LOCUS       sequence-431802-        7072 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 7072)
  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 7072)
  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: TH1531-DuET
FEATURES             Location/Qualifiers
     source          1..7072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     214..233
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(452..556)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(631..650)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        822..1201
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1202..1405
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1356..1376
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1450..1469
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1450..1468
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1507..1516
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1582..1608
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(1794..1805)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(3250..3267)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3256..3480
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3526..3954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3613..3632)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3823..3844
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3963..3983)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3968..4297
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4148..4283
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4210..4229
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4364..5158
                     /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(4418..4437)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5028..5047
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5332..5453
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5369..5388)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5423..5442
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5502..5518)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5502..5518)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5515..5537)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5526..5542
                     /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(5550..5556,5557..5574,5575..5580))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5595..5616
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5733..5750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5904..6492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5984..6003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6663..7072,1..382,383..451))
                     /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 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
       61 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      121 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      181 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      241 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      301 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
      361 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      421 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      481 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
      541 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
      601 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      661 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
      721 agctacaaca aggcaaggct tgaccgacat ttgcatgaag aatctgctta gggttaggcg
      781 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      841 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      901 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      961 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     1021 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1081 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1141 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     1201 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     1261 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     1321 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     1381 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
     1441 tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag
     1501 cttgccgcca ccatggcgct gtgtatccct caagttctgc tggccctgtt cctgagcatg
     1561 ctgacaggac ctggcgaggg ctacccttac gatgtgcctg actacgccga attcgctcct
     1621 gcagggagtc aattgttcgg cgacgaagag gaacgcagat gcgaccccat ccggatcagc
     1681 atgtgtcaga acctgggcta caacgtgacc aagatgccca acctcgtggg ccacgaactg
     1741 cagacagatg ccgaactcca gctgaccacc ttcacacccc tgatccagta cggctgtagc
     1801 tcccagctcc agttcttcct gtgctccgtg tacgtgccca tgtgcaccga gaagatcaac
     1861 atccctatcg gcccttgcgg cggcatgtgt ctgagcgtga agagaagatg cgagcccgtg
     1921 ctgaaagagt tcggctttgc ctggcctgag agcctgaact gtagcaagtt cccacctcag
     1981 aacgaccaca accacatgtg catggaaggc cctggcgacg aggaagtgcc tctgcctcac
     2041 aagacaccta tccagcctgg cgaggaatgt cacagcgtgg gcaccaacag cgaccagtac
     2101 atctgggtca agcggtccct gaactgcgtg ctgaagtgtg gctatgacgc cggcctgtat
     2161 agcagaagcg ccaaagagtt caccgacatc tggatggccg tgtgggccag cctgtgcttt
     2221 atcagcaccg cctttaccgt gctgaccttc ctgatcgaca gctccagatt cagctacccc
     2281 gagcggccta tcatcttcct gagcatgtgc tacaacatct actctatcgc ctacatcgtg
     2341 cggctgaccg tgggcagaga gagaatctcc tgcgacttcg aagaggccgc cgaacctgtg
     2401 ctgatccaag agggcctgaa gaacaccggc tgtgccatca tctttctgct gatgtacttc
     2461 ttcggcatgg ccagctccat ttggtgggtc atactgaccc tgacctggtt cctggccgct
     2521 ggactgaaat ggggacacga ggccatcgag atgcacagct cctacttcca cattgccgcc
     2581 tgggccattc ctgccgtgaa aaccatcgtg atcctgatca tgcggctggt ggacgccgat
     2641 gagctgaccg gactgtgtta cgtgggcaac cagaatctgg acgccctgac cggatttgtg
     2701 gtggcccctc tgtttaccta cctggtcatc ggcaccctgt ttatcgccgc tggtcttgtg
     2761 gccctgttca agatcagaag caacctgcag aaggacggca ccaagaccga caagctggaa
     2821 cggctgatgg tcaagatcgg cgtgttcagc gtgctgtaca ccgtgcctgc cacatgcgtg
     2881 atcgcctgct acttctacga gatcagcaac tgggccctgt ttcggtacag cgccgacgac
     2941 agcaatatgg ccgtggaaat gctgaagatc tttatgagcc tgctcgtggg catcacctcc
     3001 ggcatgtgga tttggagcgc caagacactg cacacctggc agaagtgctc caaccggctg
     3061 gtcaacagcg gcaaagtgaa gcgcgagaag cgcggcaatg gctgggttaa gcctggcaag
     3121 ggctctgaga cagtcgtggg taccgcctcc tcggatgagg ccagcacaac cgtgtctaag
     3181 accgagacat ctcaggtggc ccctgcctaa gcggccgctc tagagggccc gtttaaaccc
     3241 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3301 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3361 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3421 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3481 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     3541 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3601 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3661 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3721 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3781 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3841 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3901 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     3961 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4021 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4081 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4141 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4201 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     4261 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     4321 cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4381 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     4441 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     4501 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     4561 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     4621 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     4681 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4741 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4801 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4861 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     4921 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4981 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5041 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5101 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5161 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     5221 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5281 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt
     5341 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     5401 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5461 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5521 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5581 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5641 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5701 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5761 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5821 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5881 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5941 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6001 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6061 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6121 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6181 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6241 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6301 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6361 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6421 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6481 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6541 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6601 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6661 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6721 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6781 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6841 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6901 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6961 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7021 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gt
//
