LOCUS       sequence-441116-        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuron-specific, Cre-dependent co-expression of FLAG-tagged 
            inhibitory hM4D(Gi) DREADD receptor, and a single-cell 
            discriminating version of AausFP1 as a reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  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 6513)
  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-FLAG-hM4D(Gi)-P2A-ArgiNLS-AausFP1
FEATURES             Location/Qualifiers
     source          1..6513
                     /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(1529..1585)
                     /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(3122..3145)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3145..3154
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3161..3194
                     /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    3245..3278
                     /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(3375..3395)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   3777..3917
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3777..3906
                     /label=AAV2 ITR
     rep_origin      3992..4447
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4079..4098)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4289..4310
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4464..4483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4583..4605
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4643..4661)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4729..4833
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4834..4902,4903..5694)
                     /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(5052..5071)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5865..6452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6353..6372
                     /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 cgcgccttag gcgtaggcgg tcttgcagtc gatggctctg aacacttcca
      781 ccacgttcat gtggtcccgg gtttctttag ggtccttgct caggatggtg tgggcctgaa
      841 tgtggtgata gtgagggatg tccacggggc cacctccgag aggggtattc agctgctggt
      901 gagcagcaat gatctggcct ccgccgatca cgtcgtgcac aatgttgatg tagcacttga
      961 tgccgttgtt ctgcacgtcg ggcagcacgt agatgcagtg cgggttgtag ttgaactcaa
     1021 gtttcttgcc caggatgttg ccgcctttct tgaagccgct gccgttcagg gtcactctgt
     1081 tgtacacgga gcccttctcg taggtcactt cggctctggt cttgtacacg ccgtcgttct
     1141 cgaaggtgat ggttctctct tgcacgtagc cctcaggcat ggcgctcttg gggtagtcct
     1201 tgatgtcgtt gggatacttg gcgaagcact gggcgccgta tgtcactgtg gtcaggatgc
     1261 tgctccaagg cacaggcagt tcgccggttg tgcagatgaa gctggcctcg atctttccgg
     1321 tattggcgtc gccgtgtcct ttgcctctga cgctgaactt catgccttcc acgtcgcctt
     1381 ccatttccac cacatagggg atcttctcgc ggaacagcag ggcgccataa gacatggtgg
     1441 ccacagggga tcccgttcga cggcgccgcc gccttctcac cttctttttc tttgctatgc
     1501 tcatctgctt ctggggagcc acccttttag gaccggggtt ttcttccacg tctcctgctt
     1561 gctttaacag agagaagttc gtggctccgg atccgaccgg tggatcccgc ctggcagtgc
     1621 cgatgttccg atactggcac agcagcaggt gccggaaggt ctttttaaag gtggcgttgc
     1681 acagagcata gcaggcaggg ttgatggtgc tgttgacgta gcagagccag tagccaatgg
     1741 accacaccgt gtcagggatg cagctctggc agaaggtgtt caccaggacc atgacgttgt
     1801 agggcgtcca ggtgaggatg aaggccagca gaatggcaaa gatcgttcgt gtcactttgc
     1861 gctcccgggc cgccatctgc cgcttcttgc gcacctggtt gcgagcgatg ctggcgaact
     1921 tgcgggccac gttggccgca gggcgcatgc cagccggcgt ggcaggcaca atctcaatgg
     1981 ctgtcacaca ctcattgcct gtctgcttcg tcacaatctg gatcttggac catctggagg
     2041 ctgggttgag ggcccgcggc tgcaggggag gggcgggcat ggcgggcgtg gtggcctctg
     2101 tggtggacag ctctgtggct gggcgttcct tggtgttctg ggtggcactg cctgagctgg
     2161 actcattgga agtgtcctta tcagccacgg ggcgcggtgg cggtggcagc gctggcgggg
     2221 gggcctcctc cagcttgcca ttgcgcagct cctcccgggc ggcctccccg ggcgggggct
     2281 tcttgacgct ctgcttcatt agtgggctct tgaggaaggc cagcgtcttg gctttcttct
     2341 ccttcgggcc ctcgggccgg tgcttgtgga ctcggctgcg actggccagg gagatgtgga
     2401 tgtacagcac cgtcatgatg accacaggca ggtagaagcc agcaatggct gtgccaaagg
     2461 tcactgctgg gttggacagg aactggatga agcactggtt gtcgggcacc gtccgcttac
     2521 ccaccacaaa ctgccagaac aagatggcag gcgcccagag cacgaaggac agtacccagg
     2581 cagcagcaat catgaggcct gccatcttgg tggtgcgccg ggcagggtag gtgagaggct
     2641 tggtgacgca gaagtagcgg tcaaagctga tgatgagaag gttcatgacg gaggcgttgc
     2701 tcaccacgca gtccagggcc agccacaggt cgcagaccac ggcgcccagg ggccagtagc
     2761 ccttgatgat gtacacggtg tagaggttca tggagaaggc gcctatgatg agatcagcac
     2821 acgccaggct gaagaggaag tagttgttga ctgtctgcag ctgcctgttg accttgatgg
     2881 acagcatcac caggatgttg cccacgacag tcaccaggct cagggagcct gtcactgtgg
     2941 caatgaagac catttccacc gtctcatagc gattgtggga tgatgacgtg accaggcgca
     3001 cggactgatt gcccgagctg ccattgacag gtgtgaagtt ggcatttaaa tcttcttctg
     3061 ataataattt ttgttctaat gcatctcgag tggatccgag ctctgaattc tcaccgatat
     3121 ctttatcatc atcatcttta taatccatgg tggcgctagc ataacttcgt ataaagtatc
     3181 ctatacgaag ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca
     3241 aagaataact tcgtataatg tatgctatac gaagttatga attcgatatc aagcttatcg
     3301 atatcgaatt ccgctcgaga taatcaacct ctggattaca aaatttgtga aagattgact
     3361 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3421 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggtta
     3481 gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3541 ctgttgggca ctgacaattc cgtggtgttt atttgtgaaa tttgtgatgc tattgcttta
     3601 tttgtaacca tctagcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     3661 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3721 gggggagatg tgggaggttt tttaaagcgg ccgccacgtg cggaccgagc ggccgcagga
     3781 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     3841 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     3901 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     3961 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4021 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4081 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4141 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4201 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4261 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4321 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4381 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4441 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4501 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4561 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4621 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     4681 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4741 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4801 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     4861 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     4921 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     4981 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5041 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5101 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5161 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5221 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5281 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5341 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5401 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5461 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5521 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5581 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5641 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5701 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5761 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5821 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5881 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5941 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6001 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6061 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6121 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6181 tgaacggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6241 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6301 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6361 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6421 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6481 tcctggcctt ttgctggcct tttgctcaca tgt
//
