LOCUS       sequence-361682-        6311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is for use with neuronal cell-type selective activity 
            tagging. The ChR2-EYFP expression requires both neural activity and 
            Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6311)
  AUTHORS   Yu X, Zhao G, Wang D, Wang S, Li R, Li A, Wang H, Nollet M, Chun YY,
            Zhao T, Yustos R, Li H, Zhao J, Li J, Cai M, Vyssotski AL, Li Y, 
            Dong H, Franks NP, Wisden W
  TITLE     A specific circuit in the midbrain detects stress and induces 
            restorative sleep.
  JOURNAL   Science. 2022 Jul;377(6601):63-72. doi: 10.1126/science.abn0853. 
            Epub 2022 Jun 30.
  PUBMED    35771921
REFERENCE   2  (bases 1 to 6311)
  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: 
            "10.1126/science.abn0853"; journalName: "Science"; date: "2022-07"; 
            volume: "377"; issue: "6601"; pages: "63-72"
COMMENT     Sequence Label: pAAV-TRE-DIO-ChR2-EYFP
FEATURES             Location/Qualifiers
     source          1..6311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        113..427
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    121..139
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    157..175
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    192..210
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    228..246
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    264..282
                     /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"
     primer_bind     400..424
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(467..500)
                     /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(551..584)
                     /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(596..1309,1310..1312,1313..1315))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(641..662)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     990..1009
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1249..1270
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1312..1321
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1331..2257)
                     /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
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      2254..2263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2268..2301
                     /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    2352..2385
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2410..2998
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2463..2483)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2881..2892)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3030..3506
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3229..3248)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3546..3686
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3546..3675
                     /label=AAV2 ITR
     rep_origin      3761..4216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3848..3867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4058..4079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4233..4252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4352..4374
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4412..4430)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4498..4602
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4603..4671,4672..5463)
                     /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(4821..4840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5634..6222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6123..6142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6284..6311,1..102)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
       61 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtttac
      121 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgat
      181 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
      241 tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttatc
      301 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
      361 tcgaggtagg cgtgtacggt gggaggccta tataagcaga gctcgtttag tgaaccgtca
      421 gatcgcctgg agaattcgag ctcggtaccc gggggtcgac tccggaataa cttcgtatag
      481 gatactttat acgaagttat gcagaatggt agctggattg tagctgctat tagcaatatg
      541 aaacctctta ataacttcgt atagcataca ttatacgaag ttatggcgcg ccctattact
      601 tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca
      661 tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctggtagctc aggtagtggt
      721 tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga
      781 gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct
      841 tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc
      901 ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg
      961 tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga
     1021 tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca
     1081 tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc gaaggtggtc acgagggtgg
     1141 gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg
     1201 tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca
     1261 gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc accatggtgg
     1321 cggccgctgg cacggctccg gcctcggctt cgtcttcgac gagagtctcg acctcgatct
     1381 ccgttccgcc gatgttcagt ttggtggttt tgcggatatc tccgtgaatc aatatgtgct
     1441 cgtggatcag gacgcgcagg tagtgtccca acaaccccca acaattttta ctcatcagat
     1501 caataatcgt gtgacctacg gtggagccat agacgctcag gacgccaaaa ccttcgggcc
     1561 ccaaaatgaa gagaattggg aacatacccc agctcacgaa aaacagccat gccatgccgg
     1621 tcacgacctg gcggcaccga ccctttggca cagtatgata accctcgata tatgctttgg
     1681 cggcgtgaaa aaatgtgttc gcgccatagc acaatccaag acaaaagaag atgactttaa
     1741 catagccggt tgccatggcg ctggtagccc cccacacgat agtcccgatg tctgagacaa
     1801 ggagtcccat ggttctcctg ctgtagtcgt tgctcaggcc ggtgaggttg ctcaggcgga
     1861 taaggatgac aggacaagtg agcagccact ctgcatagcg cagccactgc acccggtgtc
     1921 ctgtggcaag gtagagcata gagggattct taaactcaaa aaagaactcg agaatcacct
     1981 taaccatttc aatggcgcac acatagatct cctcccagcc gcatgtagat ttccaggttt
     2041 ggtaggcata gaacatcagc agcaaaatgc tgaatcctgc tgcaagccac tgcaggacat
     2101 ttgacgcggt ctgagcgccg ttcgtgccgc gagattcaat ccatccggca cagtaacatt
     2161 gatcctcagg gaccaggacg gacccgttca ccaccacagg attagtaacg aacaaaagtt
     2221 cgcgtccgac ggcagacaaa gcgccgccat agtccatggt ggctagcata acttcgtata
     2281 aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta ttctttagaa
     2341 tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt cgatatcaag
     2401 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     2461 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     2521 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     2581 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     2641 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     2701 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     2761 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     2821 tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     2881 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     2941 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     3001 cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc tccccagtgc
     3061 ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa
     3121 gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg
     3181 tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac
     3241 caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag
     3301 cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca
     3361 gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca
     3421 actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt
     3481 gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg
     3541 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3601 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3661 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     3721 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     3781 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     3841 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     3901 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     3961 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4021 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4081 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     4141 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4201 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4261 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4321 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4381 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     4441 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     4501 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4561 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4621 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4681 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4741 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     4801 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     4861 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     4921 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     4981 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5041 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5101 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5161 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5221 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5281 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5341 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5401 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5461 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5521 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5581 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5641 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5701 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5761 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5821 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5881 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5941 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6001 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6061 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc
     6301 gctcgctcgc t
//
