LOCUS       sequence-430868-        6503 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 6503)
  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 6503)
  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: MRGPRD-DuET
FEATURES             Location/Qualifiers
     source          1..6503
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     506..525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(744..848)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(923..942)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1114..1493
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1494..1697
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1648..1668
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1742..1761
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1742..1760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2973..2990)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2979..3203
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3249..3677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3336..3355)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3546..3567
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3686..3706)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3691..4020
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3871..4006
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3933..3952
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4087..4881
                     /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(4141..4160)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4751..4770
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5055..5176
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5092..5111)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5146..5165
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5225..5241)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5225..5241)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5238..5260)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5249..5265
                     /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(5273..5279,5280..5297,5298..5303))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5318..5339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5456..5473)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5627..6215)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5707..5726)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6386..6503,1..674,675..743))
                     /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"
ORIGIN
        1 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
       61 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
      121 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      181 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      241 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
      301 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
      361 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
      421 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
      481 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
      541 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
      601 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
      661 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
      721 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
      781 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
      841 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
      901 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
      961 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     1021 caaggcaagg cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc
     1081 tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact agttattaat
     1141 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     1201 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     1261 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     1321 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     1381 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     1441 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     1501 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     1561 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     1621 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
     1681 tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa
     1741 ttaatacgac tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgc
     1801 caccatgaag acgatcatcg ccctgagcta catcttctgc ctggtattcg ccgactacaa
     1861 ggacgatgat gacgccagca tcgatatgaa ccagacgctg aactccagcg gcactgttga
     1921 gtctgctctg aactactcac gggggagtac tgtgcatacc gcctacctgg ttcttagctc
     1981 acttgccatg tttacatgct tgtgcggcat ggccgggaac agtatggtca tctggttgct
     2041 gggttttaga atgcatcgaa atcctttctg catctatata ctgaacctgg ccgccgccga
     2101 tctgctgttc ctttttagca tggctagcac cctgtcactt gaaactcaac ccctcgtaaa
     2161 tactaccgat aaggtgcacg agttgatgaa gaggctcatg tattttgcct acacagtggg
     2221 gctgagcctg ttgactgcta tctctacaca gcggtgtctt agcgtcttgt tccctatctg
     2281 gtttaaatgt caccggccaa gacaccttag tgcctgggtc tgcgggctgc tgtggaccct
     2341 gtgcctcctc atgaacggtc tgacaagcag tttttgttct aaatttctga agtttaatga
     2401 agaccgctgc ttccgggtag acatggttca ggcagctctg ataatgggag tgcttacacc
     2461 agttatgacc ctgtcctctc tcacgctctt cgtatgggtg cgcagaagta gccaacaatg
     2521 gcggagacag ccgaccagac ttttcgtcgt ggtgcttgca agtgtcctgg tatttctgat
     2581 atgttctttg ccgctcagta tatattggtt tgtcctgtac tggctttcat tgccccccga
     2641 aatgcaggta ttgtgtttct ctctttcccg gttgagctct tccgtctcat caagtgctaa
     2701 tcccgtgatt tactttctgg tagggtctag gaggtcccac cggctgccaa ccagatctct
     2761 gggcaccgtg ttgcagcagg ctcttaggga ggagccagaa cttgagggag gggaaacccc
     2821 cacagtcgga actaacgaaa tgggagcaat cgataccggt ggacgcaccg gtgaattcga
     2881 tgaggccagc accaccgtgt ctaagaccga gacttctcag gtggcccctg cctaactcga
     2941 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     3001 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     3061 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     3121 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     3181 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     3241 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3301 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3361 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3421 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3481 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3541 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3601 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3661 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3721 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3781 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3841 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3901 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3961 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4021 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     4081 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4141 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4201 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4261 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4321 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4381 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4441 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4501 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4561 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4621 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4681 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4741 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4801 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4861 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4921 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4981 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5041 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5101 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5161 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5221 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5281 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5341 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5401 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5461 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5521 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5581 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5641 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5701 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5761 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5821 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5881 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5941 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6001 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6061 ctagaagaac ggtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6121 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6181 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6241 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6301 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6361 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6421 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6481 tacgggaggg cttaccatct ggc
//
