LOCUS       sequence-441203-        6828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuron-specific, Cre-dependent co-expression of 3x HA-tagged 
            excitatory hM3D(Gq) DREADD receptor, and a single-cell 
            discriminating version of oScarlet as a reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6828)
  AUTHORS   Szelenyi ER, Navarrete JS, Murry AD, Zhang Y, Girven KS, Kuo L, 
            Cline MM, Bernstein MX, Burdyniuk M, Bowler B, Goodwin NL, Juarez B,
            Zweifel LS, Golden SA
  TITLE     An arginine-rich nuclear localization signal (ArgiNLS) strategy for 
            streamlined image segmentation of single cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Aug 6;121(32):e2320250121. doi: 
            10.1073/pnas.2320250121. Epub 2024 Jul 29.
  PUBMED    39074275
REFERENCE   2  (bases 1 to 6828)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-08-6"; volume: "121(32):e2320250121. 
            doi"; pages: " 10.1073/pnas.2320250121. Epub 2024 Jul 29"
COMMENT     Sequence Label: pAAV-hSyn-DIO-3xHA-hM3D(Gq)-P2A-ArgiNLS-oScarlet
FEATURES             Location/Qualifiers
     source          1..6828
                     /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        145..592
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(611..644)
                     /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(695..728)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(740..1432)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1015..1034)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1284..1302
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1508..1564)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(3362..3388)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3392..3418)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(3422..3448)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      3460..3469
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3476..3509
                     /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    3560..3593
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3690..3710)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4092..4232
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4092..4221
                     /label=AAV2 ITR
     rep_origin      4307..4762
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4394..4413)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4604..4625
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4779..4798)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4898..4920
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5044..5148
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5149..5217,5218..6009)
                     /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(5367..5386)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6180..6767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6668..6687
                     /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 gcgtagtgca agtgggtttt aggaccagga tgaggcgggg
      181 tgggggtgcc tacctgacga ccgaccccga cccactggac aagcacccaa cccccattcc
      241 ccaaattgcg catcccctat cagagagggg gaggggaaac aggatgcggc gaggcgcgtg
      301 cgcactgcca gcttcagcac cgcggacagt gccttcgccc ccgcctggcg gcgcgcgcca
      361 ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc ggtcccccgc aaactcccct
      421 tcccggccac cttggtcgcg tccgcgccgc cgccggccca gccggaccgc accacgcgag
      481 gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc ggcgccggcg actcagcgct
      541 gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc ctgagagcgc agtctagagt
      601 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
      661 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
      721 gaagttatgg cgcgcctcac ttgtacagct cgtccatgcc gccggtggag tggcggccct
      781 cggagcgttc gtactgttcc accacggtgt agtcctcgtt gtgggaggtg atgtccaact
      841 tgcggtccac gttgtaggcg ccgggcatct gcacgggctt cttggccttg taggtggtct
      901 tgaagtccgc caggtagcgc ccgccgtcct tcaggcgcag ggccatctta atgtcgccct
      961 tcagcacgcc gtcctcgggg tacaaccgct cggtggacgc ttcccagccc attgtcttct
     1021 tctgcattac ggggccgtca ggagggaagt tggtgccgcg gagcttcacc ttgtagatca
     1081 gggtgccgtc ctccagggag gtgtcctggg tcacggtcac ggcgccgccg tcctcgaagt
     1141 tcatcacgcg gtcccacttg aagccctcgg ggaaggactg cttatagtag tcggggatgt
     1201 cggcggggtg cttggtgaag gccctggagc cgtacatgaa ctgaggggac aggatgtccc
     1261 aggagaaggg cagggggcca cccttggtca ccttcagctt ggcggtctgg gtgccctcgt
     1321 aggggcggcc ctcgccctcg ccctcgatct cgaactcgtg gccgttcatg gagccctcca
     1381 tgtgcacctt gaaccgcatg aactccttga tcactgcctc gcccttgctc actcgacggc
     1441 gccgccgcct tctcaccttc tttttctttg ctatgctcat ctgcttctgg ggagccaccc
     1501 ttttcatagg accggggttt tcttccacgt ctcctgcttg ctttaacaga gagaagttcg
     1561 tggctccgga tccggtggcg accgggggat ccttcaaggc ctgctcgggt gcgcgcttgt
     1621 gaaaaatgac cgactgtctc tgctggtact gctgcttgcg cctctttttt ttgtcacact
     1681 ggcacagcag cagcatcttg aaagtggttc tgaatgtttt gttgcacaga gcatagcaca
