LOCUS       sequence-214301-        7599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double floxed Gi-coupled hM4D-IRES-mCitrine under the control of 
            human synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7599)
  AUTHORS   Bryan Roth
  TITLE     Roth lab DREADDs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7599)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-hSyn-DIO-HA-hM4D(Gi)-IRES-mCitrine
FEATURES             Location/Qualifiers
     source          1..7599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        148..595
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(633..666)
                     /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(717..750)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(765..1475,1476..1478,1479..1481))
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(807..828)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1156..1175
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1415..1436
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1478..1487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1488..2061
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1648..1667)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1877..1894
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(3517..3543)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    3557..3590
                     /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    3641..3674
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3699..4287
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3752..3772)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4170..4181)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4319..4795
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4518..4537)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4835..4975
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4835..4964
                     /label=AAV2 ITR
     rep_origin      5050..5505
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5137..5156)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5347..5368
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5522..5541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5641..5663
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5701..5719)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5787..5891
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5892..5960,5961..6752)
                     /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(6110..6129)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6923..7511
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7412..7431
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(7573..7599,1..103)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
       61 gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg cacgcgtgtg
      121 tctagactgc agagggccct gcgtatgagt gcaagtgggt tttaggacca ggatgaggcg
      181 gggtgggggt gcctacctga cgaccgaccc cgacccactg gacaagcacc caacccccat
      241 tccccaaatt gcgcatcccc tatcagagag ggggagggga aacaggatgc ggcgaggcgc
      301 gtgcgcactg ccagcttcag caccgcggac agtgccttcg cccccgcctg gcggcgcgcg
      361 ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc gccggtcccc cgcaaactcc
      421 ccttcccggc caccttggtc gcgtccgcgc cgccgccggc ccagccggac cgcaccacgc
      481 gaggcgcgag ataggggggc acgggcgcga ccatctgcgc tgcggcgccg gcgactcagc
      541 gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag cgcagtcgag
      601 aaggtaccgg atcctctaga gtcgactccg gaataacttc gtataggata ctttatacga
      661 agttatgcag aatggtagct ggattgtagc tgctattagc aatatgaaac ctcttaataa
      721 cttcgtatag catacattat acgaagttat ggcgcgccct atcacttgta cagctcgtcc
      781 atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc
      841 tcgttggggt ctttgctcag tttggactgg tagctcaggt agtggttgtc gggcagcagc
      901 acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg
      961 tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc
     1021 atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg
     1081 tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac
     1141 ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg
     1201 acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag
     1261 cgggcgaagc acatcaggcc gtagccgaag gtggtcacga gggtgggcca gggcacgggc
     1321 agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg
     1381 ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg
     1441 ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tggtggctta tcatcgtgtt
     1501 tttcaaagga aaaccacgtc cccgtggttc ggggggccta gacgtttttt taacctcgac
     1561 taaacacatg taaagcatgt gcaccgaggc cccagatcag atcccataca atggggtacc
     1621 ttctgggcat ccttcagccc cttgttgaat acgcttgagg agagccattt gactctttcc
     1681 acaactatcc aactcacaac gtggcactgg ggttgtgccg cctttgcagg tgtatcttat
     1741 acacgtggct tttggccgca gaggcacctg tcgccaggtg gggggttccg ctgcctgcaa
     1801 agggtcgcta cagacgttgt ttgtcttcaa gaagcttcca gaggaactgc ttccttcacg
     1861 acattcaaca gaccttgcat tcctttggcg agaggggaaa gacccctagg aatgctcgtc
     1921 aagaagacag ggccaggttt ccgggccctc acattgccaa aagacggcaa tatggtggaa
     1981 aataacatat agacaaacgc acaccggcct tattccaagc ggcttcggcc agtaacgtta
     2041 gggggggggg agggagaggg gcgatatcgc ggccgcctac ctggcagtgc cgatgttccg
     2101 atactggcac agcagcaggt gccggaaggt ctttttaaag gtggcgttgc acagagcata
     2161 gcaggcaggg ttgatggtgc tgttgacgta gcagagccag tagccaatgg accacaccgt
     2221 gtcagggatg cagctctggc agaaggtgtt caccaggacc atgacgttgt agggcgtcca
     2281 ggtgaggatg aaggcaagca gaatggcaaa gatcgttcgt gtcactttgc gctcccgggc
     2341 cgccatctgc cgcttcttgc gcacctggtt gcgagcgatg ctggcgaact tgcgggccac
     2401 gttggccgca gggcgcatgc cagccggcgt ggcaggcaca atctcaatgg ctgtcacaca
     2461 ctcattgcct gtctgcttcg tcacaatctg gatcttggac catctggagg ctgggttgag
     2521 ggcccgcggc tgcaggggag gggcgggcat ggcgggcgtg gtggcctctg tggtggacag
     2581 ctctgtggct gggcgttcct tggtgttctg ggtggcactg cctgagctgg actcattgga
     2641 agtgtcctta tcagccacgg ggcgcggtgg cggtggcagc gctggcgggg gggcctcctc
     2701 cagcttgcca ttgcgcagct cctcccgggc ggcctccccg ggcgggggct tcttgacgct
     2761 ctgcttcatt agtgggctct tgaggaaggc cagcgtcttg gctttcttct ccttcgggcc
     2821 ctcgggccgg tgcttgtgga ctcggctgcg actggccagg gagatgtgga tgtacagcac
     2881 cgtcatgatg accacaggca ggtagaagcc agcaatggct gtgccaaagg tcactgctgg
     2941 gttggacagg aactggatga agcactggtt gtcgggcacc gtccgcttac ccaccacaaa
     3001 ctgccagaac aagatggcag gcgcccagag cacgaaggac agtacccagg cagcagcaat
     3061 catgaggcct gccatcttgg tggtgcgccg ggcagggtag gtgagaggct tggtgacgca
     3121 gaagtagcgg tcaaagctga tgatgagaag gttcatgacg gaggcgttgc tcaccacgca
     3181 gtccagggcc agccacaggt cgcagaccac ggcgcccagg ggccagtagc ccttgatgat
     3241 gtacacggtg tagaggttca tggagaaggc gcctatgatg agatcagcac acgccaggct
     3301 gaagaggaag tagttgttga ctgtctgcag ctgcctgttg accttgatgg acagcatcac
     3361 caggatgttg cccacgacag tcaccaggct cagggagcct gtcactgtgg caatgaagac
     3421 catttccacc gtctcatagc gattgtggga tgatgacgtg accaggcgca cggactgatt
     3481 gcccgagctg ccattgacag gtgtgaagtt ggccatagcg taatctggaa catcgtatgg
     3541 gtacatggtg gctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     3601 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg
     3661 ctatacgaag ttatgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     3721 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3781 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3841 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3901 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3961 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     4021 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     4081 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     4141 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     4201 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     4261 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     4321 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     4381 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     4441 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     4501 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     4561 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     4621 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     4681 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     4741 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     4801 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     4861 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4921 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4981 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     5041 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5101 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     5161 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5221 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     5281 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5341 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     5401 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     5461 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     5521 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5581 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5641 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5701 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5761 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5821 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5881 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5941 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6001 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6061 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6121 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6181 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6241 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6301 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6361 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6421 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6481 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6541 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6601 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6661 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6721 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6781 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6841 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6901 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6961 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     7021 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     7081 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7141 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7201 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7261 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7321 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7381 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7441 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7501 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7561 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgc
//
