LOCUS       40924_3092        8095 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Double floxed Gq-coupled hM3D DREADD fused with mCherry under the 
            control of EF1a promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8095)
  AUTHORS   Bryan Roth
  TITLE     Roth lab DREADDs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8095)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8095)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     protein_bind    complement(1440..1473)
                     /label=lox2272
                     /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(1524..1557)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1569..2276)
                     /codon_start=1
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             complement(2280..4067)
                     /codon_start=1
                     /label=hM3D(Gq)
                     /note="A modified form of human Gq-coupled M3 muscarinic 
                     receptor, belonging to a class of chemogenetic receptors 
                     called designer receptors exclusively activated by designer
                     drugs (DREADD)"
                     /translation="MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRA
                     AGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNN
                     YFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDCVASNASVMNLLVIS
                     FDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECF
                     IQFLSEPTITFGTAIAGFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFV
                     HPTGSSRSCSSYELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNN
                     DAAASLENSASSDEEDIGSETRAIYSIVLKLPGHSTILNSTKLPSSDNLQVPEEELGMV
                     DLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKSTATLPLSF
                     KEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFC
                     DSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQR
                     QSVIFHKRAPEQALKDPPVA"
     protein_bind    4080..4113
                     /label=lox2272
                     /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    4164..4197
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4222..4810
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     polyA_signal    4842..5318
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     repeat_region   5358..5498
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5573..6028
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6045..6064)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     6164..6186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6224..6242)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        6310..6414
                     /label=AmpR promoter
     CDS             6415..7272
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7446..8034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
     1621 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
     1681 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
     1741 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     1801 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     1861 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     1921 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     1981 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     2041 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     2101 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     2161 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     2221 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatggtg
     2281 gcgaccgggg gatccttcaa ggcctgctcg ggtgcgcgct tgtgaaaaat gaccgactgt
     2341 ctctgctggt actgctgctt gcgcctcttt tttttgtcac actggcacag cagcagcatc
     2401 ttgaaagtgg ttctgaatgt tttgttgcac agagcatagc acacggggtt cacggtgctg
     2461 ttgatgtagc acagccagta gcccagattc caaaaggttt tgggtatgca gctgtcacaa
     2521 aaggtgttca ccagaaccat gatgttgtat ggggtccaag tgatgatgaa ggcaagcaag
     2581 atcgcactga gggtctgggc cgctttcttc tccttgacca gggacatcct tttccgctta
     2641 gtgatctgac ttctggtctt cagagcaaac ctcttggcca gagtggcttc cttgaaggac
     2701 agaggtagag tggccgtgct cttacccact gaggagttga cgtcagaagt cttagctgtg
     2761 tccacggctg actctagctg gatgggaagc ttggagaagc tttttggaaa actgcctcca
     2821 tcgtccacgc tcttctgggc ctgcagcttg tcggctttcc tctccaagtc caccatcccc
     2881 agctcctcct caggcacctg caggttgtcc gatgagggta acttggtgga gttgaggatg
     2941 gtgctgtgac ccggaagctt gagcacgatg gagtagatgg ctctcgtctc ggagccaatg
     3001 tcctcctcgt cggaggaggc ggagttctcc agggaggcag cagcatcatt gttgttccaa
     3061 ctgtcactgc tgctgtggtc ttggtccatc tgctcggagc tgggtttcca gctcttggtt
     3121 gtgaaccaga agtggcagcg gccatacttc ctcctgttgg agcgtttcat gctttgctgt
     3181 tgaagttcgt aactgctgca gcttcgagaa ctgcccgtgg ggtggacaaa gttttctgtc
     3241 tctgcctctg tcccagaggc ttgcaggcca gcaagctctt tggtacgctt ttcagtttcc
     3301 ttatagatcc tccagtataa aatagtcata atggtgacag gcatataaaa accagcgatg
     3361 gctgtgccaa aagtaatggt gggctcactg aggaactgaa tgaagcactc tcccggaggc
     3421 acagttctct ttccaacaaa gtattgccag aacaagatgg caggagccca aaggacaaag
     3481 gagatgaccc aagccagacc gatcatcaca ccggctctct ttgttgttcg tttggctcgg
     3541 tacgtgagcg gcctcgtgat ggaaaagtat ctgtcaaagc tgatgaccag aagattcata
     3601 acagaggcat tgctggctac gcagtcaatg gcaagccaga ggtcacaggc caagttccct
     3661 aaggcccatc gattcatgat gatgtaggtc gtaaacagat tcattgaaat gaccccgata
     3721 atcagatcgg cacaggccag gcttaagagg aagtagttgt tgaccgtctt cagctgcttg
     3781 ttgaccttaa atgacacaat taccaggatg ttgccgatga tggtcaccaa ggccaggatg
     3841 cccgttaaga aagcgatgaa gaccacttgc cagacggtat gacctcccag agggtcatcg
     3901 gtggtaccgt ctggagagga gaaattgcca gctgctcgag aaacattgta gctgccgaaa
     3961 tgagtgacgg ttcccggggg cagccctgca tcggaggggc tgtgtatcca ggaggagctg
     4021 atgtttggaa acaaaggcga ggttgtactg ttattgtgca aggtcatggt ggcgctagca
     4081 taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt
     4141 tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa
     4201 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     4261 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4321 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4381 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4441 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4501 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4561 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     4621 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc
     4681 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4741 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4801 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     4861 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     4921 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     4981 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     5041 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     5101 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     5161 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     5221 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     5281 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     5341 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     5401 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     5461 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     5521 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     5581 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5641 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5701 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5761 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     5821 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5881 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5941 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     6001 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     6061 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     6121 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     6181 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     6241 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     6301 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     6361 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     6421 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     6481 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6541 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6601 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6661 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6721 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6781 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6841 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6901 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6961 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     7021 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     7081 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     7141 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     7201 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     7261 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     7321 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     7381 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     7441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     7501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     7561 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     7621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     7681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     7741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     7801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     8041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
