LOCUS       sequence-75401-e        7610 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 7610)
  AUTHORS   Bryan Roth
  TITLE     Roth lab DREADDs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7610)
  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            "Unpublished"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..7610
                     /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        186..633
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(671..704)
                     /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(755..788)
                     /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(803..1513,1514..1516,1517..1519))
                     /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(845..866)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1194..1213
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1453..1474
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1516..1525
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1526..2099
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1686..1705)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     1915..1932
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(3555..3581)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    3595..3628
                     /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    3679..3712
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3737..4325
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3790..3810)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4208..4219)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4357..4833
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4556..4575)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4873..5013
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4873..5002
                     /label=AAV2 ITR
     rep_origin      5088..5543
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5175..5194)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5385..5406
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5560..5579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5679..5701
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5739..5757)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5825..5929
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5930..5998,5999..6790)
                     /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(6148..6167)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6961..7549
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7450..7469
                     /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 cgcgtgtgtc tagactgcag agggccctgc
      181 gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg
      241 accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta
      301 tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca
      361 ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa
      421 ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc
      481 gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac
      541 gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc
      601 agcggaggag tcgtgtcgtg cctgagagcg cagtcgagaa ggtaccggat cctctagagt
      661 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
      721 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
      781 gaagttatgg cgcgccctat cacttgtaca gctcgtccat gccgagagtg atcccggcgg
      841 cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct ttgctcagtt
      901 tggactggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg
      961 tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg tggcggatct
     1021 tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg ttgtggctgt
     1081 tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag tcgatgccct
     1141 tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt
     1201 agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg ggcatggcgg
     1261 acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac atcaggccgt
     1321 agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg gtgcagatga
     1381 acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac acgctgaact
     1441 tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg gtgaacagct
     1501 cctcgccctt gctcaccatg gtggcttatc atcgtgtttt tcaaaggaaa accacgtccc
     1561 cgtggttcgg ggggcctaga cgttttttta acctcgacta aacacatgta aagcatgtgc
     1621 accgaggccc cagatcagat cccatacaat ggggtacctt ctgggcatcc ttcagcccct
     1681 tgttgaatac gcttgaggag agccatttga ctctttccac aactatccaa ctcacaacgt
     1741 ggcactgggg ttgtgccgcc tttgcaggtg tatcttatac acgtggcttt tggccgcaga
     1801 ggcacctgtc gccaggtggg gggttccgct gcctgcaaag ggtcgctaca gacgttgttt
     1861 gtcttcaaga agcttccaga ggaactgctt ccttcacgac attcaacaga ccttgcattc
     1921 ctttggcgag aggggaaaga cccctaggaa tgctcgtcaa gaagacaggg ccaggtttcc
     1981 gggccctcac attgccaaaa gacggcaata tggtggaaaa taacatatag acaaacgcac
     2041 accggcctta ttccaagcgg cttcggccag taacgttagg ggggggggag ggagaggggc
     2101 gatatcgcgg ccgcctacct ggcagtgccg atgttccgat actggcacag cagcaggtgc
     2161 cggaaggtct ttttaaaggt ggcgttgcac agagcatagc aggcagggtt gatggtgctg
     2221 ttgacgtagc agagccagta gccaatggac cacaccgtgt cagggatgca gctctggcag
     2281 aaggtgttca ccaggaccat gacgttgtag ggcgtccagg tgaggatgaa ggcaagcaga
     2341 atggcaaaga tcgttcgtgt cactttgcgc tcccgggccg ccatctgccg cttcttgcgc
     2401 acctggttgc gagcgatgct ggcgaacttg cgggccacgt tggccgcagg gcgcatgcca
     2461 gccggcgtgg caggcacaat ctcaatggct gtcacacact cattgcctgt ctgcttcgtc
     2521 acaatctgga tcttggacca tctggaggct gggttgaggg cccgcggctg caggggaggg
     2581 gcgggcatgg cgggcgtggt ggcctctgtg gtggacagct ctgtggctgg gcgttccttg
     2641 gtgttctggg tggcactgcc tgagctggac tcattggaag tgtccttatc agccacgggg
     2701 cgcggtggcg gtggcagcgc tggcgggggg gcctcctcca gcttgccatt gcgcagctcc
     2761 tcccgggcgg cctccccggg cgggggcttc ttgacgctct gcttcattag tgggctcttg
     2821 aggaaggcca gcgtcttggc tttcttctcc ttcgggccct cgggccggtg cttgtggact
     2881 cggctgcgac tggccaggga gatgtggatg tacagcaccg tcatgatgac cacaggcagg
     2941 tagaagccag caatggctgt gccaaaggtc actgctgggt tggacaggaa ctggatgaag
     3001 cactggttgt cgggcaccgt ccgcttaccc accacaaact gccagaacaa gatggcaggc
     3061 gcccagagca cgaaggacag tacccaggca gcagcaatca tgaggcctgc catcttggtg
     3121 gtgcgccggg cagggtaggt gagaggcttg gtgacgcaga agtagcggtc aaagctgatg
     3181 atgagaaggt tcatgacgga ggcgttgctc accacgcagt ccagggccag ccacaggtcg
     3241 cagaccacgg cgcccagggg ccagtagccc ttgatgatgt acacggtgta gaggttcatg
     3301 gagaaggcgc ctatgatgag atcagcacac gccaggctga agaggaagta gttgttgact
     3361 gtctgcagct gcctgttgac cttgatggac agcatcacca ggatgttgcc cacgacagtc
     3421 accaggctca gggagcctgt cactgtggca atgaagacca tttccaccgt ctcatagcga
     3481 ttgtgggatg atgacgtgac caggcgcacg gactgattgc ccgagctgcc attgacaggt
     3541 gtgaagttgg ccatagcgta atctggaaca tcgtatgggt acatggtggc tagcataact
     3601 tcgtataaag tatcctatac gaagttattt gccttaaccc agaaattatc actgttattc
     3661 tttagaatgg tgcaaagaat aacttcgtat aatgtatgct atacgaagtt atgaattcga
     3721 tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     3781 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     3841 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     3901 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     3961 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     4021 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     4081 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     4141 ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt ccttctgcta
     4201 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     4261 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     4321 cccgcatcga taccgagcgc tgctcgagag atctacgggt ggcatccctg tgacccctcc
     4381 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     4441 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     4501 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     4561 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     4621 gttcaagcga ttctcctgcc tcagcctccc gagttgttgg gattccaggc atgcatgacc
     4681 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     4741 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     4801 caggcgtgaa ccactgctcc cttccctgtc cttctgattt tgtaggtaac cacgtgcgga
     4861 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
     4921 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
     4981 cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta
     5041 cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag
     5101 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5161 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5221 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5281 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     5341 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5401 aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     5461 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5521 caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc
     5581 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     5641 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     5701 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt
     5761 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     5821 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5881 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5941 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     6001 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     6061 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     6121 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     6181 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     6241 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6301 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6361 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6421 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6481 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6541 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6601 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6661 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6721 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6781 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6841 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6901 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6961 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     7021 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     7081 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     7141 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     7201 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     7261 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     7321 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     7381 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     7441 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     7501 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     7561 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
//
