LOCUS       sequence-430872-        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: MRGPRX1-DuET
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        109..312
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     263..283
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     357..376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        357..375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1591..1608)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1597..1821
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1867..2295
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1954..1973)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2164..2185
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2304..2324)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2309..2638
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2489..2624
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2551..2570
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2705..3499
                     /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(2759..2778)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3369..3388
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3673..3794
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3710..3729)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3764..3783
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3843..3859)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3843..3859)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3856..3878)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3867..3883
                     /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(3891..3897,3898..3915,3916..3921))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3936..3957
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4074..4091)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4245..4833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4325..4344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5004..5795,5796..5864))
                     /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     5627..5646
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5865..5969)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6044..6063)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6235..6506,1..108)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
       61 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      121 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      181 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      241 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
      301 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
      361 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt agcgccacca
      421 tgaagacgat catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg
      481 atgatgacgc cagcatcgat atggacccga caatcagcac actcgacact gaactgacgc
      541 ccatcaatgg cacagaggaa acactttgtt acaagcaaac actgtccttg actgtcctca
      601 cctgtattgt aagtctggtg gggctgaccg gaaatgcggt agttctctgg ctgctgggtt
      661 gccggatgcg acggaatgct ttctccattt acatactgaa cctcgccgct gctgatttcc
      721 ttttcctctc agggagactt atctacagcc ttttgtcttt tatcagcatt ccacatacaa
      781 tttccaagat cctgtacccg gtcatgatgt tttcttactt cgccggcctg tcctttttga
      841 gtgcagtctc cacggaaaga tgtctgtctg tcctctggcc gatttggtat aggtgtcata
      901 ggccgactca cctgagtgcc gtcgtgtgtg tgctgctgtg ggccctctcc cttctgcggt
      961 ctatactgga gtggatgctg tgtggatttc tctttagtgg tgccgactct gcatggtgtc
     1021 agaccagtga ttttatcact gtcgcctggc ttattttcct ctgtgtggtg ctgtgtgggt
     1081 catctttggt gctgctcatt aggatactgt gtggcagccg caaaatccca ctgacacgcc
     1141 tgtacgtgac catccttctg acggtattgg tgttcctcct gtgtggtttg cctttcggga
     1201 tccaattctt cctcttcctc tggatacacg tggatcggga agtgcttttc tgccacgttc
     1261 acctggtgag tatctttctg tccgcactga atagcagcgc aaatcccatt atctattttt
     1321 tcgtagggtc atttagacag aggcagaata gacagaacct taaacttgtt ctccaacgcg
     1381 ctttgcaaga cgcctctgaa gtggatgagg ggggtggcca actgcccgag gagatcttgg
     1441 aactgtccgg cagccggttg gaacagatcg ataccggtgg acgcaccggt gaattcgatg
     1501 aggccagcac caccgtgtct aagaccgaga cttctcaggt ggcccctgcc taactcgagt
     1561 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1621 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1681 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1741 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1801 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     1861 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     1921 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     1981 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2041 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2101 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     2161 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     2221 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     2281 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2341 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     2401 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2461 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     2521 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     2581 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     2641 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     2701 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     2761 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     2821 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     2881 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     2941 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     3001 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     3061 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     3121 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     3181 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     3241 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     3301 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     3361 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     3421 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     3481 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     3541 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     3601 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     3661 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     3721 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     3781 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     3841 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3901 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3961 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4021 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4081 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4141 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4201 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4261 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4321 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4381 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4441 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4501 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4561 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4621 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4681 agaagaacgg tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4741 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4801 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4861 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4921 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4981 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5041 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5101 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5161 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5221 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5281 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5341 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5401 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5461 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5521 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5581 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5641 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5701 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5761 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5821 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5881 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5941 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6001 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
     6061 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
     6121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga catttgcatg aagaatctgc
     6181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
     6241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
     6301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
     6361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
     6421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
     6481 atcatatgcc aagtacgccc cctatt
//
