LOCUS       sequence-430889-        6833 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 6833)
  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 6833)
  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: QRFP-DuET
FEATURES             Location/Qualifiers
     source          1..6833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    75..196
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(112..131)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     166..185
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(245..261)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(245..261)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(258..280)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    269..285
                     /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(293..299,300..317,318..323))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    338..359
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(476..493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(647..1235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(727..746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1406..2197,2198..2266))
                     /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     2029..2048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2267..2371)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2446..2465)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2637..3016
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3017..3220
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3171..3191
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3265..3284
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3265..3283
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4826..4843)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4832..5056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5102..5530
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5189..5208)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5399..5420
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5539..5559)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5544..5873
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5724..5859
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5786..5805
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5940..6734
                     /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(5994..6013)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6604..6623
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
       61 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
      121 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
      181 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
      241 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
      301 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
      361 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      421 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
      481 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
      541 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
      601 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
      661 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
      721 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
      781 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
      841 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
      901 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
      961 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     1021 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     1081 ctagaagaac ggtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     1141 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     1201 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     1261 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     1321 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     1381 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     1441 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     1501 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     1561 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     1621 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     1681 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     1741 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     1801 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     1861 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     1921 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     1981 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     2041 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     2101 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     2161 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     2221 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     2281 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2341 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     2401 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     2461 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     2521 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacatttgca tgaagaatct
     2581 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     2641 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     2701 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     2761 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     2821 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     2881 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     2941 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     3001 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     3061 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     3121 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     3181 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
     3241 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
     3301 gcgtttaaac ttaagcttag cggccgcgcc accatgaaga cgatcatcgc cctgagctac
     3361 atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat cgatatgcag
     3421 gcgttgaaca ttaccccaga acagttctct aggctcctta gagatcacaa cctgacacga
     3481 gagcagttca tcgctctcta tcgcctgaga ccactcgtgt acacaccaga actccccgga
     3541 cgagcaaagc tggctctggt gctgacaggc gtgcttatct tcgctcttgc cctgttcggc
     3601 aacgctttgg tcttctacgt ggtgactcgg tccaaggcta tgcgcactgt gaccaacatc
     3661 ttcatatgct ccctggccct gtccgacctg ctgatcacct tcttctgtat tcctgtcacg
     3721 atgctgcaaa atatctcaga caactggctc ggtggcgcat tcatttgcaa gatggtgcct
     3781 tttgtacagt caactgctgt ggtgacggaa atccttacaa tgacctgtat cgctgtggaa
     3841 cgacatcagg ggttggtgca tccattcaag atgaagtggc agtataccaa cagacgggcg
     3901 tttacaatgc ttggagtcgt ctggctggta gccgttatcg tgggcagccc aatgtggcac
     3961 gtacagcagc tggaaatcaa atatgatttt ctgtacgaaa aagagcacat ttgctgcctt
     4021 gaagagtgga cctccccagt acaccaaaag atctacacaa cattcatcct ggttattctg
     4081 tttctgcttc cacttatggt aatgttgatt ttgtactcca aaatagggta tgagctgtgg
     4141 atcaagaaac gggtcggaga tggctccgtg ttgcgaacta tccacggaaa ggaaatgtcc
     4201 aagattgcaa ggaagaagaa gcgcgcagtc attatgatgg ttaccgttgt tgcgctgttt
     4261 gctgtgtgct gggccccctt ccatgttgtg cacatgatga ttgagtactc taatttcgag
     4321 aaggagtatg acgacgtgac cattaaaatg atatttgcta ttgtgcagat cattggcttc
     4381 agcaattcta tttgcaatcc cattgtttat gctttcatga acgaaaactt taagaagaac
     4441 gtcctgtcag ctgtgtgcta ctgtattgtg aacaagacgt tcagcccggc tcagagacac
     4501 ggaaatagtg gtattaccat gatgaggaaa aaggcgaagt tttctctgag ggaaaacccc
     4561 gtggaagaga caaaaggcga agccttctca gacggcaaca ttgaggtgaa actgtgcgag
     4621 caaaccgagg agaagaaaaa actgaagcgg caccttgccc tgtttcgatc cgagttggct
     4681 gagaactctc cactggacag cggacatatc gataccggtg gacgcaccgg tgaattcgat
     4741 gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc ctaatctaga
     4801 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     4861 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4921 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4981 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     5041 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     5101 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5161 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5221 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5281 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5341 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5401 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5461 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5521 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     5581 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     5641 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     5701 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     5761 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     5821 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     5881 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     5941 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     6001 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     6061 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     6121 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     6181 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     6241 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     6301 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     6361 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     6421 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     6481 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     6541 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     6601 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     6661 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     6721 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     6781 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcg
//
