LOCUS       sequence-430888-        6566 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 6566)
  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 6566)
  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: PTAFR-DuET
FEATURES             Location/Qualifiers
     source          1..6566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      270..698
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(357..376)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     567..588
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(707..727)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        712..1041
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      892..1027
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     954..973
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1108..1902
                     /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(1162..1181)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1772..1791
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2076..2197
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2113..2132)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2167..2186
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2246..2262)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2246..2262)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2259..2281)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2270..2286
                     /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(2294..2300,2301..2318,2319..2324))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2339..2360
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2477..2494)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2648..3236)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2728..2747)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3407..4198,4199..4267))
                     /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     4030..4049
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4268..4372)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4447..4466)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4638..5017
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5018..5221
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5172..5192
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5266..5285
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5266..5284
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(join(6560..6566,1..11))
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6566,1..224)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
       61 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
      121 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
      181 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
      241 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
      301 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
      361 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
      421 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
      481 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
      541 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
      601 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
      661 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
      721 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      781 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
      841 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
      901 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
      961 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     1021 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     1081 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     1141 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     1201 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     1261 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     1321 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     1381 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     1441 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     1501 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     1561 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     1621 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     1681 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     1741 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     1801 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     1861 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     1921 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     1981 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     2041 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     2101 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2161 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     2221 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     2281 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     2341 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     2401 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     2461 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     2521 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2581 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2641 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2701 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2761 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2821 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2881 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2941 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3001 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3061 ggtggcctaa ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc
     3121 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3181 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3241 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3301 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3361 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3421 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3481 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3541 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3601 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3661 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3721 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3781 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3841 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3901 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3961 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4021 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4081 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4141 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4201 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4261 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4321 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4381 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
     4441 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     4501 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     4561 cgacatttgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     4621 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     4681 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     4741 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     4801 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     4861 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     4921 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     4981 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     5041 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     5101 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     5161 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     5221 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
     5281 tagggagacc caagctggct agcgtttaaa cttaagctta gcggccgcgc caccatgaag
     5341 acgatcatcg ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat
     5401 gacgccagca tcgatatgga gccacatgac agttctcaca tggattcaga gttccggtat
     5461 acgctctttc ccatcgtcta cagcattatc ttcgtcttgg gcgtgattgc caatgggtac
     5521 gtactctggg ttttcgccag actctatccg tgcaaaaagt ttaatgaaat caaaatcttc
     5581 atggtgaatc tcaccatggc agacatgttg ttcctcatca cactgcccct gtggattgtg
     5641 tactatcaga atcagggcaa ttggattctt cctaaatttc tgtgcaatgt cgcaggctgc
     5701 ctgtttttca tcaatacata ttgtagcgtc gcatttctgg gggtgatcac atataaccgc
     5761 ttccaggcag tgacgcggcc aataaaaacc gcacaggcaa atacaaggaa gagagggata
     5821 agcctgtctc tcgttatctg ggtcgccatt gttggggcag cgtcctactt cctgatactg
     5881 gatagcacca acacagttcc cgacagcgcc ggtagcggga atgttaccag gtgttttgag
     5941 cactacgaga agggctccgt tcctgttctg ataatacata tatttatagt tttttcattc
     6001 ttcttggtgt ttttgatcat attgttttgt aatctggtga tcattcgcac acttctgatg
     6061 cagcccgtgc agcaacaacg caatgctgaa gtaaagcgcc gggcgctttg gatggtatgt
     6121 acagtgctcg cagtgtttat catatgtttc gtaccccatc atgtggtgca gctgccgtgg
     6181 accttggccg aactcggctt ccaggattct aagtttcacc aggctataaa cgatgcccac
     6241 caagtaactc tctgcctgct gagtaccaac tgcgtgttgg atcccgttat ttattgcttc
     6301 ctcacaaaga agtttcgcaa gcacttgacg gaaaaattct actctatgcg aagcagtcgg
     6361 aaatgtagta gggccactac cgatacagtc accgaggtgg tggtgccgtt caatcagata
     6421 ccagggaaca gtctgaagaa tatcgatacc ggtggacgca ccggtgaatt cgatgaggcc
     6481 agcaccaccg tgtctaagac cgagacttct caggtggccc ctgcctaatc tagagggccc
     6541 gtttaaaccc gctgatcagc ctcgac
//