     1741 cggggttcac ggtgctgttg atgtagcaca gccagtagcc cagattccaa aaggttttgg
     1801 gtatgcagct gtcacaaaag gtgttcacca gaaccatgat gttgtatggg gtccaagtga
     1861 tgatgaaggc aagcaagatc gcactgaggg tctgggccgc tttcttctcc ttgaccaggg
     1921 acatcctttt ccgcttagtg atctgacttc tggtcttcag agcaaacctc ttggccagag
     1981 tggcttcctt gaaggacaga ggtagagtgg ccgtgctctt acccactgag gagttgacgt
     2041 cagaagtctt agctgtgtcc acggctgact ctagctggat gggaagcttg gagaagcttt
     2101 ttggaaaact gcctccatcg tccacgctct tctgggcctg cagcttgtcg gctttcctct
     2161 ccaagtccac catccccagc tcctcctcag gcacctgcag gttgtccgat gagggtaact
     2221 tggtggagtt gaggatggtg ctgtgacccg gaagcttgag cacgatggag tagatggctc
     2281 tcgtctcgga gccaatgtcc tcctcgtcgg aggaggcgga gttctccagg gaggcagcag
     2341 catcattgtt gttccaactg tcactgctgc tgtggtcttg gtccatctgc tcggagctgg
     2401 gtttccagct cttggttgtg aaccagaagt ggcagcggcc atacttcctc ctgttggagc
     2461 gtttcatgct ttgctgttga agttcgtaac tgctgcagct tcgagaactg cccgtggggt
     2521 ggacaaagtt ttctgtctct gcctctgtcc cagaggcttg caggccagca agctctttgg
     2581 tacgcttttc agtttcctta tagatcctcc agtataaaat agtcataatg gtgacaggca
     2641 tataaaaacc agcgatggct gtgccaaaag taatggtggg ctcactgagg aactgaatga
     2701 agcactctcc cggaggcaca gttctctttc caacaaagta ttgccagaac aagatggcag
     2761 gagcccaaag gacaaaggag atgacccaag ccagaccgat catcacaccg gctctctttg
     2821 ttgttcgttt ggctcggtac gtgagcggcc tcgtgatgga aaagtatctg tcaaagctga
     2881 tgaccagaag attcataaca gaggcattgc tggctacgca gtcaatggca agccagaggt
     2941 cacaggccaa gttccctaag gcccatcgat tcatgatgat gtaggtcgta aacagattca
     3001 ttgaaatgac cccgataatc agatcggcac aggccaggct taagaggaag tagttgttga
     3061 ccgtcttcag ctgcttgttg accttaaatg acacaattac caggatgttg ccgatgatgg
     3121 tcaccaaggc caggatgccc gttaagaaag cgatgaagac cacttgccag acggtatgac
     3181 ctcccagagg gtcatcggtg gtaccgtctg gagaggagaa attgccagct gctcgagaaa
     3241 cattgtagct gccgaaatga gtgacggttc ccgggggcag ccctgcatcg gaggggctgt
     3301 gtatccagga ggagctgatg tttggaaaca aaggcgaggt tgtactgtta ttgtgcaagg
     3361 tagcgtagtc tgggacgtcg tatgggtagc cagcgtagtc tgggacgtcg tatgggtagc
     3421 cagcgtaatc cggaacatca tacgggtatc cttgttgccc catggtggcg ctagcataac
     3481 ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt
     3541 ctttagaatg gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg
     3601 atatcaagct tatcgatatc gaattccgct cgagataatc aacctctgga ttacaaaatt
     3661 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3721 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3781 tataaatcct ggttagttct tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3841 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgtttatttg tgaaatttgt
     3901 gatgctattg ctttatttgt aaccatctag ctttatttgt gaaatttgtg atgctattgc
     3961 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
     4021 tatgtttcag gttcaggggg agatgtggga ggttttttaa agcggccgcc acgtgcggac
     4081 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4141 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     4201 agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac
     4261 gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc
     4321 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4381 gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4441 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4501 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     4561 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     4621 actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg
     4681 atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     4741 aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca
     4801 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4861 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4921 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
     4981 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5041 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5101 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5161 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5221 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5281 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5341 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5401 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5461 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5521 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5581 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5641 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5701 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5761 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5821 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5881 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     5941 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6001 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6061 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6481 gcgcagcggt cgggctgaac gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6781 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
