LOCUS       sequence-430885-        6617 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 6617)
  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 6617)
  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: P2RY8-DuET
FEATURES             Location/Qualifiers
     source          1..6617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(572..591)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(573..591)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(636..839)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(665..685)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        840..1219
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1391..1410
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1485..1589
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1590..1658,1659..2450)
                     /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(1808..1827)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2621..3209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3110..3129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3363..3380
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3497..3518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3533..3538,3539..3556,3557..3563)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3571..3587
                     /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     3576..3598
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3595..3611
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3595..3611
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3660..3781
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3671..3690)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3725..3744
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3955..4749)
                     /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(4066..4085)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4676..4695
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4816..5145)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4830..4965
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4884..4903)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5130..5150
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5159..5587)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5269..5290)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5481..5500
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5633..5857
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5846..5863
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 agtccccgcg cacgcagcga ctgcgtacct tcgtcgccgc caccgcttag aagagagggg
       61 gtaaagcacc cccagaaatc gttccacgct aatgcacgtc atagtgagga tactagagta
      121 catattggca taaaatgcca cggttacgac gttgcacagc agcaccccaa acacccagtg
      181 gtgcctatta cagtgataat agatttgaaa gggcaggact gaggccagca tgaggtccgt
      241 gacagacagg ttgatcatga agataactga gggtgatctg ggacccattc tccggcagag
      301 cacccacaat gagaacaaat ttcctggaat tgaaacggct gccaccaggc tatagactac
      361 cgggagggcg acggcaatgg caggattccg cagcatctgc agcgtagcat tatcggggcc
      421 agtgctgttg ggtacctgca tatcgatgct ggcgtcatca tcgtccttgt agtcggcgaa
      481 taccaggcag aagatgtagc tcagggcgat gatcgtcttc atggtggcgc ggccgctaag
      541 cttaagttta aacgctagcc agcttgggtc tccctatagt gagtcgtatt aatttcgata
      601 agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata gacctcccac
      661 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
      721 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
      781 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
      841 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
      901 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
      961 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     1021 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     1081 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     1141 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     1201 ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa
     1261 cgcctaaccc taagcagatt cttcatgcaa atgtcggtca agccttgcct tgttgtagct
     1321 taaattttgc tcgcgcacta ctcagcgacc tccaacacac aagcagggag cagatactgg
     1381 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatagggg atcgggagat
     1441 ctcccgatcc gtcgacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1501 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1561 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1621 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1681 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1741 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1801 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1861 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1921 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     1981 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2041 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2101 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     2161 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2221 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2281 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2341 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2401 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2461 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2521 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2761 ataccgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3241 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3301 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3361 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     3421 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3481 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     3541 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     3601 acagctatga ccatgattac gccaagctct agctagaggt cgacggtata cagacatgat
     3661 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3721 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3781 tggggtgggc gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt
     3841 cccggaaaac gattccgaag cccaaccttt catagaaggc ggcggtggaa tcgaaatctc
     3901 gtgatggcag gttgggcgtc gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa
     3961 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     4021 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     4081 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     4141 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     4201 atcctcgccg tcgggcatgc gcgccttgag cctggcgaac agttcggctg gcgcgagccc
     4261 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     4321 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     4381 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     4441 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     4501 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     4561 ctcgtcctgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     4621 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     4681 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     4741 ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gatccgaaaa tggatataca
     4801 agctcccggg agctttttgc aaaagcctag gcctccaaaa aagcctcctc actacttctg
     4861 gaatagctca gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc
     4921 atggggcgga gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg
     4981 cgggactatg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     5041 gcctggggac tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct
     5101 gcctgctggg gagcctgggg actttccaca ccctaactga cacacattcc acagaattaa
     5161 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5221 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     5281 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     5341 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     5401 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     5461 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     5521 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     5581 ggcgcgtggg gataccccct agagccccag ctggttcttt ccgcctcaga agccatagag
     5641 cccaccgcat ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc
     5701 cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca
     5761 ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag
     5821 gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgggtttaa
     5881 acgggccctc tagattaggc aggggccacc tgagaagtct cggtcttaga cacggtggtg
     5941 ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga tgaacacgga ctcctggcgc
     6001 tggaggccag gtcgagtagc gccctccatg ccctcagggt gcgcgcccgc ctcactccgt
     6061 acgctcgtag tccgtgcgct gaaaagggac tccctccgag tgtccagagt atctcttgga
     6121 accctcctgc atccaaggta ctctctcagg cggagctgaa actccctgga agcaaaataa
     6181 tacacaaacg gatccagaca attgttgagg catgacagac acaaggtaag tttataaacg
     6241 tggtaataag atttgccgta gaacaaccta ctcacgatgt gggccagcag cacgaaatta
     6301 ttgggcgcga agcaagtcac aaaggccagc agaacgactg cggcgaggcc tacggctcgt
     6361 cttctttgct ctctcccgtg ggcctcctct gttctgagca gtttcagaat agtggcagtg
     6421 taacaagcta ctgtgatgac aaagggtatc aagaagagca gtatgaagat tgtaaacagg
     6481 aacactgccc acattgccac tgaggggagc attgtccact tcagcacgtc aaagcaggtg
     6541 atgatgccga gtgcgtggac tggatatgtc agatcagttc gggcaagagg gctgagggcg
     6601 gtcagcagca acagcca
//
