LOCUS       sequence-431825-        6506 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 6506)
  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 6506)
  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: MRGPRX4-DuET
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     317..336
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        411..515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(516..584,585..1376)
                     /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(734..753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1547..2135
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2036..2055
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2289..2306
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2423..2444
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2459..2464,2465..2482,2483..2489)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2497..2513
                     /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     2502..2524
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2521..2537
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2521..2537
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2586..2707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2597..2616)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2651..2670
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2881..3675)
                     /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(2992..3011)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3602..3621
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3742..4071)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3756..3891
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3810..3829)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4056..4076
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4085..4513)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4195..4216)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4407..4426
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    4559..4783
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4772..4789
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(6004..6023)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6005..6023)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6068..6271)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6097..6117)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(6272..6506,1..145)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
       61 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
      121 taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc
      181 gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc ttgccttgtt
      241 gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga
      301 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg
      361 ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
      421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
      481 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
      541 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
      601 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
      661 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
      721 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
      781 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
      841 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
      901 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
      961 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     1021 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     1081 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     1141 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     1201 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     1261 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     1321 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     1381 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1441 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1501 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1561 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1621 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1681 taccaaatac cgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     1741 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     1801 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1861 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     1921 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     1981 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     2041 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     2101 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     2161 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2221 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2281 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2341 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     2401 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     2461 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     2521 caggaaacag ctatgaccat gattacgcca agctctagct agaggtcgac ggtatacaga
     2581 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     2641 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     2701 acaagttggg gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc
     2761 cggcgtcccg gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga
     2821 aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc
     2881 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     2941 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     3001 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     3061 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     3121 gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
     3181 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     3241 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     3301 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     3361 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     3421 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     3481 cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     3541 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     3601 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     3661 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc cgaaaatgga
     3721 tatacaagct cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta
     3781 cttctggaat agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag
     3841 tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt
     3901 taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc
     3961 tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat
     4021 acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca cattccacag
     4081 aattaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4141 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4201 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4261 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4321 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4381 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4441 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4501 ctacagggcg cgtggggata ccccctagag ccccagctgg ttctttccgc ctcagaagcc
     4561 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
     4621 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
     4681 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
     4741 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
     4801 gtttaaacgg gccctctaga ctcgagttag gcaggggcca cctgagaagt ctcggtctta
     4861 gacacggtgg tgctggcctc atcgaattca ccggtgcgtc caccggtatc gatgggtccg
     4921 agtcttgaac cagacagttc cagggactcc tcaggcagct ggccctcccc tttatccact
     4981 tcgggtttat cttggagtgc tcgctgaaga acgagtttca agttttggcg gttctgtcgc
     5041 tgccggaagc tccccacaaa gaaatagatg attgggttag cgctagagtt gaggctagac
     5101 agagacatac acaccaggta aacgtgacag tagagcacct ccaggttcag gtgcatcctg
     5161 tagatgaggg ctccgagaat cccgaacgga agcccacaca gaagaaatac cagaactgtg
     5221 agcaaaattg tgacgtacaa ccgtgtgagc ggcatctttc tggacccaca cagaattctg
     5281 acgagcagta ccaaggatga cacacaaagt actacacaaa gaaagatgag ccaagcgacc
     5341 ggaataaaat cggaggtctc gcaccagctg gagtcagctc cggagaagag aaagtcacag
     5401 aagcgccatt ccagcatgga gaacagcagt gacaggcccc acagaagcac acaaaccact
     5461 gcggacaggt gagtgggtct tctgcatcgg taccatatgg gccacaggac actcaaacac
     5521 cgttcggtgc taatggctga gagcatgctc agacctgtga aatagggaaa tgtcatcact
     5581 gacacgagga tcttccgaat aaggtgagag atattaatga gtcgcagagg caggcggatg
     5641 atttggaagg agagaaacag aaaatctgcg gctgccaaat taaggatata aatactcacg
     5701 gcattgcgtc gcattctata ccccagcaac caaagtacca ctgcgttgcc tgtcagcccg
     5761 accagactga tgatacatgt gagcacggtg aaagagaggg tctgattata gcagggagtt
     5821 tcctctctcc cgttaatagg ggtgagcttt gtaccaaaca caggcacagt aggatccata
     5881 tcgatgctgg cgtcatcatc gtccttgtag tcggcgaata ccaggcagaa gatgtagctc
     5941 agggcgatga tcgtcttcat ggtgccgcta agcttaagtt taaacgctag ccagcttggg
     6001 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     6061 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     6121 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     6181 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     6241 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     6301 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     6361 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     6421 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     6481 gcgttactat gggaacatac gtcatt
//
