LOCUS       sequence-441114-        7662 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  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 7662)
  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 7662)
  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-EF1a-DIO-3xHA-hM3D(Gq)-P2A-ArgiNLS-oScarlet
FEATURES             Location/Qualifiers
     source          1..7662
                     /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        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1445..1478)
                     /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(1529..1562)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1574..2266)
                     /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(1849..1868)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2118..2136
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2342..2398)
                     /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(4196..4222)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4226..4252)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4256..4282)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      4294..4303
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4310..4343
                     /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    4394..4427
                     /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(4524..4544)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4926..5066
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4926..5055
                     /label=AAV2 ITR
     rep_origin      5141..5596
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5228..5247)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5438..5459
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5613..5632)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5732..5754
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5878..5982
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5983..6051,6052..6843)
                     /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(6201..6220)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7014..7601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7502..7521
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccgtacc ggatcctcta gagtcgactc
     1441 cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta
     1501 gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt
     1561 atggcgcgcc tcacttgtac agctcgtcca tgccgccggt ggagtggcgg ccctcggagc
     1621 gttcgtactg ttccaccacg gtgtagtcct cgttgtggga ggtgatgtcc aacttgcggt
     1681 ccacgttgta ggcgccgggc atctgcacgg gcttcttggc cttgtaggtg gtcttgaagt
     1741 ccgccaggta gcgcccgccg tccttcaggc gcagggccat cttaatgtcg cccttcagca
     1801 cgccgtcctc ggggtacaac cgctcggtgg acgcttccca gcccattgtc ttcttctgca
     1861 ttacggggcc gtcaggaggg aagttggtgc cgcggagctt caccttgtag atcagggtgc
     1921 cgtcctccag ggaggtgtcc tgggtcacgg tcacggcgcc gccgtcctcg aagttcatca
     1981 cgcggtccca cttgaagccc tcggggaagg actgcttata gtagtcgggg atgtcggcgg
     2041 ggtgcttggt gaaggccctg gagccgtaca tgaactgagg ggacaggatg tcccaggaga
     2101 agggcagggg gccacccttg gtcaccttca gcttggcggt ctgggtgccc tcgtaggggc
     2161 ggccctcgcc ctcgccctcg atctcgaact cgtggccgtt catggagccc tccatgtgca
     2221 ccttgaaccg catgaactcc ttgatcactg cctcgccctt gctcactcga cggcgccgcc
     2281 gccttctcac cttctttttc tttgctatgc tcatctgctt ctggggagcc acccttttca
     2341 taggaccggg gttttcttcc acgtctcctg cttgctttaa cagagagaag ttcgtggctc
     2401 cggatccggt ggcgaccggg ggatccttca aggcctgctc gggtgcgcgc ttgtgaaaaa
     2461 tgaccgactg tctctgctgg tactgctgct tgcgcctctt ttttttgtca cactggcaca
     2521 gcagcagcat cttgaaagtg gttctgaatg ttttgttgca cagagcatag cacacggggt
     2581 tcacggtgct gttgatgtag cacagccagt agcccagatt ccaaaaggtt ttgggtatgc
     2641 agctgtcaca aaaggtgttc accagaacca tgatgttgta tggggtccaa gtgatgatga
     2701 aggcaagcaa gatcgcactg agggtctggg ccgctttctt ctccttgacc agggacatcc
     2761 ttttccgctt agtgatctga cttctggtct tcagagcaaa cctcttggcc agagtggctt
     2821 ccttgaagga cagaggtaga gtggccgtgc tcttacccac tgaggagttg acgtcagaag
     2881 tcttagctgt gtccacggct gactctagct ggatgggaag cttggagaag ctttttggaa
     2941 aactgcctcc atcgtccacg ctcttctggg cctgcagctt gtcggctttc ctctccaagt
     3001 ccaccatccc cagctcctcc tcaggcacct gcaggttgtc cgatgagggt aacttggtgg
     3061 agttgaggat ggtgctgtga cccggaagct tgagcacgat ggagtagatg gctctcgtct
     3121 cggagccaat gtcctcctcg tcggaggagg cggagttctc cagggaggca gcagcatcat
     3181 tgttgttcca actgtcactg ctgctgtggt cttggtccat ctgctcggag ctgggtttcc
     3241 agctcttggt tgtgaaccag aagtggcagc ggccatactt cctcctgttg gagcgtttca
     3301 tgctttgctg ttgaagttcg taactgctgc agcttcgaga actgcccgtg gggtggacaa
     3361 agttttctgt ctctgcctct gtcccagagg cttgcaggcc agcaagctct ttggtacgct
     3421 tttcagtttc cttatagatc ctccagtata aaatagtcat aatggtgaca ggcatataaa
     3481 aaccagcgat ggctgtgcca aaagtaatgg tgggctcact gaggaactga atgaagcact
     3541 ctcccggagg cacagttctc tttccaacaa agtattgcca gaacaagatg gcaggagccc
     3601 aaaggacaaa ggagatgacc caagccagac cgatcatcac accggctctc tttgttgttc
     3661 gtttggctcg gtacgtgagc ggcctcgtga tggaaaagta tctgtcaaag ctgatgacca
     3721 gaagattcat aacagaggca ttgctggcta cgcagtcaat ggcaagccag aggtcacagg
     3781 ccaagttccc taaggcccat cgattcatga tgatgtaggt cgtaaacaga ttcattgaaa
     3841 tgaccccgat aatcagatcg gcacaggcca ggcttaagag gaagtagttg ttgaccgtct
     3901 tcagctgctt gttgacctta aatgacacaa ttaccaggat gttgccgatg atggtcacca
     3961 aggccaggat gcccgttaag aaagcgatga agaccacttg ccagacggta tgacctccca
     4021 gagggtcatc ggtggtaccg tctggagagg agaaattgcc agctgctcga gaaacattgt
     4081 agctgccgaa atgagtgacg gttcccgggg gcagccctgc atcggagggg ctgtgtatcc
     4141 aggaggagct gatgtttgga aacaaaggcg aggttgtact gttattgtgc aaggtagcgt
     4201 agtctgggac gtcgtatggg tagccagcgt agtctgggac gtcgtatggg tagccagcgt
     4261 aatccggaac atcatacggg tatccttgtt gccccatggt ggcgctagca taacttcgta
     4321 taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag
     4381 aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatgaa ttcgatatca
     4441 agcttatcga tatcgaattc cgctcgagat aatcaacctc tggattacaa aatttgtgaa
     4501 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     4561 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     4621 tcctggttag ttcttgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4681 ggggctcggc tgttgggcac tgacaattcc gtggtgttta tttgtgaaat ttgtgatgct
     4741 attgctttat ttgtaaccat ctagctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4801 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4861 tcaggttcag ggggagatgt gggaggtttt ttaaagcggc cgccacgtgc ggaccgagcg
     4921 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     4981 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     5041 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
     5101 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
     5161 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
     5221 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5281 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5341 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5401 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     5461 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
     5521 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     5581 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     5641 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5701 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5761 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
     5821 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     5881 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5941 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     6001 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     6061 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     6121 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     6181 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     6241 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     6301 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     6361 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     6421 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     6481 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     6541 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     6601 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6661 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     6721 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6781 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6841 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6901 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6961 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7021 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7081 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7141 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     7201 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7261 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7321 cggtcgggct gaacgggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7381 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7441 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7501 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7561 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7621 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
