LOCUS       sequence-431806-        7252 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 7252)
  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 7252)
  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: TH1534-DuET
FEATURES             Location/Qualifiers
     source          1..7252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     124..143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(362..466)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(541..560)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        732..1111
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1112..1315
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1266..1286
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1360..1379
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1360..1378
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1417..1426
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1492..1518
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3340..3357)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3346..3570
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3616..4044
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3703..3722)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3913..3934
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4053..4073)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4058..4387
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4238..4373
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4300..4319
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4454..5248
                     /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(4508..4527)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5118..5137
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5422..5543
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5459..5478)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5513..5532
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5592..5608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5592..5608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5605..5627)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5616..5632
                     /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(5640..5646,5647..5664,5665..5670))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5685..5706
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5823..5840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5994..6582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6074..6093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6753..7252,1..292,293..361))
                     /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 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
       61 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
      121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
      181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
      241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
      301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
      361 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
      421 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
      481 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
      541 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
      601 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacat
      661 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      721 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      781 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      841 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      901 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      961 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     1021 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     1081 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     1141 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     1201 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     1261 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     1321 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     1381 gacccaagct ggctagcgtt taaacttaag cttgccgcca ccatggcgct gtgtatccct
     1441 caagttctgc tggccctgtt cctgagcatg ctgacaggac ctggcgaggg ctacccttac
     1501 gatgtgcctg actacgccga attcgctcct gcagggagtc aattgatctc cagcatggac
     1561 atggaaagac ccggcgacgg caagtgccag cctatcgaga ttcccatgtg caaggacatc
     1621 ggctacaaca tgaccagaat gcccaacctg atgggccacg agaaccagag agaggccgcc
     1681 attcagctcc acgagtttgc ccctctggtg gaatatggct gtcacggcca cctgagattc
     1741 tttctgtgct ccctgtacgc ccctatgtgc accgagcagg tttccacacc tattccagcc
     1801 tgcagagtga tgtgcgagca ggccagactg aagtgtagcc ccatcatgga acagttcaac
     1861 ttcaagtggc ccgacagcct ggactgccgg aaactgccca acaagaacga ccccaactac
     1921 ctgtgcatgg aagcccctaa caacggctcc gacgagccta caagaggctc cggactgttc
     1981 cctccactgt tcagacctca gaggcctcac tctgcccaag agcaccctct gaaagatggc
     2041 ggacctggca gaggcggctg tgacaatcct ggcaagttcc accacgtgga aaagtccgcc
     2101 tcttgcgccc ctttgtgtac acctggtgtc gatgtgtact ggtccagaga ggacaagaga
     2161 ttcgccgtcg tgtggctggc catttgggcc gtgctgtgct ttttcagctc cgcctttacc
     2221 gtgctgacct tcctgatcga ccccgccaga ttcagatacc ccgagcggcc tatcatcttc
     2281 ctgagcatgt gctactgcgt gtacagcgtg ggctacctga tcagactgtt tgccggcgct
     2341 gagagcattg cctgcgatag agatagcggc cagctgtacg tgatccaaga aggcctggaa
     2401 agcaccggct gtactctggt gttcctggtg ctgtactact tcggcatggc ctccagcctt
     2461 tggtgggttg tgctgaccct gacctggttt ctggccgctg gaaagaagtg gggacacgag
     2521 gccatcgagg ccaacagctc ctattttcat ctggccgcct gggctatccc cgccgtgaaa
     2581 acaatcctga tcctggtcat gcggagagtg gccggcgacg aacttacagg cgtgtgttac
     2641 gtgggctcta tggacgtgaa cgccctgacc ggctttgtgc tgattcccct ggcctgttac
     2701 ctggtcatcg gcaccagctt tatcctgagc ggattcgtgg ccctgttcca cattcggaga
     2761 gtgatgaaga caggcggcga gaacaccgac aagctggaaa agctgatggt ccgaatcggc
     2821 ctgttcagcg tgctgtacac agtgcctgcc acctgtgtga tcgcctgcta cttctacgag
     2881 cggctgaaca tggactactg gaagatcctg gccgctcagc acaagtgcaa gatgaacaac
     2941 cagaccaaga cactcgactg cctgatggcc gcctctattc ctgccgtgga aatcttcatg
     3001 gtcaagatct tcatgctcct ggtcgtgggc atcaccagcg gcatgtggat ctggacaagc
     3061 aagaccctgc agagctggca acaagtgtgc tccagacggc tgaagaagaa gtccagacgg
     3121 aagcccgcca gcgtgatcac aagcggcggc atctataaga aggcccagca tcctcagaaa
     3181 acccaccacg ggaagtacga gatccccgct cagagcccta catgcgttgg taccgcctcc
     3241 tcggatgagg ccagcacaac cgtgtctaag accgagacat ctcaggtggc ccctgcctaa
     3301 gcggccgctc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3361 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3421 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3481 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     3541 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     3601 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3661 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3721 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3781 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     3841 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3901 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3961 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4021 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     4081 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4141 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4201 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     4261 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     4321 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     4381 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     4441 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4501 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4561 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4621 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4681 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4741 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     4801 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     4861 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     4921 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     4981 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     5041 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     5101 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5161 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5221 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5281 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5341 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5401 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     5461 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5521 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     5581 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5641 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5701 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5761 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5821 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5881 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5941 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6001 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6061 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6121 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6181 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6241 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6301 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6361 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6421 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6481 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6541 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6601 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6661 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6721 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6781 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6841 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6901 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6961 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7021 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7081 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7141 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7201 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gc
//
