LOCUS       sequence-430901-        6638 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 6638)
  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 6638)
  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: GHSR-DuET
FEATURES             Location/Qualifiers
     source          1..6638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     436..455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(674..778)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(853..872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1044..1423
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1424..1627
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1578..1598
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1672..1691
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1672..1690
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3038..3055)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3044..3268
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3314..3742
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3401..3420)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3611..3632
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3751..3771)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3756..4085
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3936..4071
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3998..4017
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4152..4946
                     /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(4206..4225)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4816..4835
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5120..5241
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5157..5176)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5211..5230
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5290..5306)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5290..5306)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5303..5325)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5314..5330
                     /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(5338..5344,5345..5362,5363..5368))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5383..5404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5521..5538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5692..6280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5772..5791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6451..6638,1..604,605..673))
                     /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 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
       61 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
      121 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
      181 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
      241 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
      301 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
      361 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
      421 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
      481 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
      541 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
      601 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
      661 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
      721 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
      781 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
      841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
      901 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
      961 cttgaccgac atttgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     1021 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     1081 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     1141 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1201 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1261 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     1321 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     1381 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     1441 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     1501 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     1561 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     1621 cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac
     1681 tcactatagg gagacccaag ctggctagcg tttaaactta agcttagcgg ccgcgccacc
     1741 atgaagacga tcatcgccct gagctacatc ttctgcctgg tattcgccga ctacaaggac
     1801 gatgatgacg ccagcatcga tatgtggaat gccaccccca gcgaagaacc cggatttaat
     1861 ctgaccttgg ccgatttgga ttgggacgcg agtcccggca atgattcact gggcgatgaa
     1921 cttctgcagc tttttcccgc ccctctgctt gccggagtga ctgcaacctg cgtggccttg
     1981 tttgttgtcg gaattgctgg aaacctgctt acgatgctgg tagtctctcg attccgggag
     2041 ctcaggacaa caacaaacct gtatctctcc agtatggcct tttctgacct gctgattttt
     2101 ctgtgtatgc ccctcgacct ggtgcggttg tggcaatatc gcccctggaa cttcggagac
     2161 ctgctttgca agctctttca gttcgtgagc gagtcctgta cctatgcaac cgtgctgact
     2221 ataaccgccc tgagtgtgga gcgatacttc gcaatctgtt tccccctgag agctaaggtg
     2281 gtcgtgacaa aagggcgcgt caagctcgtg atcttcgtaa tctgggccgt tgctttctgc
     2341 tcagctggac cgattttcgt cctggtggga gtggaacacg aaaatggaac cgatccatgg
     2401 gataccaacg agtgcaggcc aacagagttt gccgtcaggt cagggctgct gaccgtcatg
     2461 gtttgggtct cctccatatt tttctttctc cccgtgtttt gccttactgt gctgtacagc
     2521 ttgataggta ggaagctgtg gagaagacgc cgaggggacg ccgtggttgg ggcaagcctc
     2581 cgcgatcaga atcacaaaca aacagtcaaa atgttggcag tcgtcgtgtt cgccttcatc
     2641 ttgtgctggc ttccgtttca cgtaggacgc tacttgtttt ctaaaagttt cgagcctgga
     2701 agcctggaga tcgctcagat ttcccagtat tgcaacctcg tgtcctttgt cctgttctac
     2761 ctttccgcag cgataaatcc tatcctgtat aacatcatga gcaagaaata cagagtcgcg
     2821 gtcttccgac tgcttggttt cgagccgttc tctcaacgga agctttccac acttaaggat
     2881 gaatcctccc gagcttggac ggagagttca ataaacacta tcgataccgg tggacgcacc
     2941 ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct
     3001 gcctaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     3061 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     3121 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     3181 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     3241 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     3301 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3361 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3421 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3481 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3541 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3601 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3661 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3721 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3781 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3841 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3901 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3961 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     4021 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     4081 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     4141 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4201 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4261 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4321 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4381 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4441 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4501 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4561 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4621 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4681 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4741 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4801 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4861 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4921 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4981 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5041 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5101 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     5161 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     5221 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5281 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5341 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5401 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5461 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5521 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5581 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5641 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5701 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5761 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5821 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5881 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5941 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6001 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6061 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6121 ctacactaga agaacggtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6181 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6241 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6301 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6361 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6421 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6481 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6541 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6601 ctcaccggct ccagatttat cagcaataaa ccagccag
//
