LOCUS       sequence-430819-        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: GPR182-DuET
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(593..1384,1385..1453))
                     /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     1216..1235
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1454..1558)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1633..1652)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1824..2203
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2204..2407
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2358..2378
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2452..2471
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2452..2470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3932..3949)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3938..4162
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4208..4636
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4295..4314)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4505..4526
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4645..4665)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4650..4979
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4830..4965
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4892..4911
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5046..5840
                     /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(5100..5119)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5710..5729
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6014..6135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6051..6070)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6105..6124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6184..6200)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6184..6200)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6197..6219)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6208..6224
                     /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)."
     promoter        complement(join(6232..6238,6239..6256,6257..6262))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6277..6298
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6415..6432)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6586..6752,1..422))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6666..6685)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
       61 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      121 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      181 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      241 tgaagtggtg gcctaactac ggctacacta gaagaacggt atttggtatc tgcgctctgc
      301 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      361 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      421 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      481 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      541 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
      601 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
      661 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
      721 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
      781 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
      841 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
      901 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
      961 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1021 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1081 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1141 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1201 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1261 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1321 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1381 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1441 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1501 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1561 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     1621 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     1681 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     1741 cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     1801 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     1861 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     1921 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1981 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     2041 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2101 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2161 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2221 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2281 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2341 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2401 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
     2461 tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgc caccatgaag
     2521 acgatcatcg ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat
     2581 gacgccagca tcgatatgtc agtcaagccc tcctggggac caggtccatc agagggggtg
     2641 acagcagtgc cgacaagtga tcttggcgag atccacaatt ggacagagct cctcgatctg
     2701 ttcaatcaca cactctccga gtgtcacgtt gagctttcac agtccaccaa gagagtcgtg
     2761 ctgtttgccc tttatcttgc catgttcgtc gtcgggttgg tcgagaatct gctcgtgatt
     2821 tgtgtcaatt ggaggggaag cggtagagcc ggactcatga acctgtatat tcttaatatg
     2881 gcaatcgccg acctggggat cgtcttgagt ctgcccgtgt ggatgcttga ggttacactg
     2941 gattatactt ggctgtgggg gagcttctct tgtaggttta cccattattt ctattttgtg
     3001 aacatgtaca gctcaatttt tttcctcgtc tgtctgagcg tcgaccgata cgtaaccctt
     3061 accagcgcgt ctccaagctg gcagagatat caacacaggg tgaggcgcgc catgtgcgcg
     3121 ggcatctggg tgcttagcgc tatcatcccg ctgccagaag tagtgcacat tcagcttgtg
     3181 gaagggcccg agcccatgtg cctttttatg gccccgttcg agacttattc cacctgggct
     3241 cttgccgtgg ccctctccac tacaattctg ggtttcttgc tgcccttccc tctgattaca
     3301 gtgtttaatg tgctcacagc ttgcaggctg cggcaaccgg ggcaacccaa gagccgccgc
     3361 cattgcttgc tgctgtgcgc ttatgtcgcc gtcttcgtta tgtgctggct cccttatcat
     3421 gtgaccctgt tgctcctgac cctgcacggg actcatatct ccctgcattg tcacctcgtt
     3481 cacctgctct atttcttcta tgatgttatt gattgtttta gtatgttgca ctgtgtaatt
     3541 aacccgatcc tgtacaactt tctgtcaccg cattttagag gcagactgct gaacgcagtc
     3601 gtgcactacc tgccgaagga ccagactaag gctggaacct gcgccagctc ttcaagctgc
     3661 tccacacagc attccattat tatcaccaag ggggattccc aacctgccgc tgccgctccc
     3721 caccccgagc cttctcttag cttccaggct caccacctgc tgcccaacac tagtcccatc
     3781 tcccctaccc agcctctgac cccaagtatc gataccggtg gacgcaccgg tgaattcgat
     3841 gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc ctaactcgag
     3901 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     3961 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4021 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4081 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4141 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     4201 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4261 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4321 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4381 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4441 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4501 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4561 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4621 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4681 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     4741 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4801 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4861 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     4921 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     4981 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     5041 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     5101 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     5161 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     5221 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     5281 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     5341 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     5401 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     5461 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     5521 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     5581 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     5641 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     5701 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     5761 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     5821 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     5881 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     5941 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     6001 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     6061 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     6121 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     6181 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     6241 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     6301 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6361 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6421 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6481 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6541 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6601 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6661 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6721 cctgccgctt accggatacc tgtccgcctt tc
//
