LOCUS       sequence-366066-        6017 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  his plasmid is for use with neuronal cell-type selective activity 
            tagging. The GCaMP6 expression requires both neural activity and Cre
            recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6017)
  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 6017)
  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-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..6017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..33
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    51..69
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    87..105
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    122..140
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    158..176
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     223..247
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(290..323)
                     /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(374..407)
                     /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(417..860,861..866,867..1301,1302..1319,
                     1320..1589,1590..1595,1596..1652,1653..1679,1680..1703,
                     1704..1706,1707..1739,1740..1742,1743..1760,1761..1769))
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     976..995
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1235..1256
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1362..1383)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1664..1673
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1766..1775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1797..1830
                     /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    1881..1914
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1939..2527
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1992..2012)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2410..2421)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2559..3035
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2758..2777)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3075..3215
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3075..3204
                     /label=AAV2 ITR
     rep_origin      3290..3745
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3377..3396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3587..3608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3762..3781)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3881..3903
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3941..3959)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4027..4131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4132..4200,4201..4992)
                     /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(4350..4369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5163..5751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5652..5671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5813..5942
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(5953..6017,1..250)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    5961..5979
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5997..6015
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 gatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
       61 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      121 atccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt
      181 atgtcgaggt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt tagtgaaccg
      241 tcagatcgcc tggagaattc gagctcggta cccgggggtc gactccggaa taacttcgta
      301 taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc tattagcaat
      361 atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc gcgccctcac
      421 ttcgctgtca tcatttgtac aaactcttcg tagtttacct gaccatcccc atcgatgtct
      481 gcttccctga tcatttcatc aacctcttca tctgttaact tctctccaag gtttgtcatc
      541 acgtggcgaa gctctgctgc actgatgtag ccattgccat ccttatcaaa cacaccgaac
      601 gcttctctaa tttcttcttc cgtgtccctg tatttcattt ttcttgccat cattgtcagg
      661 aactcaggga agtcgattgt gccgtcaccg tcggcatcta cttcattgat catgtcctgc
      721 agctctgctt ctgtggggtt ctgccccaga gaccgcatca ccgtccccag ctccttggtt
      781 gttattgtcc catccccgtc cttgtcaaat agggagaaag cctctttaaa ttctgcgatc
      841 tgctcttcag tcagttggtc cggcaggttg tactccagct tgtgccccag gatgttgccg
      901 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
      961 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1021 atgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1081 cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc
     1141 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctca
     1201 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     1261 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tgctccctcc ggtaccgccc
     1321 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
     1381 atgtgatcgc gcttctcgtt ggggtctttc gaaagtttgg actgcacgct caggtagtgg
     1441 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtaggcg
     1501 agctgcacgc cgccgtcctc gatgttgtgg cggatgtgga agttcgcctt gatgccgttc
     1561 ttctgcttgt cggccttgat atagacgttc tcgagtgagc tcagccgacc tatagctctg
     1621 actgcgtgac ctgtcttatt ccacttacga cgtgatgagt ccaccatggt ggcgagatcc
     1681 ttatcgtcat cgtcgtacag atcccgaccc atttgctgtc caccagtcat gctcgccata
     1741 ccatgatgat gatgatgatg agaacccatg gtggcgagat ctgagtcgag gctagcataa
     1801 cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat
     1861 tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatgaattc
     1921 gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     1981 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     2041 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     2101 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2161 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     2221 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2281 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg
     2341 tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc
     2401 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     2461 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     2521 tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc tgtgacccct
     2581 ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa
     2641 taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga
     2701 ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct
     2761 attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct
     2821 gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga
     2881 ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc
     2941 tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat
     3001 tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg
     3061 gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3121 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     3181 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     3241 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3301 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3361 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3421 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3481 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     3541 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3601 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     3661 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3721 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     3781 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3841 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3901 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3961 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     4021 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     4081 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     4141 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     4201 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     4261 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4321 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4381 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4441 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4501 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4561 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4621 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4681 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4741 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4801 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4861 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4921 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4981 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     5041 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     5101 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     5161 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     5221 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     5281 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5341 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5401 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5461 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5521 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5581 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5641 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5701 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5761 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gtcctgcagg
     5821 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc
     5881 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
     5941 ctgcggccgc acgcgtttac tccctatcag tgatagagaa cgtatgtcga gtttactccc
     6001 tatcagtgat agagaac
//
