LOCUS       sequence-430814-        6581 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 6581)
  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 6581)
  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: GPR148-DuET
FEATURES             Location/Qualifiers
     source          1..6581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(86..877,878..946))
                     /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     709..728
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(947..1051)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1126..1145)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1317..1696
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1697..1900
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1851..1871
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1945..1964
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1945..1963
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3254..3271)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3260..3484
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3530..3958
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3617..3636)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3827..3848
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3967..3987)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3972..4301
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4152..4287
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4214..4233
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4368..5162
                     /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(4422..4441)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5032..5051
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5336..5457
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5373..5392)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5427..5446
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5506..5522)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5506..5522)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5519..5541)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5530..5546
                     /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(5554..5560,5561..5578,5579..5584))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5599..5620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5737..5754)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5908..6496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5988..6007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
       61 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
      121 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
      181 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
      241 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
      301 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
      361 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
      421 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
      481 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
      541 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
      601 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
      661 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
      721 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
      781 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
      841 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
      901 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
      961 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     1021 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     1081 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     1141 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     1201 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacatttgca tgaagaatct
     1261 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     1321 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     1381 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     1441 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     1501 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     1561 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
     1621 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
     1681 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
     1741 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
     1801 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
     1861 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
     1921 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta
     1981 gcgtttaaac ttaagcttag cgccaccatg aagacgatca tcgccctgag ctacatcttc
     2041 tgcctggtat tcgccgacta caaggacgat gatgacgcca gcatcgatat gggggacgag
     2101 ttggcgcctt gccccgtagg caccaccgcc tggcctgccc tgattcagct gataagtaag
     2161 accccctgca tgccgcaggc cgcttccaat acctccctcg gacttggtga cctccgagtt
     2221 cccagctcta tgctgtattg gctgttcctg ccctccagcc tgctggccgc ggctacgctg
     2281 gctgtttccc cgctgctgct ggttactatc ctgagaaacc agcgcctccg ccaagaaccc
     2341 cattacctgc tgcctgccaa catcctgctg agtgacctgg cctatatcct gcttcacatg
     2401 ctgatctctt cctccagctt ggggggctgg gaactcggac gcatggcatg tggcattttg
     2461 acggatgctg tgtttgctgc ctgcacttca accattctca gcttcacagc catcgtgctc
     2521 cacacctatc tggcggtgat ccatccactc aggtatttga gcttcatgag ccacggggcc
     2581 gcgtggaagg ccgtggccct tatctggctt gttgcatgct gcttccccac ctttctcatt
     2641 tggctgtcta aatggcagga tgctcagttg gaggaacagg gagccagcta tattcttccc
     2701 ccaagcatgg gtacgcagcc cggttgtggg cttcttgtga ttgtgacata tacatcaata
     2761 ctttgtgtcc tgtttctgtg taccgctctg atagcaaatt gtttttggag gatatacgcg
     2821 gaggcaaaga cttctggcat ctggggacag ggctactccc gagcccgggg aactctgctg
     2881 attcactctg tcctgattac actgtacgtg agcacggggg tggttttcag ccttgatatg
     2941 gtgcttacac gctaccatca cattgatagt ggcacccaca catggctcct ggccgctaac
     3001 agcgaagtgc tgatgatgct gccaagggcc atgctcactt acctttatct gctccgctac
     3061 cggcagctgc tgggaatggt caggggacac ttgcctagca gacgacacca ggcaatcttc
     3121 accatctcta tcgataccgg tggacgcacc ggtgaattcg atgaggccag caccaccgtg
     3181 tctaagaccg agacttctca ggtggcccct gcctaactcg agtctagagg gcccgtttaa
     3241 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     3301 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3361 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3421 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3481 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     3541 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3601 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3661 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3721 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     3781 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3841 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     3901 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     3961 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4021 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     4081 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4141 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4201 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     4261 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     4321 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     4381 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4441 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4501 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     4561 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4621 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4681 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4741 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4801 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4861 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     4921 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4981 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5041 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     5101 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     5161 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     5221 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     5281 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     5341 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     5401 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     5461 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     5521 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5581 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5641 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5701 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5761 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5821 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5881 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5941 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6001 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6061 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6121 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6181 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6241 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6301 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cggtatttgg
     6361 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6421 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6481 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6541 cgaaaactca cgttaaggga ttttggtcat gagattatca a
//
