LOCUS       Exported                7150 bp ds-DNA     circular SYN 12-MAY-2021
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    pAAV-PTRE-tight-flex-hM3Dq-mCherry-WPRE-pA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7150)
  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 7150)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7150
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        141..455
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    149..167
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    185..203
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    292..310
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     428..452
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    495..528
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    579..612
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(621..1331)
                     /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(899..918)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1168..1186
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1328..1337
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     protein_bind    complement(3135..3168)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(3219..3252)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3277..3865
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3330..3350)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3748..3759)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3897..4373
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4096..4115)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4413..4553
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4413..4542
                     /label=AAV2 ITR
     rep_origin      4628..5083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4715..4734)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4925..4946
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5100..5119)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5219..5241
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5279..5297)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5365..5469
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5470..6330
                     /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(5688..5707)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6501..7089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6990..7009
                     /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 gcgtttactc cctatcagtg atagagaacg tatgtcgagt
      181 ttactcccta tcagtgatag agaacgatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca
      301 gtgatagaga acgtatgtcg agtttatccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata
      421 taagcagagc tcgtttagtg aaccgtcaga tcgcctggag aattcgagct cggtacccgg
      481 gggtcgactc cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag
      541 ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt
      601 atacgaagtt atggcgcgcc ttacttgtac agctcgtcca tgccgccggt ggagtggcgg
      661 ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct cgttgtggga ggtgatgtcc
      721 aacttgatgt tgacgttgta ggcgccgggc agctgcacgg gcttcttggc cttgtaggtg
      781 gtcttgacct cagcgtcgta gtggccgccg tccttcagct tcagcctctg cttgatctcg
      841 cccttcaggg cgccgtcctc ggggtacatc cgctcggagg aggcctccca gcccatggtc
      901 ttcttctgca ttacggggcc gtcggagggg aagttggtgc cgcgcagctt caccttgtag
      961 atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg tcaccacgcc gccgtcctcg
     1021 aagttcatca cgcgctccca cttgaagccc tcggggaagg acagcttcaa gtagtcgggg
     1081 atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca tgaactgagg ggacaggatg
     1141 tcccaggcga agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc
     1201 tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt cacggagccc
     1261 tccatgtgca ccttgaagcg catgaactcc ttgatgatgg ccatgttatc ctcctcgccc
     1321 ttgctcacca tggtggcgac cgggggatcc ttcaaggcct gctcgggtgc gcgcttgtga
     1381 aaaatgaccg actgtctctg ctggtactgc tgcttgcgcc tctttttttt gtcacactgg
     1441 cacagcagca gcatcttgaa agtggttctg aatgttttgt tgcacagagc atagcacacg
     1501 gggttcacgg tgctgttgat gtagcacagc cagtagccca gattccaaaa ggttttgggt
     1561 atgcagctgt cacaaaaggt gttcaccaga accatgatgt tgtatggggt ccaagtgatg
     1621 atgaaggcaa gcaagatcgc actgagggtc tgggccgctt tcttctcctt gaccagggac
     1681 atccttttcc gcttagtgat ctgacttctg gtcttcagag caaacctctt ggccagagtg
     1741 gcttccttga aggacagagg tagagtggcc gtgctcttac ccactgagga gttgacgtca
     1801 gaagtcttag ctgtgtccac ggctgactct agctggatgg gaagcttgga gaagcttttt
     1861 ggaaaactgc ctccatcgtc cacgctcttc tgggcctgca gcttgtcggc tttcctctcc
     1921 aagtccacca tccccagctc ctcctcaggc acctgcaggt tgtccgatga gggtaacttg
     1981 gtggagttga ggatggtgct gtgacccgga agcttgagca cgatggagta gatggctctc
     2041 gtctcggagc caatgtcctc ctcgtcggag gaggcggagt tctccaggga ggcagcagca
     2101 tcattgttgt tccaactgtc actgctgctg tggtcttggt ccatctgctc ggagctgggt
     2161 ttccagctct tggttgtgaa ccagaagtgg cagcggccat acttcctcct gttggagcgt
     2221 ttcatgcttt gctgttgaag ttcgtaactg ctgcagcttc gagaactgcc cgtggggtgg
     2281 acaaagtttt ctgtctctgc ctctgtccca gaggcttgca ggccagcaag ctctttggta
     2341 cgcttttcag tttccttata gatcctccag tataaaatag tcataatggt gacaggcata
     2401 taaaaaccag cgatggctgt gccaaaagta atggtgggct cactgaggaa ctgaatgaag
     2461 cactctcccg gaggcacagt tctctttcca acaaagtatt gccagaacaa gatggcagga
     2521 gcccaaagga caaaggagat gacccaagcc agaccgatca tcacaccggc tctctttgtt
     2581 gttcgtttgg ctcggtacgt gagcggcctc gtgatggaaa agtatctgtc aaagctgatg
     2641 accagaagat tcataacaga ggcattgctg gctacgcagt caatggcaag ccagaggtca
     2701 caggccaagt tccctaaggc ccatcgattc atgatgatgt aggtcgtaaa cagattcatt
     2761 gaaatgaccc cgataatcag atcggcacag gccaggctta agaggaagta gttgttgacc
     2821 gtcttcagct gcttgttgac cttaaatgac acaattacca ggatgttgcc gatgatggtc
     2881 accaaggcca ggatgcccgt taagaaagcg atgaagacca cttgccagac ggtatgacct
     2941 cccagagggt catcggtggt accgtctgga gaggagaaat tgccagctgc tcgagaaaca
     3001 ttgtagctgc cgaaatgagt gacggttccc gggggcagcc ctgcatcgga ggggctgtgt
     3061 atccaggagg agctgatgtt tggaaacaaa ggcgaggttg tactgttatt gtgcaaggtc
     3121 atggtggcgc tagcataact tcgtataaag tatcctatac gaagttattt gccttaaccc
     3181 agaaattatc actgttattc tttagaatgg tgcaaagaat aacttcgtat aatgtatgct
     3241 atacgaagtt atgaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt
     3301 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3361 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3421 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3481 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3541 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3601 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     3661 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg
     3721 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     3781 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     3841 atctcccttt gggccgcctc cccgcatcga taccgagcgc tgctcgagag atctacgggt
     3901 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     3961 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     4021 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     4081 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     4141 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     4201 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     4261 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     4321 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     4381 tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac
     4441 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     4501 gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct
     4561 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa
     4621 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4681 gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt
     4741 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4801 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt
     4861 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4921 aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt
     4981 gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa
     5041 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt
     5101 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac
     5161 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     5221 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc
     5281 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     5341 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5401 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5461 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5521 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5581 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5641 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5701 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5761 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5821 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5881 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5941 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6001 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6061 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6121 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6181 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6241 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6301 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6361 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6421 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     7021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     7081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7141 gctcacatgt
//
