LOCUS       Exported                6982 bp DNA     circular SYN 29-MAY-2024
DEFINITION  Double floxed Gi-coupled hM4D DREADD fused with mCherry under the 
            control of human synapsin promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-hSyn-DIO-hM4D(Gi)-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6982)
  AUTHORS   Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher DS, 
            Maratos-Flier E, Roth BL, Lowell BB
  TITLE     Rapid, reversible activation of AgRP neurons drives feeding behavior
            in mice.
  JOURNAL   J Clin Invest. 2011 Apr;121(4):1424-8. doi: 10.1172/JCI46229.
  PUBMED    21364278
REFERENCE   2  (bases 1 to 6982)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6982)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 29, 2024 from SnapGene 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "J Clin Invest"; date: "2011-04"; volume: "121"; issue: "4"; pages: 
            "1424-8"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6982
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    660..693
                     /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    744..777
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(786..1496)
                     /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(1064..1083)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1333..1351
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1493..1502
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1518..2954)
                     /codon_start=1
                     /label=Muscarinic acetylcholine receptor M4
                     /translation="MANFTPVNGSSGNQSVRLVTSSSHNRYETVEMVFIATVTGSLSLV
                     TVVGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYTVYIIKGYWPLGAVV
                     CDLWLALDCVVSNASVMNLLIISFDRYFCVTKPLTYPARRTTKMAGLMIAAAWVLSFVL
                     WAPAILFWQFVVGKRTVPDNQCFIQFLSNPAVTFGTAIAGFYLPVVIMTVLYIHISLAS
                     RSRVHKHRPEGPKEKKAKTLAFLKSPLMKQSVKKPPPGEAAREELRNGKLEEAPPPALP
                     PPPRPVADKDTSNESSSGSATQNTKERPATELSTTEATTPAMPAPPLQPRALNPASRWS
                     KIQIVTKQTGNECVTAIEIVPATPAGMRPAANVARKFASIARNQVRKKRQMAARERKVT
                     RTIFAILLAFILTWTPYNVMVLVNTFCQSCIPDTVWSIGYWLCYVNSTINPACYALCNA
                     TFKKTFRHLLLCQYRNIGTAR"
     regulatory      2951..2960
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(2967..3000)
                     /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(3051..3084)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    3109..3697
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     primer_bind     complement(3162..3182)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3580..3591)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3729..4205
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3928..3947)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4245..4385
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4245..4374
                     /label=AAV2 ITR
     rep_origin      4460..4915
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4547..4566)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4757..4778
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4932..4951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     5051..5073
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5111..5129)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        5197..5301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5302..6162
                     /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(5520..5539)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6333..6921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6822..6841
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgccttact tgtacagctc gtccatgccg ccggtggagt ggcggccctc ggcgcgttcg
      841 tactgttcca cgatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
      901 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
      961 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     1021 tcctcggggt acatccgctc ggaggaggcc tcccagccca tggtcttctt ctgcattacg
     1081 gggccgtcgg aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc
     1141 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc
     1201 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     1261 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     1321 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc
     1381 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     1441 aagcgcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatggtg
     1501 gcgaccggtg gatcccgcct ggcagtgccg atgttccgat actggcacag cagcaggtgc
     1561 cggaaggtct ttttaaaggt ggcgttgcac agagcatagc aggcagggtt gatggtgctg
     1621 ttgacgtagc agagccagta gccaatggac cacaccgtgt cagggatgca gctctggcag
     1681 aaggtgttca ccaggaccat gacgttgtag ggcgtccagg tgaggatgaa ggccagcaga
     1741 atggcaaaga tcgttcgtgt cactttgcgc tcccgggccg ccatctgccg cttcttgcgc
     1801 acctggttgc gagcgatgct ggcgaacttg cgggccacgt tggccgcagg gcgcatgcca
     1861 gccggcgtgg caggcacaat ctcaatggct gtcacacact cattgcctgt ctgcttcgtc
     1921 acaatctgga tcttggacca tctggaggct gggttgaggg cccgcggctg caggggaggg
     1981 gcgggcatgg cgggcgtggt ggcctctgtg gtggacagct ctgtggctgg gcgttccttg
     2041 gtgttctggg tggcactgcc tgagctggac tcattggaag tgtccttatc agccacgggg
     2101 cgcggtggcg gtggcagcgc tggcgggggg gcctcctcca gcttgccatt gcgcagctcc
     2161 tcccgggcgg cctccccggg cgggggcttc ttgacgctct gcttcattag tgggctcttg
     2221 aggaaggcca gcgtcttggc tttcttctcc ttcgggccct cgggccggtg cttgtggact
     2281 cggctgcgac tggccaggga gatgtggatg tacagcaccg tcatgatgac cacaggcagg
     2341 tagaagccag caatggctgt gccaaaggtc actgctgggt tggacaggaa ctggatgaag
     2401 cactggttgt cgggcaccgt ccgcttaccc accacaaact gccagaacaa gatggcaggc
     2461 gcccagagca cgaaggacag tacccaggca gcagcaatca tgaggcctgc catcttggtg
     2521 gtgcgccggg cagggtaggt gagaggcttg gtgacgcaga agtagcggtc aaagctgatg
     2581 atgagaaggt tcatgacgga ggcgttgctc accacgcagt ccagggccag ccacaggtcg
     2641 cagaccacgg cgcccagggg ccagtagccc ttgatgatgt acacggtgta gaggttcatg
     2701 gagaaggcgc ctatgatgag atcagcacac gccaggctga agaggaagta gttgttgact
     2761 gtctgcagct gcctgttgac cttgatggac agcatcacca ggatgttgcc cacgacagtc
     2821 accaggctca gggagcctgt cactgtggca atgaagacca tttccaccgt ctcatagcga
     2881 ttgtgggatg atgacgtgac caggcgcacg gactgattgc ccgagctgcc attgacaggt
     2941 gtgaagttgg ccatggtggc gctagcataa cttcgtataa agtatcctat acgaagttat
     3001 ttgccttaac ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt
     3061 ataatgtatg ctatacgaag ttatgaattc gatatcaagc ttatcgataa tcaacctctg
     3121 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3181 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3241 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3301 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     3361 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     3421 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     3481 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc
     3541 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     3601 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     3661 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag
     3721 agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg
     3781 ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact
     3841 aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg
     3901 ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac
     3961 aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc
     4021 ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt
     4081 agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct
     4141 acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg
     4201 tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     4261 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4321 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4381 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4441 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4501 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     4561 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4621 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     4681 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4741 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     4801 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     4861 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     4921 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     4981 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5041 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5101 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5161 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5221 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5281 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5341 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5401 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5461 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5521 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5581 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5641 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5701 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5761 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5821 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5881 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5941 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6001 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6061 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6121 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6181 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6241 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6301 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6361 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6421 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6481 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6541 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6601 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6661 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6721 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6781 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6841 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     6901 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     6961 tgctggcctt ttgctcacat gt
//
