LOCUS       sequence-361681-        6817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is for use with neuronal cell-type selective activity 
            tagging. The hM4Di receptor expression requires both neural activity
            and Cre recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  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 6817)
  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-hM4Di-mCherry
FEATURES             Location/Qualifiers
     source          1..6817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..59
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(102..135)
                     /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(186..219)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(228..938)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(506..525)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     775..793
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      935..944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2393..2402
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2409..2442
                     /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    2493..2526
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2551..3139
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2604..2624)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3022..3033)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3171..3647
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3370..3389)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3687..3827
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3687..3816
                     /label=AAV2 ITR
     rep_origin      3902..4357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3989..4008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4199..4220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4374..4393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4493..4515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4553..4571)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4639..4743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4744..4812,4813..5604)
                     /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(4962..4981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5775..6363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6264..6283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6425..6554
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(6565..6817,1..62)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    6573..6591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6609..6627
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6644..6662
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6680..6698
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6716..6734
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6751..6769
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6787..6805
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac cgtcagatcg
       61 cctggagaat tcgagctcgg tacccggggg tcgactccgg aataacttcg tataggatac
      121 tttatacgaa gttatgcaga atggtagctg gattgtagct gctattagca atatgaaacc
      181 tcttaataac ttcgtatagc atacattata cgaagttatg gcgcgcctta cttgtacagc
      241 tcgtccatgc cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg
      301 tagtcctcgt tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc gccgggcagc
      361 tgcacgggct tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc
      421 ttcagcttca gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc
      481 tcggaggagg cctcccagcc catggtcttc ttctgcatta cggggccgtc ggaggggaag
      541 ttggtgccgc gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg
      601 gtcacggtca ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg
      661 gggaaggaca gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag
      721 ccgtacatga actgagggga caggatgtcc caggcgaagg gcagggggcc acccttggtc
      781 accttcagct tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc
      841 tcgaactcgt ggccgttcac ggagccctcc atgtgcacct tgaagcgcat gaactccttg
      901 atgatggcca tgttatcctc ctcgcccttg ctcaccatgg tggcgaccgg tggatcccgc
      961 ctggcagtgc cgatgttccg atactggcac agcagcaggt gccggaaggt ctttttaaag
     1021 gtggcgttgc acagagcata gcaggcaggg ttgatggtgc tgttgacgta gcagagccag
     1081 tagccaatgg accacaccgt gtcagggatg cagctctggc agaaggtgtt caccaggacc
     1141 atgacgttgt agggcgtcca ggtgaggatg aaggccagca gaatggcaaa gatcgttcgt
     1201 gtcactttgc gctcccgggc cgccatctgc cgcttcttgc gcacctggtt gcgagcgatg
     1261 ctggcgaact tgcgggccac gttggccgca gggcgcatgc cagccggcgt ggcaggcaca
     1321 atctcaatgg ctgtcacaca ctcattgcct gtctgcttcg tcacaatctg gatcttggac
     1381 catctggagg ctgggttgag ggcccgcggc tgcaggggag gggcgggcat ggcgggcgtg
     1441 gtggcctctg tggtggacag ctctgtggct gggcgttcct tggtgttctg ggtggcactg
     1501 cctgagctgg actcattgga agtgtcctta tcagccacgg ggcgcggtgg cggtggcagc
     1561 gctggcgggg gggcctcctc cagcttgcca ttgcgcagct cctcccgggc ggcctccccg
     1621 ggcgggggct tcttgacgct ctgcttcatt agtgggctct tgaggaaggc cagcgtcttg
     1681 gctttcttct ccttcgggcc ctcgggccgg tgcttgtgga ctcggctgcg actggccagg
     1741 gagatgtgga tgtacagcac cgtcatgatg accacaggca ggtagaagcc agcaatggct
     1801 gtgccaaagg tcactgctgg gttggacagg aactggatga agcactggtt gtcgggcacc
     1861 gtccgcttac ccaccacaaa ctgccagaac aagatggcag gcgcccagag cacgaaggac
     1921 agtacccagg cagcagcaat catgaggcct gccatcttgg tggtgcgccg ggcagggtag
     1981 gtgagaggct tggtgacgca gaagtagcgg tcaaagctga tgatgagaag gttcatgacg
     2041 gaggcgttgc tcaccacgca gtccagggcc agccacaggt cgcagaccac ggcgcccagg
     2101 ggccagtagc ccttgatgat gtacacggtg tagaggttca tggagaaggc gcctatgatg
     2161 agatcagcac acgccaggct gaagaggaag tagttgttga ctgtctgcag ctgcctgttg
     2221 accttgatgg acagcatcac caggatgttg cccacgacag tcaccaggct cagggagcct
     2281 gtcactgtgg caatgaagac catttccacc gtctcatagc gattgtggga tgatgacgtg
     2341 accaggcgca cggactgatt gcccgagctg ccattgacag gtgtgaagtt ggccatggtg
     2401 gcgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     2461 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     2521 agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     2581 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2641 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     2701 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     2761 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2821 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     2881 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     2941 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     3001 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3061 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3121 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     3181 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     3241 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     3301 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     3361 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     3421 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     3481 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     3541 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     3601 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     3661 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     3721 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3781 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     3841 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     3901 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3961 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4021 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4081 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4141 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4201 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     4261 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     4321 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     4381 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4441 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4501 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4561 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4681 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4741 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4801 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4861 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4921 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4981 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5041 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5101 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5161 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5221 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5281 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5341 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5401 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5461 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5521 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5581 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5641 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5701 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5761 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5821 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5881 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5941 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6001 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6061 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6121 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6181 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6241 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6301 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6361 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6421 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
     6481 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
     6541 cactaggggt tcctgcggcc gcacgcgttt actccctatc agtgatagag aacgtatgtc
     6601 gagtttactc cctatcagtg atagagaacg atgtcgagtt tactccctat cagtgataga
     6661 gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
     6721 atcagtgata gagaacgtat gtcgagttta tccctatcag tgatagagaa cgtatgtcga
     6781 gtttactccc tatcagtgat agagaacgta tgtcgag
//
