572)
                     /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(1853..1855,1856..1858,1859..2569)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1898..1919)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2159..2178)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2506..2527
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    2603..2622
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     polyA_signal    2631..2752
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2642..2661)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2696..2715
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3099..3554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3186..3205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3396..3417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3621..3640)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3740..3762
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3800..3818)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3886..3990
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3991..4059,4060..4851)
                     /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(4209..4228)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5022..5610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5511..5530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5764..5781
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaatcaatat tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt
       61 atattggctc atgtccaata tgaccgccat gttggcattg attattgact agttattaat
      121 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      181 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      241 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      301 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc
      361 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac
      421 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      481 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      541 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg
      601 cgccrggsgg ggsggggsgg ggsgrggggs ggggsggggc gaggcggaga ggtgcggcgg
      661 cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc
      721 ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg
      781 tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc
      841 cacaggtgag cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat
      901 gacggcttgt ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt
      961 tgtgcggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt
     1021 gcggctccgc gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc
     1081 tccgcagtgt gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct
     1141 gcgaggggaa caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg
     1201 gcgcgtcggt cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc
     1261 cggcttcggg tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg
     1321 ggtggcggca ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg grgggctcgg
     1381 gggrggggcg cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc
     1441 attgcctttt atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg
     1501 cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt
     1561 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     1621 cccttctccc tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac
     1681 ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagacgg gctgtaatta
     1741 gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta
     1801 cctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtcgcca ccatggtgag
     1861 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1921 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1981 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2041 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     2101 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2161 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     2221 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     2281 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     2341 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     2401 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2461 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2521 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aactcgagcg
     2581 gccgcttcga gcagacatga tagttcactg ccgtataggc agctaagaaa taagatacat
     2641 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     2701 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     2761 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa
     2821 gtaaaacctc tacaaatgtg gtaaaatcga taaggatctg aggaacccct agtgatggag
     2881 ttggccactc cccccccctg cagcctggcg taatagcgaa gaggcccgca ccgatcgccc
     2941 ttcccaacag yykckmmrmm tgwrkkrmra wygrwmrgga tctgaggaac ccctagtgat
     3001 ggagttggcc actccccccc cctgcagcct ggcgtaatag cgaagaggcc cgcaccgatc
     3061 gcccttccca acagttgcgc agcctgaatg gcgaatggac gcgccctgta gcggcgcatt
     3121 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3181 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3241 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3301 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3361 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3421 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3481 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     3541 aacgcttaca atttcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3601 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3661 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3721 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3781 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3841 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3901 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3961 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4021 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4081 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4141 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4201 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4261 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4321 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4381 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4441 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4501 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4561 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4621 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4681 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4741 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4801 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4861 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4921 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4981 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5041 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5101 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5161 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5221 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5281 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5341 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5401 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5461 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5521 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5581 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5641 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5701 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5761 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     5821 gcgcgttggc cgattcatta atgcaggctg cagagaggga gtggccaact ccatcactag
     5881 gggttcctca gatcttcaat attggccatt agccatatta ttcattggtt atatagcat
//
