LOCUS       sequence-430739-        6584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The DuET construct encodes the named human GPCR engineered with a 
            cleaved hemagglutinin signal peptide, a 5' FLAG and a 3' 1D4 epitope
            tag optimized for expression in mammalian cells and proteomics 
            studies..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6584)
  AUTHORS   Dahl L, Kotliar IB, Bendes A, Dodig-Crnkovic T, Fromm S, Elofsson A,
            Uhlen M, Sakmar TP, Schwenk JM
  TITLE     Multiplexed selectivity screening of anti-GPCR antibodies.
  JOURNAL   Sci Adv. 2023 May 3;9(18):eadf9297. doi: 10.1126/sciadv.adf9297. 
            Epub 2023 May 3.
  PUBMED    37134173
REFERENCE   2  (bases 1 to 6584)
  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: "Sci Adv."; 
            date: "2023-05-3"; volume: "9(18):eadf9297. doi"; pages: " 
            10.1126/sciadv.adf9297. Epub 2023 May 3"
COMMENT     Sequence Label: TAAR9-DuET
FEATURES             Location/Qualifiers
     source          1..6584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     547..566
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(687..1016)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      701..836
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(755..774)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1001..1021
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1030..1458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1140..1161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1352..1371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1504..1728
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1717..1734
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3027..3046)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3028..3046)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3091..3294)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3120..3140)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3295..3674
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3846..3865
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3940..4044
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4045..4113,4114..4905)
                     /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(4263..4282)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5076..5664
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5565..5584
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5818..5835
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5952..5973
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5988..5993,5994..6011,6012..6018)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6026..6042
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     6031..6053
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6050..6066
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6050..6066
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6115..6236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6126..6145)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6180..6199
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6410..6584,1..620))
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6521..6540)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
       61 gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct
      121 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
      181 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
      241 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
      301 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
      361 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
      421 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
      481 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
      541 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
      601 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatccgaaa
      661 atggatatac aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct
      721 cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa
      781 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
      841 ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg
      901 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt
      961 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc
     1021 cacagaatta attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     1081 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     1141 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1201 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     1261 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     1321 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     1381 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     1441 cgccgctaca gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag
     1501 aagccataga gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct
     1561 tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg
     1621 caatttcctc attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag
     1681 gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc
     1741 agcgggttta aacgggccct ctagactcga gttaggcagg ggccacctga gaagtctcgg
     1801 tcttagacac ggtggtgctg gcctcatcga attcaccggt gcgtccaccg gtatcgatat
     1861 cggtctccac ttcctcacta aaaaggttgg tggttgaact gtcggttctc aataccttac
     1921 cggagacgat caatttgatt gcttttccga accattgata gaagaaggcg taaatcagtg
     1981 gattcattgc tgaattatag tagacgcacc acaccaggat ctcataaaca tagggtggcg
     2041 ttatgaagtt catataagcg tcaatcacgg cgtccaccaa gtacggcaac caagacacca
     2101 gaaaggcggc catggcgata cccagggtct tagccgcttt tctctcgcgc ttagcgaccc
     2161 tttccttata agactcgctt gatgactgag cctgagaagc tgtagactcg attttccttg
     2221 cttggtgctt agcaacgaga aagatcttgg agtaaataaa caccatagcg acgttcggaa
     2281 taaagaaaag caggaaacag agcagcaccc aattctgatt caatggagcc tggcatccgc
     2341 ccacgcatgt aagtgccacc acaagctctt cgatcccttc ctcgttggca cctgtgtaaa
     2401 aaatgctgaa ggagtatgtc acactgaaaa accaggacag gacgatacaa atgccggaga
     2461 cgctcacggt aaattttgtg gggtaagtaa gtgggtctgt gacagcgata tatcggtcga
     2521 cggagataca gcagaggtga aacaatgaag caaagcagaa acttgtgtca aagcaggtat
     2581 gaaacttaca gtatgagtca ccgaaatacc agcagctctc aacacttcga acggtgctaa
     2641 aaggcattac agtaaccccg accaggaagt cagcacaagc gagggacgct atcaggaaat
     2701 tggtgggggt gtgcagctgc ttgaaatgca ggatcgcaat catcaccaag agattgccaa
     2761 atgcggccag cacggcaccg aagcccagga ccgcgtacag tatactcctc ggccctgggg
     2821 agtagggggt cttgatacaa gattcgttca cgttcttata gcacaactca acggcttcgg
     2881 cctgggagaa attattcacc atatcgatgc tggcgtcatc atcgtccttg tagtcggcga
     2941 ataccaggca gaagatgtag ctcagggcga tgatcgtctt catggtggcg ctaagcttaa
     3001 gtttaaacgc tagccagctt gggtctccct atagtgagtc gtattaattt cgataagcca
     3061 gtaagcagtg ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac
     3121 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     3181 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     3241 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     3301 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     3361 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     3421 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     3481 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     3541 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     3601 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     3661 caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct
     3721 aaccctaagc agattcttca tgcaaatgtc ggtcaagcct tgccttgttg tagcttaaat
     3781 tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa
     3841 ctatgcggca tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc
     3901 gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3961 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4021 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4081 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4141 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4201 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4261 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4321 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4381 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4441 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4501 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4561 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4621 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4681 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4741 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4801 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4861 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4921 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4981 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5041 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5101 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5161 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatacc
     5221 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5281 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5341 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5401 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5461 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5521 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5581 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5641 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5701 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     5761 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     5821 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     5881 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     5941 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     6001 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6061 tatgaccatg attacgccaa gctctagcta gaggtcgacg gtatacagac atgataagat
     6121 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     6181 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg
     6241 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     6301 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     6361 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     6421 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     6481 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     6541 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcg
//
