LOCUS       sequence-430839-        6611 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 6611)
  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 6611)
  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: GPRC5A-DuET
FEATURES             Location/Qualifiers
     source          1..6611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(377..396)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(378..396)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(441..644)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(470..490)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        645..1024
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1196..1215
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1290..1394
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1395..1463,1464..2255)
                     /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(1613..1632)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2426..3014
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2915..2934
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3168..3185
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3302..3323
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3338..3343,3344..3361,3362..3368)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3376..3392
                     /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     3381..3403
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3400..3416
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3400..3416
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3465..3586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3476..3495)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3530..3549
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3760..4554)
                     /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(3871..3890)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4481..4500
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4621..4950)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4635..4770
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4689..4708)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4935..4955
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4964..5392)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5074..5095)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5286..5305
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5438..5662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5651..5668
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 ggcaaacgtc agcccaaaga tgccaagcac gccgagcaag aacaggaact gggttggcag
       61 catcttgcgt ctattggaat cctgcacttt gcagacgagg ataggcaggg tgagcataaa
      121 tgccacggag gttactacgc cagctgtagc gaccgtttcg agcactatgc cccatgcctc
      181 tgctttgtca cagaggcgat aatacttgga tttcaatcca ttgcggcagc cgtctggcac
      241 cgtcgtggcc atatcgatgc tggcgtcatc atcgtccttg tagtcggcga ataccaggca
      301 gaagatgtag ctcagggcga tgatcgtctt catggtggcg ctaagcttaa gtttaaacgc
      361 tagccagctt gggtctccct atagtgagtc gtattaattt cgataagcca gtaagcagtg
      421 ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac acgcctaccg
      481 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
      541 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
      601 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
      661 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
      721 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
      781 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
      841 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
      901 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
      961 tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa
     1021 tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc
     1081 agattcttca tgcaaatgtc ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg
     1141 cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa ctatgcggca
     1201 tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc gatccgtcga
     1261 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1321 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1381 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1441 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1501 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1561 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1621 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1681 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1741 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1801 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1861 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1921 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1981 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2041 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2101 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2161 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2221 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2281 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2341 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2401 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2461 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2521 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatacc gttcttctag
     2581 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2641 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2701 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2761 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2821 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2881 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2941 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3001 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3061 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3121 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3181 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3241 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3301 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     3361 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     3421 attacgccaa gctctagcta gaggtcgacg gtatacagac atgataagat acattgatga
     3481 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     3541 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga
     3601 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
     3661 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
     3721 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     3781 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     3841 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     3901 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     3961 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     4021 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     4081 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     4141 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     4201 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     4261 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     4321 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     4381 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     4441 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     4501 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     4561 cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc ccgggagctt
     4621 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     4681 agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     4741 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     4801 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     4861 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     4921 tggggacttt ccacacccta actgacacac attccacaga attaattcgc gttaaatttt
     4981 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5041 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5101 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5161 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5221 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5281 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5341 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtggggatac
     5401 cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac cgcatcccca
     5461 gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc
     5521 agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg
     5581 acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat
     5641 ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaacggg ccctctagac
     5701 tcgagttagg caggggccac ctgagaagtc tcggtcttag acacggtggt gctggcctca
     5761 tcgaattcac cggtgcgtcc accggtatcg attgacccct ccttcttgac ttcatagtcc
     5821 ttgtatggag atggccaagc gtgggcgcgt ggaattgaaa attccttctg ggggggttgg
     5881 ttctgcagct ggaagtgtgt gctgtatgga gcataaagtg tatctcccgt ctcctcgaaa
     5941 ccctgtgtga tctcctcttg gctataagcc cgattctcca ccccataact cttcttgacc
     6001 agctgaggct tgcagaatgc gtcctcgact gggtagtcca tgggatttcg ctgtttcgtg
     6061 agcagccaga actcgggaga aacataagcg aggagaaaca cccaaccgtt ggcggcgagt
     6121 gcactgctca gtatggtgtc gtcccagcgg cggtcaaagt ctggcaacat cagcaaagtg
     6181 atccaggcca cccagatagc aatactcaga agcatcgtca gataaatatg cgcgccgtgc
     6241 cttttccaac cagtaaagga cccgcaaaag gtgaaggagg acatcaagaa cgtcagggcc
     6301 atcaaaaaca gtacgtaagt caggagcagc acgaagtcct cgttccgccg gggagcgctc
     6361 agttcgctga acacgttgac gttcgtgcga ttcattgtca acacgatgta ctcgattgca
     6421 atcacatctt gcaccaggga aaagccgaca gccaggccga ggatcacgag gagtgagagg
     6481 ggtttccttc ctcgcaccaa cttcgtgagt gagacggcat gagccaggag gcaggagaaa
     6541 caaatagaaa acagtatccc aaacagaaag aaccttgtgg ggccagtgga gccatccagg
     6601 ccgataataa a
//
