LOCUS       sequence-75403-e        7406 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double floxed Gs-coupled rM3D-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 7406)
  AUTHORS   Bryan Roth
  TITLE     Roth lab DREADDs
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7406)
  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            "Unpublished"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-rM3D(Gs)-IRES-mCitrine
FEATURES             Location/Qualifiers
     source          1..7406
                     /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(3351..3377)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    3391..3424
                     /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    3475..3508
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3533..4121
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3586..3606)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4004..4015)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4153..4629
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4352..4371)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4669..4809
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4669..4798
                     /label=AAV2 ITR
     rep_origin      4884..5339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4971..4990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5181..5202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5356..5375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5475..5497
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5535..5553)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5621..5725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5726..5794,5795..6586)
                     /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(5944..5963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6757..7345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7246..7265
                     /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 ccgcctacaa ggcctgctcc ggcactcgct tgtgaaaaat gaccgactgt
     2161 ctctgctggt actgctgttt gcgcctcttc cttttgtcac actggcacaa gaggagcgtc
     2221 ttgaaggtgg ttctgaatgt tttgttgcac agggcatagc acacagggtt cacggtgctg
     2281 ttgatatagc acagccagta gcccagattc caataggttt tgggtatgca gctgtcacag
     2341 aaggtgttca ccaggaccat gatgttgtag ggggtccacg tgatgatgaa ggcaagcaag
     2401 atggcactga gcgtctgcag agctttgtgt tccctcatgg ccatgacacg ggacgtcttc
     2461 ctcttgctgg cacggccgtt gccgaggatg ggctggtgtt gggggagcgg gggtggctgc
     2521 ggctgctcct ggctgccata gaaccggccc tcgcagcggt cgatcttcct gatctgctcc
     2581 ttggcctccc ggtacacccg ccagtataaa atagtcatga tggtgacagg catgtaaaag
     2641 ccagcgatcg ccgtgccgaa ggtgatggtg ggctcactca gaaactgaat gaaacattct
     2701 cctgggggca cagttctctt ccctacaaag tattgccaga acaagatggc aggagcccat
     2761 aggacaaagg agatgaccca agccagacca atcatcacac cagctcgagc cctggtcatc
     2821 aggctctggt agcggaatgg agaagtgatg gaaaagtacc tgtcaaagct gatgaccagc
     2881 agattcatga cagaggcatt gctggccaca cagtcaatgg agagccagag gtcgcaggct
     2941 aagttcccca gtgcccaacg gttcataatg atgtaggtag tgaacaggtt catggaaatg
     3001 accccgatga tcaggtctgc acaggccagg cttaagagga agtagttgtt gactgtcttc
     3061 agctgtttgt tgaccttgaa ggccacaatg acaaggatgt tgccaatgat ggtcaccaat
     3121 gccaggaagc cagttaagaa ggcaatgaag accacttgcc agatggtgtg acccccgaga
     3181 gggtcgctgg aggtgtcgtt tgaggagaaa ttcccagttt cttgtgaaat gttgtagctg
     3241 cccaactgag tgactgtccc caagggcagc cctgcctccg agggactgtg cacccaggaa
     3301 gagctgatgt tgggaaacaa aggcgaggtt gtactgttac tgtgcaaggt ggcgtagtcg
     3361 gggacgtcgt aggggtacat ggtggctagc ataacttcgt ataaagtatc ctatacgaag
     3421 ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagaataact
     3481 tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg ataatcaacc
     3541 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     3601 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     3661 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     3721 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     3781 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     3841 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     3901 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     3961 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     4021 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     4081 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     4141 cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa
     4201 gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct
     4261 gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa
     4321 gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg
     4381 gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag
     4441 cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt
     4501 tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg
     4561 atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc
     4621 cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag gaacccctag
     4681 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4741 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4801 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4861 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4921 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4981 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5041 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5101 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5161 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5221 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5281 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5401 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5461 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5521 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5581 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5641 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5701 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5761 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5821 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5881 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5941 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6001 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6061 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6121 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6181 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6241 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6301 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6361 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6421 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6481 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6541 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6601 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6661 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6721 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6781 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6841 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6901 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6961 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7021 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7081 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7141 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7201 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7261 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7321 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7381 cttttgctgg ccttttgctc acatgt
//
