LOCUS       sequence-268585-        6354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expression of TRE(TRE3G)-driven, Cre-dependent, humanized 
            channelrhodopsin E123T/T159C mutant fused to EYFP for optogenetic 
            activation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  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 6354)
  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-ChR2 (E123T/T159C)-EYFP
FEATURES             Location/Qualifiers
     source          1..6354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        147..525
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    155..173
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    191..209
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    227..245
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    263..281
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    299..317
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    335..353
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    371..389
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     434..458
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(538..571)
                     /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(622..655)
                     /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(667..1380,1381..1383,1384..1386))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(712..733)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1061..1080
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1320..1341
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1383..1392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1402..2328)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYATWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGCIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      2325..2334
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2339..2372
                     /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    2423..2456
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2481..3069
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2534..2554)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2952..2963)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3101..3577
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3300..3319)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3617..3757
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3617..3746
                     /label=AAV2 ITR
     rep_origin      3832..4287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3919..3938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4129..4150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4304..4323)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4423..4445
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4483..4501)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4569..4673
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4674..4742,4743..5534)
                     /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(4892..4911)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5705..6293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6194..6213
                     /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 gcggccgcac gcgttcgagt ttactcccta tcagtgatag agaacgtatg
      181 aagagtttac tccctatcag tgatagagaa cgtatgcaga ctttactccc tatcagtgat
      241 agagaacgta taaggagttt actccctatc agtgatagag aacgtatgac cagtttactc
      301 cctatcagtg atagagaacg tatctacagt ttactcccta tcagtgatag agaacgtata
      361 tccagtttac tccctatcag tgatagagaa cgtataagct ttaggcgtgt acggtgggcg
      421 cctataaaag cagagctcgt ttagtgaacc gtcagatcgc ctggagcaat tccacaacac
      481 ttttgtctta taccaacttt ccgtaccact tcctaccctc gtaaagtcga ctccggaata
      541 acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta
      601 ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc
      661 gccctattac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
      721 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actggtagct
      781 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
      841 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
      901 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
      961 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
     1021 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
     1081 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
     1141 gtgctgcttc atgtggtcgg ggtagcgggc gaagcactgc aggccgtagc cgaaggtggt
     1201 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
     1261 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
     1321 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
     1381 caccatggtg gcggccgctg gcacggctcc ggcctcggct tcgtcttcga cgagagtctc
     1441 gacctcgatc tccgttccgc cgatgttcag tttggtggtt ttgcggatat ctccgtgaat
     1501 caatatgtgc tcgtggatca ggacgcgcag gtagtgtccc aacaaccccc aacaattttt
     1561 actcatcaga tcaataatcg tgtgacctac ggtggagcca tagacgctca ggacgccaaa
     1621 accttcgggc cccaaaatga agagaattgg gaacataccc cagctcacga aaaacagcca
     1681 tgccatgccg gtcacgacct ggcggcaccg accctttggc acagtatgat aaccctcgat
     1741 atatgctttg gcggcgtgaa aaaatgtgtt cgcgccatag cacaatccaa gacaaaagaa
     1801 gatgacttta acatagccgg ttgccatggc gctggtagcc ccccacacga tacacccgat
     1861 gtctgagaca aggagtccca tggttctcct gctgtagtcg ttgctcaggc cggtgaggtt
     1921 gctcaggtgg ataaggatga caggacaagt gagcagccag gttgcatagc gcagccactg
     1981 cacccggtgt cctgtggcaa ggtagagcat agagggattc ttaaactcaa aaaagaactc
     2041 gagaatcacc ttaaccattt caatggcgca cacatagatc tcctcccagc cgcatgtaga
     2101 tttccaggtt tggtaggcat agaacatcag cagcaaaatg ctgaatcctg ctgcaagcca
     2161 ctgcaggaca tttgacgcgg tctgagcgcc gttcgtgccg cgagattcaa tccatccggc
     2221 acagtaacat tgatcctcag ggaccaggac ggacccgttc accaccacag gattagtaac
     2281 gaacaaaagt tcgcgtccga cggcagacaa agcgccgcca tagtccatgg tggctagcat
     2341 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt
     2401 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat
     2461 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     2521 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     2581 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2641 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2701 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2761 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2821 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     2881 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     2941 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3001 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3061 cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc
     3121 ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct
     3181 aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg
     3241 gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt
     3301 ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc
     3361 ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat
     3421 gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag
     3481 gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg
     3541 attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg
     3601 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3661 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3721 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3781 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3841 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3901 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3961 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4021 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     4081 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4141 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     4201 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4261 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     4321 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4381 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4441 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4501 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4561 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4621 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4681 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4741 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4801 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4861 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4921 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4981 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5041 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5101 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5161 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5221 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5281 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5341 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5401 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5461 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     5521 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     5581 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5641 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5701 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5761 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5821 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5881 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5941 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6001 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6061 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6121 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6181 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6241 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6301 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
