LOCUS       sequence-430813-        6752 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 6752)
  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 6752)
  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: GPR143-DuET
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      154..742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     643..662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     896..913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1030..1051
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1066..1071,1072..1089,1090..1096)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1104..1120
                     /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     1109..1131
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1128..1144
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1128..1144
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1193..1314
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1204..1223)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1258..1277
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1488..2282)
                     /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(1599..1618)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2209..2228
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2349..2678)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2363..2498
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2417..2436)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2663..2683
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2692..3120)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2802..2823)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3014..3033
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3166..3390
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3379..3396
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4857..4876)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4858..4876)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4921..5124)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4950..4970)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5125..5504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5676..5695
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5770..5874
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5875..5943,5944..6735)
                     /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(6093..6112)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
       61 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
      121 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
      181 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      241 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaataccgt
      301 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      361 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      421 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      481 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
      541 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
      601 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
      661 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
      721 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
      781 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
      841 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      901 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
      961 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     1021 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     1081 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     1141 tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac
     1201 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1261 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg
     1321 ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa
     1381 aacgattccg aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg
     1441 caggttgggc gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg
     1501 tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg
     1561 aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct
     1621 atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg
     1681 ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg
     1741 ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc
     1801 tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg
     1861 atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc
     1921 cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga
     1981 tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg
     2041 agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc
     2101 tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc
     2161 gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag
     2221 ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc
     2281 atgcgaaacg atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc
     2341 gggagctttt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     2401 tcagaggcag aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc
     2461 ggagaatggg cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact
     2521 atggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     2581 gactttccac acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
     2641 ggggagcctg gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt
     2701 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2761 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     2821 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     2881 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     2941 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3001 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3061 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3121 ggggataccc cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg
     3181 catccccagc atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc
     3241 caccccccag aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt
     3301 aggaaaggac agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa
     3361 caacagatgg ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaacgggcc
     3421 ctctagactc gagttaggca ggggccacct gagaagtctc ggtcttagac acggtggtgc
     3481 tggcctcatc gaattcaccg gtgcgtccac cggtatcgat gagatcgcca tgggtgggca
     3541 acgccgggtc tccttcattc ttattgcatg actcgcttgc ggtatgaatc tcgatcgttg
     3601 aagcatcaga gccctcggac agcatggaca gtgcctcatc ggatgtctgc cctcctacct
     3661 gagaaacctt gccagaggcg gggttttcat gtggcatcag gggggatgga tgggcccctt
     3721 cggcagcact ggtggtgagg gattcccatt gaatctcttt gcgggggctc tggaacccga
     3781 ggctgcagcc ggtccaaccg taaaaagcca agctgaggag aaaaccttgc gcagggttca
     3841 ggatccccat gataaaccag gtagtcttgg cggcggttct gactggcttg agggaccctc
     3901 cattaatatc tgtttgcatt tccaaataaa agagcaagga ttcgtttatg atgttgctga
     3961 gccaacagat gataagtaca agcatgattt tgaagaaccg aattttgata actgcgccca
     4021 tcctcctctc gttttcagtg tagatcccct ggcgtccttt aagcagactt gcgacggcgg
     4081 taaccgtttt ttggaaaagg ataggattgg ccaccagaac aagcagcaac ggcaggtaca
     4141 tggtcacata gtgtgggatt gcatggtcga gccctctctc gcatcgacta acagatggat
     4201 agtagagcat cgcggcgcct tccacgcaca gcagagtggc aagcccccag gccataatgt
     4261 gatacaacag tatggtagac agtccggcag agcgcctaat cacaagatat gcatcgacgg
     4321 cgtaacaaaa gagccaccag aagcaggcgg agtagagcaa ctgaatccac atagcgctgc
     4381 ctacgcagaa agctgctggc cagatctcgg tatgattcat gtcggacaca gaatctacga
     4441 aattggggaa tcccagccac actgtactcc gtatcaccat cccgaggcag cccaggagat
     4501 cacacgctgc ggctgccctc aagatgcgca cagaagccgg tggggaggtt gcgggggatc
     4561 ctgggccagc aggccttctc ccaggcagca gctgaaggag acccagagca agtctaagtc
     4621 cgccactccc aaggcacaag gcgtgaaagg cgcgtggctg gaaggacagc accaactggg
     4681 tagctgcgtc gcgagtggga caacaaaaag tgcccagcct ggggcttgcc atatcgatgc
     4741 tggcgtcatc atcgtccttg tagtcggcga ataccaggca gaagatgtag ctcagggcga
     4801 tgatcgtctt catggtggcg ctaagcttaa gtttaaacgc tagccagctt gggtctccct
     4861 atagtgagtc gtattaattt cgataagcca gtaagcagtg ggttctctag ttagccagag
     4921 agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc
     4981 ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt
     5041 gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt
     5101 gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg
     5161 ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc
     5221 gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg
     5281 ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac
     5341 tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg
     5401 cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat gaactaatga
     5461 ccccgtaatt gattactatt aataactagt caataatcaa tgtcaacgcg tatatctggc
     5521 ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc agattcttca tgcaaatgtc
     5581 ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg cactactcag cgacctccaa
     5641 cacacaagca gggagcagat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     5701 agtgcaccat aggggatcgg gagatctccc gatccgtcga cgtcaggtgg cacttttcgg
     5761 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5821 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5881 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5941 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     6001 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     6061 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     6121 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     6181 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     6241 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6301 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6361 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6421 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6481 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6541 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6601 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6661 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6721 attaagcatt ggtaactgtc agaccaagtt ta
//
