LOCUS       sequence-268586-        5431 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of TRE(TRE3G)-driven, Cre-dependent, Clover 
            expression for fluorescent labling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5431)
  AUTHORS   Ebina T, Obara K, Watakabe A, Masamizu Y, Terada SI, Matoba R, 
            Takaji M, Hatanaka N, Nambu A, Mizukami H, Yamamori T, Matsuzaki M
  TITLE     Arm movements induced by noninvasive optogenetic stimulation of the 
            motor cortex in the common marmoset.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Nov 5;116(45):22844-22850. doi: 
            10.1073/pnas.1903445116. Epub 2019 Oct 21.
  PUBMED    31636197
REFERENCE   2  (bases 1 to 5431)
  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: 
            "10.1073/pnas.1903445116"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2019-11-5- 5"; volume: "116"; issue: "45"; pages: 
            "22844-22850"
COMMENT     Sequence Label: pAAV-TRE-flex-Clover
FEATURES             Location/Qualifiers
     source          1..5431
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     144..161
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     145..161
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        162..540
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    170..188
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    206..224
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    242..260
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    278..296
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    314..332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    350..368
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    386..404
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     449..473
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(546..579)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(630..663)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(693..1403,1404..1406,1407..1409))
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(735..756)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1343..1364
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1406..1415
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1432..1465
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1516..1549
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1585..2173
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1638..1658)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2056..2067)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2179..2655
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2378..2397)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2694..2834
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2694..2823
                     /label=AAV2 ITR
     rep_origin      2909..3364
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2996..3015)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3206..3227
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3381..3400)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3500..3522
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3560..3578)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3646..3750
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3751..3819,3820..4611)
                     /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(3969..3988)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4782..5370
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5271..5290
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcccgtc gactgtaaaa cgacggccag tgagtttact ccctatcagt
      181 gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta
      241 ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt
      301 atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt
      361 gatagagaac gtatatccag tttactccct atcagtgata gagaacgtat aagctttagg
      421 cgtgtacggt gggcgcctat aaaagcagag ctcgtttagt gaaccgtcag atcgcctgga
      481 gcaattccac aacacttttg tcttatacca actttccgta ccacttccta ccctcgtaaa
      541 gctagataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
      601 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
      661 tattggcgcg ccctattact tgcggccgct tacttgtaca gctcgtccat gccatgtgta
      721 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
      781 ttgctcaggg cggactgatg gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
      841 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcaacgttg
      901 tggcggatct tgaagttagc cttgatgccg ttcttctgct tgtcggccgt gatatagacg
      961 ttgtggctgt tgaagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     1021 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     1081 cgggtcttgt aggtaccgtc gtccttgaaa gagatggtgc gctcctggac gtagccttcg
     1141 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg gctgaagcag
     1201 gccacgccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     1261 gtgcagatga acttcagggt cagcttgccg ttggtggcat cgccctcgcc ctcgccgcgg
     1321 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     1381 gtgaacagct cctcgccctt gctcaccatg gtggcggcgc tagcgaattc gataacttcg
     1441 tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt
     1501 agaatggtgc aaagaataac ttcgtataat gtatgctata cgaagttatg gccgcggatc
     1561 agtcgaggga tcgagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     1621 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     1681 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     1741 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     1801 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     1861 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     1921 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     1981 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     2041 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     2101 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     2161 gccgcctccc cgcatcacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc
     2221 ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt
     2281 ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag
     2341 gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt
     2401 gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg
     2461 cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg
     2521 tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct
     2581 caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct
     2641 cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagggc cgcaggaacc
     2701 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     2761 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     2821 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2881 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     2941 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     3001 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3061 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3121 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3181 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3241 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     3301 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     3361 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     3421 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3481 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3541 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3601 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3661 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3721 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3781 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3841 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3901 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3961 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4021 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4081 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4141 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4201 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4261 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4321 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4381 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4441 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4501 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4561 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4621 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4681 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4741 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4801 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4861 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4921 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4981 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5041 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5101 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5161 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5221 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5281 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5341 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5401 ctggcctttt gctggccttt tgctcacatg t
//
