LOCUS       sequence-430842-        6575 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 6575)
  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 6575)
  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: GPRC5D-DuET
FEATURES             Location/Qualifiers
     source          1..6575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     578..597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(816..920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(995..1014)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1186..1565
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1566..1769
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1720..1740
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1814..1833
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1814..1832
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3117..3134)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3123..3347
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3393..3821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3480..3499)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3690..3711
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3830..3850)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3835..4164
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4015..4150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4077..4096
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4231..5025
                     /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(4285..4304)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4895..4914
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5199..5320
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5236..5255)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5290..5309
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5369..5385)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5369..5385)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5382..5404)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5393..5409
                     /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(5417..5423,5424..5441,5442..5447))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5462..5483
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5600..5617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5771..6359)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5851..5870)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6530..6575,1..746,747..815))
                     /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 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
       61 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
      121 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
      181 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
      241 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
      301 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
      361 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
      421 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
      481 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
      541 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
      601 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
      661 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
      721 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
      781 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
      841 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
      901 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
      961 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     1021 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     1081 tttaagctac aacaaggcaa ggcttgaccg acatttgcat gaagaatctg cttagggtta
     1141 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     1201 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     1261 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     1321 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     1381 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     1441 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     1501 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     1561 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     1621 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     1681 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     1741 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
     1801 ggcttatcga aattaatacg actcactata gggagaccca agctggctag cgtttaaact
     1861 taagcttagc gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt
     1921 cgccgactac aaggacgatg atgacgccag catcgatatg tataaagatt gtattgagtc
     1981 tactggtgac tatttcctgc tgtgtgacgc cgagggccca tggggaatca ttctggaatc
     2041 tctggccatc ctgggaatcg tggtgaccat tttgctcctt cttgcttttc tgtttctgat
     2101 gcgcaaaatc caggattgca gccagtggaa tgtcctgccc actcagcttt tgttcttgct
     2161 tagcgttctt ggcctgtttg ggctggcttt cgcctttatt atcgaactga accagcaaac
     2221 agcccccgtg cgctactttc tcttcggcgt gctgtttgcc ctctgcttct catgtcttct
     2281 ggcccatgcc agtaacctgg ttaagctggt ccggggatgt gtatcatttt catggactac
     2341 catcctgtgt atcgctatag gctgtagtct gcttcagatt atcatcgcga cagaatacgt
     2401 gaccctgatc atgaccagag gcatgatgtt cgtgaacatg actccatgcc aactgaacgt
     2461 agactttgtt gtactccttg tgtacgttct gtttctgatg gccctgactt tcttcgtcag
     2521 taaagctacg ttttgcggtc cgtgcgagaa ttggaagcag cacggtcgcc tgattttcat
     2581 tacggttctc ttctctatca ttatttgggt cgtgtggatt agtatgctgc tgcgcggaaa
     2641 tccacagttt cagcgccagc cccagtggga cgatcctgtc gtttgtatcg ctctcgtgac
     2701 taacgcctgg gttttcctgc tcctgtacat tgttccagag ctgtgcattc tctacagatc
     2761 ttgccgacag gaatgtcccc tccaggggaa cgcatgtccc gtgacagcct atcaacatag
     2821 tttccaagtg gaaaaccagg agctgtcacg ggccagggac tctgacggag ccgaagaaga
     2881 tgtggcgctg acgagttatg gcacacccat ccaaccacag accgtggatc cgacccaaga
     2941 atgcttcatc ccacaggcca agctgagccc gcagcaggat gctggcggtg taatcgatac
     3001 cggtggacgc accggtgaat tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc
     3061 tcaggtggcc cctgcctaac tcgagtctag agggcccgtt taaacccgct gatcagcctc
     3121 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3181 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3241 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3301 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3361 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3421 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3481 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3541 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3601 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3661 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3721 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3781 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     3841 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3901 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3961 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4021 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4081 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4141 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4201 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4261 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4321 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4381 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4441 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4501 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4561 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4621 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4681 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4741 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4801 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     4861 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     4921 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     4981 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5041 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5101 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5161 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5221 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5281 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5341 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5401 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5461 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5521 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5581 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5641 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5701 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5761 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5821 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5881 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5941 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6001 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6061 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6121 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6181 agtggtggcc taactacggc tacactagaa gaacggtatt tggtatctgc gctctgctga
     6241 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6301 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6361 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6421 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6481 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6541 taatcagtga ggcacctatc tcagcgatct gtcta
//
