LOCUS       sequence-430838-        6614 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 6614)
  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 6614)
  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: GPR87-DuET
FEATURES             Location/Qualifiers
     source          1..6614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     430..449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     683..700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    817..838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(853..858,859..876,877..883)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    891..907
                     /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     896..918
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     915..931
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     915..931
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    980..1101
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(991..1010)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1045..1064
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1275..2069)
                     /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(1386..1405)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1996..2015
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2136..2465)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2150..2285
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2204..2223)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2450..2470
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2479..2907)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2589..2610)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2801..2820
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2953..3177
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3166..3183
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4506..4525)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4507..4525)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4570..4773)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4599..4619)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4774..5153
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5325..5344
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5419..5523
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5524..5592,5593..6384)
                     /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(5742..5761)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6555..6614,1..529)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
       61 cagcagagcg cagataccaa ataccgttct tctagtgtag ccgtagttag gccaccactt
      121 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      181 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      241 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      301 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      361 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      421 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      481 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      541 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      601 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      661 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      721 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      781 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      841 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      901 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
      961 cgacggtata cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     1021 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     1081 ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat ccccgcgctg
     1141 gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt catagaaggc
     1201 ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa
     1261 ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     1321 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     1381 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     1441 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     1501 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac
     1561 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     1621 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     1681 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     1741 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     1801 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     1861 agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc
     1921 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     1981 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     2041 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     2101 gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag gcctccaaaa
     2161 aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc ctctgcataa
     2221 ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg agttaggggc
     2281 gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg catgctttgc
     2341 atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga ctaattgaga
     2401 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca ccctaactga
     2461 cacacattcc acagaattaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     2521 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     2581 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2641 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2701 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2761 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2821 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2881 cgcttaatgc gccgctacag ggcgcgtggg gataccccct agagccccag ctggttcttt
     2941 ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg tcttcccaat
     3001 cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac
     3061 aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg
     3121 tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg
     3181 aggctgatca gcgggtttaa acgggccctc tagactcgag ttaggcaggg gccacctgag
     3241 aagtctcggt cttagacacg gtggtgctgg cctcatcgaa ttcaccggtg cgtccaccgg
     3301 tatcgatcac atctgtatag tcgtagtaaa tccgcacttc ggaccttctt acagactgca
     3361 agcttcgaat gctctcagat cgcgttctga tattactttt cttgaaaagt cttcttgaga
     3421 agctccgaca catgaagaaa taaataatag gatcgaggca cacattgcag gcagacagga
     3481 acagcgtgat ctccttacaa tagtacaaga ttttttgagc gctctcgtcg agcaaccgat
     3541 cgagatggct gaaggtaaat gggatccgac acaggtggta aggcagaaaa catgtaaaga
     3601 atactgcgac caccactctg atgctctgat tatgtttcct ctttctggat gactgactta
     3661 taaactgcct tgaggacttg tgaatgtacc tagagatagc gatataacag ccaatcagaa
     3721 tgacaagtac ggcgacaaac aggcaagaat taacgtatgt cacggcagta tgccacttca
     3781 ccccaagcgg agatttaagc tttgagcaat cgtggatgtt gtcctcagtg ggctgcccat
     3841 ttgtcagaat tatgttaggc agactaagaa cagccatgat cacccacacg catacactga
     3901 ggaccttcgt gaatgtgata ctgtacattc gactgtcccc gaacggtttt acgactttca
     3961 ggtatctgtc gatagagatc agtcccagga aaacgataga tgtgtacata ttagcgtaga
     4021 acaggacgct agtatagcga cacagtatga acttgaaata ccaggggcca aagccggcgt
     4081 cgtgtactat tcgaaagggg aaagtgaggg tcattatcag atcagccacg actatgtttt
     4141 tgaggtaaaa tatgaaagat gtcttgttcc ttatgtggaa aaaaatccac actgcgaggc
     4201 cgttaagcag gatggatgca acgaagatga tgagatacag tacagggagc acgatggtgt
     4261 caaactcatt gtggagtgtt gtatttttcc ccgggccgtc ggatctattg ccgctgttat
     4321 gactctcctg cccgtgaagc tcattattcg gaagtttggc caaagtcaaa ttaaagccca
     4381 tatcgatgct ggcgtcatca tcgtccttgt agtcggcgaa taccaggcag aagatgtagc
     4441 tcagggcgat gatcgtcttc atggtggcgc taagcttaag tttaaacgct agccagcttg
     4501 ggtctcccta tagtgagtcg tattaatttc gataagccag taagcagtgg gttctctagt
     4561 tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt
     4621 caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt gccaaaacaa
     4681 actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa accgctatcc
     4741 acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga ctaatacgta
     4801 gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat gccaggcggg
     4861 ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca cttgatgtac
     4921 tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga aagtccctat
     4981 tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt cgttgggcgg
     5041 tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat atgggctatg
     5101 aactaatgac cccgtaattg attactatta ataactagtc aataatcaat gtcaacgcgt
     5161 atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat
     5221 gcaaatgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc
     5281 gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat
     5341 tgtactgaga gtgcaccata ggggatcggg agatctcccg atccgtcgac gtcaggtggc
     5401 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5461 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5521 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5581 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5641 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5701 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5761 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5821 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5881 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5941 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6001 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6061 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6121 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6181 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6241 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6301 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6361 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6421 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6481 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6541 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6601 aaaccaccgc tacc
//
