LOCUS       sequence-431824-        6530 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 6530)
  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 6530)
  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: MRGPRX2-DuET
FEATURES             Location/Qualifiers
     source          1..6530
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(903..920)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    909..1133
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1179..1607
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1266..1285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1476..1497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1616..1636)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1621..1950
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1801..1936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1863..1882
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2017..2811
                     /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(2071..2090)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2681..2700
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2985..3106
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3022..3041)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3076..3095
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3155..3171)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3155..3171)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3168..3190)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3179..3195
                     /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(3203..3209,3210..3227,3228..3233))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3248..3269
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3386..3403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3557..4145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3637..3656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4316..5107,5108..5176))
                     /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     4939..4958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5177..5281)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5356..5375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5547..5926
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5927..6130
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6081..6101
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6175..6194
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6175..6193
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggcaggcgcc gatttcctgt tcctttgctt ccagataatt aattgcctcg tttacttgtc
       61 caatttcttc tgctccattt ccataaattt ccctagtttt ttcactactg ttatgacctg
      121 tgcatatctg gccggcctgt ctatgttgag tactgtgagc acagagcgat gcctgagtgt
      181 gctttggcct atatggtatc ggtgtcgcag acctagacac ttgtccgcag tggtgtgtgt
      241 tcttctgtgg gccctttcac tgctcctgag catcctggag ggaaaattct gtgggttcct
      301 gttcagcgac ggggactccg gctggtgtca gacttttgac tttatcaccg cggcatggct
      361 gatcttcttg ttcatggtgc tgtgcggaag ctcactggcg ctgctggttc gcatcctctg
      421 cggatcccga ggccttccac tcactcggct gtatctgacg atcctgctga cagtactcgt
      481 gttcctcctg tgcggcctcc ctttcggcat tcaatggttc cttatactgt ggatttggaa
      541 agattccgac gtgctgtttt gccacattca tcccgttagc gtggtgctgt cttcactgaa
      601 tagttctgcc aaccccatta tctacttttt tgtaggttct tttcggaaac agtggcggct
      661 tcagcagcct atccttaaac tggccctgca aagggccttg caagacatcg ccgaagtcga
      721 ccatagcgag ggttgcttta ggcagggtac cccagagatg tctaggtcca gcttggtaat
      781 cgataccggt ggacgcaccg gtgaattcga tgaggccagc accaccgtgt ctaagaccga
      841 gacttctcag gtggcccctg cctaactcga gtctagaggg cccgtttaaa cccgctgatc
      901 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
      961 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     1021 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     1081 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     1141 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     1201 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1261 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1321 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1381 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1441 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1501 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1561 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     1621 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1681 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     1741 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     1801 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     1861 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     1921 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     1981 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2041 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2101 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2161 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     2221 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2281 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2341 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2401 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2461 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2521 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     2581 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2641 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2701 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2761 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     2821 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     2881 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     2941 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     3001 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     3061 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     3121 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     3181 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3241 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3301 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3361 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3421 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     3481 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     3541 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3601 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3661 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3721 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3781 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3841 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3901 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3961 tcttgaagtg gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc
     4021 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     4081 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     4141 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     4201 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     4261 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     4321 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4381 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4441 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4501 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4561 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4621 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4681 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4741 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4801 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4861 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4921 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4981 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5041 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5101 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5161 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     5221 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5281 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     5341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     5401 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     5461 aggcttgacc gacatttgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     5521 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     5581 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     5641 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     5701 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     5761 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     5821 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     5881 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     5941 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     6001 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     6061 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     6121 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
     6181 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttag cgccaccatg
     6241 aagacgatca tcgccctgag ctacatcttc tgcctggtat tcgccgacta caaggacgat
     6301 gatgacgcca gcatcgatat ggatcccaca accccggcat ggggaaccga atctacaaca
     6361 gtgaacggaa acgatcaggc actgctcctg ctgtgtggca aggaaaccct cataccagta
     6421 tttcttattc tctttattgc actggtgggg ctggtgggga atggttttgt gttgtggctt
     6481 ctgggatttc ggatgcggcg caacgctttc agcgtctacg tactgtccct
//
