LOCUS       sequence-374527-        6278 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flpo dependent tTA expression under the control of TRE promotor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6278)
  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 6278)
  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-TRE-FRT-2xtTA
FEATURES             Location/Qualifiers
     source          1..6278
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             390..500
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
     protein_bind    complement(614..661)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     complement(738..762)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    784..802
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    821..839
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    858..876
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    895..913
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    932..950
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    969..987
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1006..1024
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1043..1061
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     repeat_region   1096..1225
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(1287..1875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1367..1386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2046..2837,2838..2906))
                     /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     2669..2688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2907..3011)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3079..3097
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3135..3157)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3257..3276
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3293..3748)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3430..3451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3642..3661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3823..3963
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3834..3963
                     /label=AAV2 ITR
     polyA_signal    4003..4479
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     4261..4280
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    4511..5099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4617..4628
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     5026..5046
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    5196..5243
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             5912..6022
                     /codon_start=1
                     /product="3 tandem repeats of the minimal activation domain
                     of herpes simplex virus VP16 (Cheng et al., 2013)"
                     /label=VP48
                     /translation="DALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML"
ORIGIN
        1 gcaacaacgc aaagagcttc cggtgtgcct tgctgagtca cagagacggc gctaaggttc
       61 atctgggcac acgccccacc gaaaaacaat atgaaaccct ggagaatcag ctcgcattcc
      121 tgtgtcagca gggttttagt ctcgaaaacg ccctctatgc tctttctgcc gttggccatt
      181 ttacactcgg ctgcgtactt gaggagcaag aacatcaagt tgctaaagag gagcgcgaaa
      241 caccaaccac tgattccatg ccacctctct tgcggcaggc cattgaactc tttgatcgac
      301 aaggagcaga gcctgctttt cttttcggcc tcgagcttat catatgtgga cttgaaaagc
      361 aactgaagtg tgagtctgga ggacctgccg acgcccttga cgattttgac ttagacatgc
      421 tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg
      481 attttgacct tgacatgctc cccgggtaag tcgacacgcg tttaattaag tttaaacgaa
      541 gttcctatac ttagttcaga ataggaactt cggaatagga acttcagagc gctttggccg
      601 ctccggccaa agggaagttc ctatactttc tagagaatag gaacttcgga ataggaactt
      661 cggcgcgccg gatccaggct ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt
      721 ggatggcgtc tccaggcgat ctgacggttc actaaacgag ctctgcttat atagggggta
      781 gcttctctat cactgatagg gagctcgaga cgtgtcgact tctctatcac tgatagggag
      841 acgttctaga cgtaccttct ctatcactga tagggagctc gagacgtgtc gacttctcta
      901 tcactgatag ggagacgttc tagacgtacc ttctctatca ctgataggga gctcgagacg
      961 tgtcgacttc tctatcactg atagggagac gttctagacg taccttctct atcactgata
     1021 gggagctcga gacgtgtcga cttctctatc actgataggg agacgtcagg tggcacttga
     1081 tacgcgtgcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     1141 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcggc ctcagtgagc
     1201 gagcgagcgc gcagctgcct gcaggacatg tgagcaaaag gccagcaaaa ggccaggaac
     1261 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1321 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1381 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1441 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1501 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1561 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1621 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1681 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1741 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1801 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1861 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1921 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2041 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2101 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2161 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2221 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2281 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2341 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     2401 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     2461 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     2521 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     2581 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     2641 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     2701 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     2761 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     2821 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     2881 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     2941 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3001 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     3061 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     3121 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     3181 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     3241 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca taaaattgta
     3301 aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     3361 caatagaccg aaatcggcaa aatcccttat aaatcaaaag aatagcccga gatagagttg
     3421 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     3481 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc caaatcaagt
     3541 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     3601 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     3661 gcgggcgcta aggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     3721 gcgcttaatg cgccgctaca gggcgcgtac tatggttgct ttgacgtatg cggtgtgaaa
     3781 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg cccctgcagg cagctgcgcg
     3841 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     3901 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     3961 cctgcggccg ctcggtccgc acgtggttac ctacaaaatc agaaggacag ggaagggagc
     4021 agtggttcac gcctgtaatc ccagcaattt gggaggccaa ggtgggtaga tcacctgaga
     4081 ttaggagttg gagaccagcc tggccaatat ggtgaaaccc cgtctctacc aaaaaaacaa
     4141 aaattagctg agcctggtca tgcatgcctg gaatcccaac aactcgggag gctgaggcag
     4201 gagaatcgct tgaacccagg aggcggagat tgcagtgagc caagattgtg ccactgcact
     4261 ccagcttggt tcccaataga ccccgcaggc cctacaggtt gtcttcccaa cttgcccctt
     4321 gctccatacc acccccctcc accccataat attatagaag gacacctagt cagacaaaat
     4381 gatgcaactt aattttatta ggacaaggct ggtgggcact ggagtggcaa cttccagggc
     4441 caggagaggc actggggagg ggtcacaggg atgccacccg tagatctctc gagcagcgct
     4501 cggtatcgat gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga
     4561 agacgcggaa gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg
     4621 agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacataggcg
     4681 agcagccaag gaaaggacga tgatttcccc gacaacacca cggaattgtc agtgcccaac
     4741 agccgagccc ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata
     4801 gggaggggga aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca
     4861 gtgggggttg cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac
     4921 aactcctcat aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata
     4981 cgggaagcaa tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga
     5041 gcaacatagt taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta
     5101 tcgataagct tcctgcaggg aagttcctat tccgaagttc ctattctgaa ctaagtatag
     5161 gaacttcttg gatcaaaata ggagcgatcg caatggaagt tcctattccg aagttcctat
     5221 tctctagaaa gtataggaac ttcgtttaaa ctggccggcc actagtagat ctgaattcgc
     5281 caccatgagt cggctggata aatctaaagt cataaacggc gctctggaat tactcaatga
     5341 agtcggtatc gaaggcctga cgacaaggaa actcgctcaa aagctgggag ttgagcagcc
     5401 taccctgtac tggcacgtga agaacaagcg ggccctgctc gatgccctgg ccatcgagat
     5461 gctggacagg catcataccc acttctgccc cctggaaggc gagtcatggc aagactttct
     5521 gcggaacaac gccaagtcat tccgctgtgc tctcctctca catcgcgacg gggctaaagt
     5581 gcatctcggc acccgcccaa cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt
     5641 cctgtgtcag caaggcttct ccctggagaa cgcactgtac gctctgtccg ccgtgggcca
     5701 ctttacactg ggctgcgtat tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga
     5761 gacacctacc accgattcta tgcccccact tctgagacaa gcaattgagc tgttcgaccg
     5821 gcagggagcc gaacctgcct tccttttcgg cctggaacta atcatatgtg gcctggagaa
     5881 acagctaaag tgcgaaagcg gcgggccggc cgacgccctt gacgattttg acttagacat
     5941 gctcccagcc gatgcccttg acgactttga ccttgatatg ctgcctgctg acgctcttga
     6001 cgattttgac cttgacatgc tccccggggg ttccaccggt atgtccagac ttgataaatc
     6061 caaagtcata aacggtgccc tggagcttct taatgaggtg ggaattgagg gactgactac
     6121 caggaaactt gcccaaaaac ttggtgtcga gcagccaact ctgtactggc acgtaaagaa
     6181 taaacgggca cttcttgacg cattggctat cgaaatgctt gatagacatc atacacactt
     6241 ctgccctctt gagggggaat cttggcagga tttcttgc
//
