LOCUS       sequence-216901-        7161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is for use with neuronal cell-type selective activity 
            tagging. The hM3Dq 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 7161)
  AUTHORS   Harding EC, Yu X, Miao A, Andrews N, Ma Y, Ye Z, Lignos L, Miracca 
            G, Ba W, Yustos R, Vyssotski AL, Wisden W, Franks NP
  TITLE     A Neuronal Hub Binding Sleep Initiation and Body Cooling in Response
            to a Warm External Stimulus.
  JOURNAL   Curr Biol. 2018 Jul 23;28(14):2263-2273.e4. doi: 
            10.1016/j.cub.2018.05.054. Epub 2018 Jul 12.
  PUBMED    30017485
REFERENCE   2  (bases 1 to 7161)
  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: 
            "10.1016/j.cub.2018.05.054"; journalName: "Curr Biol"; date: 
            "2018-07-23- 23"; volume: "28"; issue: "14"; pages: "2263-2273.e4"
COMMENT     Sequence Label: pAAV-PTRE-tight-flex-hM3Dq-mCherry-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        152..466
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    160..178
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    196..214
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    231..249
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    267..285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    338..356
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     439..463
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(506..539)
                     /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(590..623)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(632..1342)
                     /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(910..929)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1179..1197
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1339..1348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3146..3179
                     /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    3230..3263
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3288..3876
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3341..3361)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3759..3770)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3908..4384
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4107..4126)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4424..4564
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4424..4553
                     /label=AAV2 ITR
     rep_origin      4639..5094
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4726..4745)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4936..4957
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5111..5130)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5230..5252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5376..5480
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5481..5549,5550..6341)
                     /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(5699..5718)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6512..7100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7001..7020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtttact ccctatcagt gatagagaac
      181 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      241 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      301 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      421 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattcgagc
      481 tcggtacccg ggggtcgact ccggaataac ttcgtatagg atactttata cgaagttatg
      541 cagaatggta gctggattgt agctgctatt agcaatatga aacctcttaa taacttcgta
      601 tagcatacat tatacgaagt tatggcgcgc cttacttgta cagctcgtcc atgccgccgg
      661 tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc tcgttgtggg
      721 aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg ggcttcttgg
      781 ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc ttcagcctct
      841 gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag gaggcctccc
      901 agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg ccgcgcagct
      961 tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg gtcaccacgc
     1021 cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag gacagcttca
     1081 agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac atgaactgag
     1141 gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc agcttggcgg
     1201 tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac tcgtggccgt
     1261 tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg gccatgttat
     1321 cctcctcgcc cttgctcacc atggtggcga ccggtggatc cttcaaggcc tgctcgggtg
     1381 cgcgcttgtg aaaaatgacc gactgtctct gctggtactg ctgcttgcgc ctcttttttt
     1441 tgtcacactg gcacagcagc agcatcttga aagtggttct gaatgttttg ttgcacagag
     1501 catagcacac ggggttcacg gtgctgttga tgtagcacag ccagtagccc agattccaaa
     1561 aggttttggg tatgcagctg tcacaaaagg tgttcaccag aaccatgatg ttgtatgggg
     1621 tccaagtgat gatgaaggca agcaagatcg cactgagggt ctgggccgct ttcttctcct
     1681 tgaccaggga catccttttc cgcttagtga tctgacttct ggtcttcaga gcaaacctct
     1741 tggccagagt ggcttccttg aaggacagag gtagagtggc cgtgctctta cccactgagg
     1801 agttgacgtc agaagtctta gctgtgtcca cggctgactc tagctggatg ggaagcttgg
     1861 agaagctttt tggaaaactg cctccatcgt ccacgctctt ctgggcctgc agcttgtcgg
     1921 ctttcctctc caagtccacc atccccagct cctcctcagg cacctgcagg ttgtccgatg
     1981 agggtaactt ggtggagttg aggatggtgc tgtgacccgg aagcttgagc acgatggagt
     2041 agatggctct cgtctcggag ccaatgtcct cctcgtcgga ggaggcggag ttctccaggg
     2101 aggcagcagc atcattgttg ttccaactgt cactgctgct gtggtcttgg tccatctgct
     2161 cggagctggg tttccagctc ttggttgtga accagaagtg gcagcggcca tacttcctcc
     2221 tgttggagcg tttcatgctt tgctgttgaa gttcgtaact gctgcagctt cgagaactgc
     2281 ccgtggggtg gacaaagttt tctgtctctg cctctgtccc agaggcttgc aggccagcaa
     2341 gctctttggt acgcttttca gtttccttat agatcctcca gtataaaata gtcataatgg
     2401 tgacaggcat ataaaaacca gcgatggctg tgccaaaagt aatggtgggc tcactgagga
     2461 actgaatgaa gcactctccc ggaggcacag ttctctttcc aacaaagtat tgccagaaca
     2521 agatggcagg agcccaaagg acaaaggaga tgacccaagc cagaccgatc atcacaccgg
     2581 ctctctttgt tgttcgtttg gctcggtacg tgagcggcct cgtgatggaa aagtatctgt
     2641 caaagctgat gaccagaaga ttcataacag aggcattgct ggctacgcag tcaatggcaa
     2701 gccagaggtc acaggccaag ttccctaagg cccatcgatt catgatgatg taggtcgtaa
     2761 acagattcat tgaaatgacc ccgataatca gatcggcaca ggccaggctt aagaggaagt
     2821 agttgttgac cgtcttcagc tgcttgttga ccttaaatga cacaattacc aggatgttgc
     2881 cgatgatggt caccaaggcc aggatgcccg ttaagaaagc gatgaagacc acttgccaga
     2941 cggtatgacc tcccagaggg tcatcggtgg taccgtctgg agaggagaaa ttgccagctg
     3001 ctcgagaaac attgtagctg ccgaaatgag tgacggttcc cgggggcagc cctgcatcgg
     3061 aggggctgtg tatccaggag gagctgatgt ttggaaacaa aggcgaggtt gtactgttat
     3121 tgtgcaaggt catggtggcg ctagcataac ttcgtataaa gtatcctata cgaagttatt
     3181 tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa taacttcgta
     3241 taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgataat caacctctgg
     3301 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat
     3361 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     3421 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     3481 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     3541 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     3601 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     3661 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca
     3721 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     3781 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     3841 agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga
     3901 gatctacggg tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc
     3961 cactccagtg cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta
     4021 ggtgtccttc tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg
     4081 gaagacaacc tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca
     4141 atcttggctc actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc
     4201 cgagttgttg ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta
     4261 gagacggggt ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta
     4321 cccaccttgg cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt
     4381 ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg
     4441 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc
     4501 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg
     4561 caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4621 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     4681 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     4741 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     4801 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     4861 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     4921 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     4981 gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5041 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5101 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5161 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5221 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5281 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5341 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5401 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5461 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5521 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5581 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5641 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5701 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5761 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5821 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5881 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5941 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6001 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6061 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6121 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6181 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6241 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6301 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6361 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6421 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6481 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6541 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6601 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     6661 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6721 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6781 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6841 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6901 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6961 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7021 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7081 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7141 gctggccttt tgctcacatg t
//
