LOCUS       sequence-430824-        6623 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 6623)
  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 6623)
  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: GPR25-DuET
FEATURES             Location/Qualifiers
     source          1..6623
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..25
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9..25
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    74..195
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(85..104)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     139..158
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(369..1163)
                     /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(480..499)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1090..1109
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1230..1559)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1244..1379
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1298..1317)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1544..1564
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1573..2001)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1683..1704)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1895..1914
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2047..2271
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2260..2277
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3609..3628)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3610..3628)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3673..3876)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3702..3722)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3877..4256
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4428..4447
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4522..4626
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4627..4695,4696..5487)
                     /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     complement(4845..4864)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5658..6246
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6147..6166
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6400..6417
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6534..6555
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6570..6575,6576..6593,6594..6600)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(6608..6623,1)
                     /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)."
     primer_bind     join(6613..6623,1..12)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag aggtcgacgg
       61 tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
      121 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
      181 tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
      241 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
      301 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag
      361 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
      421 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
      481 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
      541 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
      601 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
      661 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
      721 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
      781 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
      841 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
      901 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
      961 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     1021 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     1081 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     1141 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatccg
     1201 aaaatggata tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc aaaaaagcct
     1261 cctcactact tctggaatag ctcagaggca gaggcggcct cggcctctgc ataaataaaa
     1321 aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg
     1381 ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt
     1441 ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg
     1501 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca
     1561 ttccacagaa ttaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     1621 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     1681 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     1741 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     1801 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     1861 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     1921 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     1981 atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt ctttccgcct
     2041 cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc
     2101 ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg
     2161 atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg
     2221 aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg
     2281 atcagcgggt ttaaacgggc cctctagact cgagttaggc aggggccacc tgagaagtct
     2341 cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga
     2401 tccatgatgc gcttgcggtg ttggccgctt gcgctctgca ccggaaaact gagctatcgt
     2461 ctcgtgacaa tgaggaagcg gaagagattc tccttgccag tctcccggtc cgaccacatg
     2521 caccatccag agctctagct ctgaaggacc tatccagcaa caaatagatc aaggggttag
     2581 cacatgagtt gacgaatgcg aggcaagtgg caatagtcag accccaccgg agtgcgagca
     2641 acaggggaca tggcagaggc aatgctccga gccgtgccag gtggaagaca gcccgaagag
     2701 cactaaatgg cagccatgat ccaacaaagg tggattcaat cgcaaagatg atgcgcaatg
     2761 aattgcggcg agcccgaccc acgtggggag gcctcctcag ccttcgtgaa atcctgcaat
     2821 agcagaacag agtcacaaca agtgggagaa caaaagtcaa caagaggagc aacagggaca
     2881 ggccctggaa cgcgtgtgat ggttcttctc cgcattgtga atcctgaccc ccaggcagag
     2941 gctggaggcc gcggtacaca agagacggca gacccgcgag aagagccact gcccacaccc
     3001 cacagcaact agccagcgca cacctgggtg tcctgagggg tctagcttcc agcagcttga
     3061 caactgccaa atagcggtcc acagacatgc cggccaacag aagagctcct gcacagcgtg
     3121 taccagccag agcgaaagaa gacagtttac acaatccgtc gccgaaaggc cacctgcctc
     3181 cgagagctgc tgcagcggcc caaagcggca gtgtaagaac gaatccgagg tcagcggcag
     3241 ccaaatggag cacgaaggtg tcaaccagcc tgcgagggcc tctcctcccg gcgaggagcc
     3301 acaccacaaa agcgttacca aggagaccca cggcgaaggc ggcgaggtac agggctggga
     3361 tataaacgta gccatacggc aagtcgcctg ctggacacag ttccagctcc tccaaaccgt
     3421 ccaatccaga gtaatcccat ggagcagacc ctggagaagg actccatggc tcggtagggg
     3481 ccatatcgat gctggcgtca tcatcgtcct tgtagtcggc gaataccagg cagaagatgt
     3541 agctcagggc gatgatcgtc ttcatggtgg cgctaagctt aagtttaaac gctagccagc
     3601 ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct
     3661 agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg
     3721 cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa
     3781 caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta
     3841 tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac
     3901 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     3961 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     4021 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     4081 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     4141 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     4201 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg
     4261 cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt
     4321 catgcaaatg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc
     4381 agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg catcagagca
     4441 gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt
     4501 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4561 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4621 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4681 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4741 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     4801 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     4861 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     4921 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     4981 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5041 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5101 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5161 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5221 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5281 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     5341 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     5401 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     5461 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5521 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5581 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5641 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5701 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5761 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ccgttcttct agtgtagccg
     5821 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5881 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5941 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6001 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6061 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6121 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6181 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6241 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6301 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6361 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6421 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     6481 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     6541 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     6601 tgtggaattg tgagcggata aca
//
