LOCUS       sequence-430826-        6497 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 6497)
  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 6497)
  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: GPR31-DuET
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(224..328)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(403..422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        594..973
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        974..1177
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1128..1148
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1222..1241
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1222..1240
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2447..2464)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2453..2677
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2723..3151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2810..2829)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3020..3041
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3160..3180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3165..3494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3345..3480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3407..3426
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3561..4355
                     /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(3615..3634)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4225..4244
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4529..4650
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4566..4585)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4620..4639
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4699..4715)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4699..4715)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4712..4734)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4723..4739
                     /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(4747..4753,4754..4771,4772..4777))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4792..4813
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4930..4947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5101..5689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5181..5200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5860..6497,1..154,155..223))
                     /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     join(6483..6497,1..5)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
       61 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
      121 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
      181 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
      241 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
      301 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
      361 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
      421 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      481 gagcaaaatt taagctacaa caaggcaagg cttgaccgac atttgcatga agaatctgct
      541 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      601 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      661 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      721 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      781 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      841 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      901 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      961 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     1021 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     1081 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     1141 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
     1201 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg
     1261 tttaaactta agcttagcgc caccatgaag acgatcatcg ccctgagcta catcttctgc
     1321 ctggtattcg ccgactacaa ggacgatgat gacgccagca tcgatatgcc attccctaac
     1381 tgtagtgcgc caagcaccgt ggttgccaca gctgtgggcg tcctcctggg ccttgagtgc
     1441 ggtctgggcc tcctgggaaa tgcagtggcc ctttggactt tcctctttag agtacgggta
     1501 tggaaaccgt acgccgtgta tctgctgaac ctggcattgg cggacctttt gctcgcagct
     1561 tgtctcccat tcctggcggc tttttacctg agtctgcagg cctggcatct cggtcgggtc
     1621 ggttgttggg ctttgcattt cctgttggac ctctcccgga gcgtggggat ggcattcctt
     1681 gctgctgtcg ctctggatag atacttgagg gtggtgcacc ctcggctgaa agtgaatctg
     1741 ctgtcacctc aggctgctct cggcgtctct ggcctggttt ggctcctcat ggtcgccctc
     1801 acttgtcctg gcttgctgat tagtgaagca gcccagaact ctacgagatg ccacagtttc
     1861 tacagtcgcg cggacgggtc cttttcaatt atttggcagg aggctttgtc atgcttgcaa
     1921 tttgtcctgc ccttcggtct tatagttttt tgcaatgccg gaatcattcg ggcacttcag
     1981 aaaagactga gagaacccga gaagcagcca aaactccagc gggcccaggc ccttgttact
     2041 cttgtggtgg tactgtttgc cctgtgcttc ctcccttgct tcctggctag agtcctgatg
     2101 catatatttc agaacctggg atcctgtcgg gcgttgtgcg ccgtggccca taccagtgac
     2161 gtgacgggct ccctcaccta tctgcatagc gtattgaacc cggtagtgta ttgcttctcc
     2221 tctccgacgt ttcggtccag ctaccgacgg gtcttccata cccttcgagg caagggacag
     2281 gctgccgaac cccccgattt caaccccaga gattcttact caatcgatac cggtggacgc
     2341 accggtgaat tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc tcaggtggcc
     2401 cctgcctaac tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     2461 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2521 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2581 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2641 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     2701 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2761 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2821 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2881 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2941 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3001 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3061 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3121 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3241 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3301 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3361 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3421 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3481 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     3541 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3601 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     3661 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     3721 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     3781 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     3841 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     3901 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     3961 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4021 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4081 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4141 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4201 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4261 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4321 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4381 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4441 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4501 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     4561 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4621 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     4681 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4741 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4801 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4861 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     4921 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4981 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5041 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5101 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5161 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5221 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5281 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5341 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     5401 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     5461 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     5521 taactacggc tacactagaa gaacggtatt tggtatctgc gctctgctga agccagttac
     5581 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5641 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5701 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5761 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5821 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5881 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5941 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6001 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6061 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6121 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6181 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6241 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6301 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     6361 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     6421 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     6481 ggataatacc gcgccac
//
