LOCUS       sequence-374526-        6746 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP expression under the control of TRE promotor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6746)
  AUTHORS   Glaser AK, Bishop KW, Barner LA, Susaki EA, Kubota SI, Gao G, 
            Serafin RB, Balaram P, Turschak E, Nicovich PR, Lai H, Lucas LAG, Yi
            Y, Nichols EK, Huang H, Reder NP, Wilson JJ, Sivakumar R, Shamskhou 
            E, Stoltzfus CR, Wei X, Hempton AK, Pende M, Murawala P, Dodt HU, 
            Imaizumi T, Shendure J, Beliveau BJ, Gerner MY, Xin L, Zhao H, True 
            LD, Reid RC, Chandrashekar J, Ueda HR, Svoboda K, Liu JTC
  TITLE     A hybrid open-top light-sheet microscope for versatile multi-scale 
            imaging of cleared tissues.
  JOURNAL   Nat Methods. 2022 May;19(5):613-619. doi: 
            10.1038/s41592-022-01468-5. Epub 2022 May 11.
  PUBMED    35545715
REFERENCE   2  (bases 1 to 6746)
  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: 
            "10.1038/s41592-022-01468-5"; journalName: "Nat Methods"; date: 
            "2022-05"; volume: "19"; issue: "5"; pages: "613-619"
COMMENT     Sequence Label: pAAV-7xTRE-3xeGFP
FEATURES             Location/Qualifiers
     source          1..6746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(95..114)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      908..1496
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1397..1416
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1558..1687
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    1722..1740
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1759..1777
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1796..1814
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1833..1851
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1870..1888
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1907..1925
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1944..1962
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1981..1999
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2021..2045
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2120..2129
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2126..2128,2129..2131,2132..2842)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2126..2128,2129..2131,2132..2842)
                     /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(2912..2914,2915..3625)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3695..3697,3698..4408)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    4430..5018
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4483..4503)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4901..4912)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    5050..5526
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(5249..5268)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5566..5706
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5566..5695
                     /label=AAV2 ITR
     rep_origin      5781..6236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5868..5887)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6078..6099
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6253..6272)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6372..6394
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6432..6450)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6518..6622
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6623..6691,6692..6746,1..737)
                     /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"
ORIGIN
        1 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
       61 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
      121 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
      181 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
      241 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
      301 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
      361 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
      421 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
      481 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
      541 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
      601 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
      661 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
      721 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
      781 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
      841 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      901 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      961 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1021 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     1081 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1141 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1201 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1261 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1321 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1381 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1441 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1501 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct
     1561 gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt
     1621 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     1681 ggttcctgcg gccgcacgcg tatcaagtgc cacctgacgt ctccctatca gtgatagaga
     1741 agtcgacacg tctcgagctc cctatcagtg atagagaagg tacgtctaga acgtctccct
     1801 atcagtgata gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc
     1861 tagaacgtct ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat
     1921 agagaaggta cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc
     1981 tccctatcag tgatagagaa gctaccccct atataagcag agctcgttta gtgaaccgtc
     2041 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
     2101 ccagcctgga tccactagtg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     2161 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2221 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2281 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2341 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2401 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2461 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2521 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2581 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2641 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2701 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2761 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2821 cggcatggac gagctgtaca agggttccgg acagcttttg aactttgacc ttttgaagtt
     2881 ggcaggagac gttgagtcca accccggacc cgtgtcgaaa ggggaggaac tgttcacggg
     2941 agtggtgcct atcctcgtcg aactcgacgg agatgtgaac ggacataagt tctcggtgtc
     3001 cggggaagga gaaggggacg ctacttacgg aaagctgact ctgaagttta tctgtaccac
     3061 tggaaagctc ccagtcccgt ggccaaccct tgtcaccacc ttgacttacg gagtgcaatg
     3121 tttctcccgg tacccggacc acatgaagca gcacgacttc ttcaaatccg cgatgccgga
     3181 aggatacgtg caggagcgga caatcttctt taaggacgac ggaaactata agaccagggc
     3241 cgaggtcaag tttgaggggg ataccctcgt gaacagaatt gaactgaagg gtattgactt
     3301 caaggaagat ggcaacatct tggggcataa gctcgaatac aattacaact cccacaacgt
     3361 ctacattatg gcggacaaac agaagaatgg tattaaggtc aatttcaaga tcaggcataa
     3421 cattgaggac ggatcggtgc agttggccga tcactaccaa cagaataccc cgattggaga
     3481 tgggccggtg ctcctgcctg ataaccacta cttgtccact cagtccgctc tgtcgaagga
     3541 cccaaacgaa aagcgggatc acatggtgct cctggaattc gtcactgcgg ctggtattac
     3601 cctggggatg gacgaactct acaaaggttc cggacagctt ttgaactttg accttttgaa
     3661 gttggcagga gacgttgagt ccaaccccgg acccgtgtcc aagggagagg aactcttcac
     3721 tggagtggtg ccgattctgg tggagctgga tggggacgtg aacggtcaca aattctccgt
     3781 gtcaggagaa ggagagggag atgctaccta cggaaaactt actctcaagt tcatctgcac
     3841 caccggaaag ctgccagtgc cgtggccgac acttgtgact acccttacct acggagtgca
     3901 atgcttctcg cggtatcctg accatatgaa gcaacatgat ttctttaagt ccgcaatgcc
     3961 tgaaggatat gtgcaggaaa gaactatctt tttcaaggat gatggaaact acaaaactcg
     4021 ggccgaagtc aagtttgaag gagacacgct cgtgaaccgg atcgaactta agggaattga
     4081 ctttaaggaa gatggaaaca ttctcggtca caagctggag tacaactaca actctcataa
     4141 cgtctacatc atggcagaca agcagaagaa cggtatcaag gtcaacttca aaatcaggca
     4201 caatattgaa gatggttcag tccagctggc agaccactat cagcaaaata ctccgatagg
     4261 agatggtcct gtgctgcttc ctgacaatca ttacctgtca actcagtcgg ccctgtcaaa
     4321 ggacccgaac gaaaagagag atcacatggt gttgctggag ttcgtgacgg cggcgggaat
     4381 caccctggga atggacgaat tgtacaaatg aaccggtaag cttatcgata atcaacctct
     4441 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4501 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4561 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4621 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4681 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     4741 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4801 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     4861 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4921 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4981 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     5041 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     5101 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     5161 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     5221 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     5281 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     5341 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     5401 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     5461 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     5521 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     5581 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     5641 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     5701 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     5761 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5821 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     5881 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5941 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     6001 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     6061 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     6121 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     6181 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     6241 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     6301 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6361 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     6421 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     6481 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     6541 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     6601 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     6661 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     6721 tgctgaagat cagttgggtg cacgag
//
