LOCUS       sequence-430812-        7220 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 7220)
  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 7220)
  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: GPR123-DuET
FEATURES             Location/Qualifiers
     source          1..7220
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     35..54
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     288..305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    422..443
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(458..463,464..481,482..488)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    496..512
                     /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     501..523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     520..536
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     520..536
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    585..706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(596..615)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     650..669
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(880..1674)
                     /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(991..1010)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1601..1620
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1741..2070)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1755..1890
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1809..1828)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2055..2075
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2084..2512)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2194..2215)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2406..2425
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2558..2782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2771..2788
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4717..4736)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4718..4736)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4781..4984)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4810..4830)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4985..5364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5536..5555
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5630..5734
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5735..5803,5804..6595)
                     /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(5953..5972)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6766..7220,1..134)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
       61 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      181 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      241 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      301 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
      361 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
      421 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
      481 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
      541 attacgccaa gctctagcta gaggtcgacg gtatacagac atgataagat acattgatga
      601 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
      661 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga
      721 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
      781 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
      841 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
      901 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
      961 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     1021 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     1081 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     1141 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     1201 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     1261 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     1321 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     1381 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     1441 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     1501 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     1561 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     1621 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     1681 cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc ccgggagctt
     1741 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     1801 agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     1861 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     1921 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     1981 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     2041 tggggacttt ccacacccta actgacacac attccacaga attaattcgc gttaaatttt
     2101 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2161 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2221 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2281 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2341 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2401 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2461 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtggggatac
     2521 cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac cgcatcccca
     2581 gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc
     2641 agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg
     2701 acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat
     2761 ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaacggg ccctctagac
     2821 tcgagttagg caggggccac ctgagaagtc tcggtcttag acacggtggt gctggcctca
     2881 tcgaattcac cggtgcgtcc accggtatcg atcactgtgg tctcgttctt ccatggtccg
     2941 gttcgtatat tacccgtgcc gtctgtgccg aacggcagac cctccaggag ttttccttta
     3001 ggcagcccat tctgggaagg tccgccaaag ggcaaggact gagttcgtct aagcatttcc
     3061 aagtgaccag gaccaagtgc ggcctctctc ccaggctgag ttggacatga gtacaatgct
     3121 ggagacccat cgcttccttc gggctgtgtg accataggga atgggtcgcc ctgggtgcaa
     3181 caggcaagag tgtgagtccc tgctgggcca tccagggaag atggtgggct atctgttcgg
     3241 gatgagcgtg gggagccatc gagggagcta gggatgtggt aggcatattc gggctcttcg
     3301 gcaggatggc gtgaaaaata cggtggcttt gtccgtcctt gcaggcagcc atgcagatgg
     3361 gggtcgtggc cgaacgcacc ttcttcctgc aagtgcacgt gggcttcgtc ggcggtcagt
     3421 ggctcgcagt gcatcttggc gcaacagggg gtagctgggc tcagacaact ggcatgcccc
     3481 tgagcggcct gcaggttagt cattttacag gggccggggg ggtgagcaaa gccgcggggc
     3541 tgccccagac caggtgagtg aagacaggct gctcttccaa gagcggctcc gttagcgtcc
     3601 agagctgggt gggcgtcttt ccttggaggg cagcaggccc accaacactg ccagacgtct
     3661 tcccgtttag cacaatggtg gatcaacaca aagagtccga gtgtcacgca aaaggcaccg
     3721 tacagacagg agaaaaccat atccagaaaa tgtccttgag acactgcgag agccccaaat
     3781 gcccatgtgg cggtaaagag gaagagtgta aaggcggcag ctctcagttg agcttgaaag
     3841 ctgtgctcgt tctgcaggac agatgctccg ggagcatcgt gtgcgggagg agtcccaggg
     3901 cgaatacccc tcccgccctc gggtgtagcc agcctgcgtt gctcttcagg ctgtgtccta
     3961 agctcatacc ttctgccggg gtgtctcctc agctgcacat atgtgcccag aaaatacacg
     4021 caggtcacca aagtgattat ggctgctggt ccgtaaaagg ctcccaaaga aggttcccag
     4081 gccatccagc agtaggctgt gtcctcgtcc tcggtgccat aattcctaat gtttgtggcg
     4141 gctgtcacgc cacaaattat gaagggcacc ccgccactta ccaaatagaa gcggaggagg
     4201 ggctgcctcg gatagggagg ctgatcagtg tccaggcaca gtggtgcttt cttagttacc
     4261 tgtttgtata tattgcgggc tgtgactccg atccagagca tggtgctcaa tgttgagtaa
     4321 tgcagcacga tgcccacggc ttgacacagt attgggtatt tcgtccgatt gatgccacca
     4381 gcaaagaccg taaaagtcaa ggccgcgtgg aaacagaagt tcagcagagt gtgtcggccc
     4441 ttgcgagaga ttcgtatggc actttggtgt acgatataag tcacaaagga ggcgagcaga
     4501 cacaacagca tgacggcagt gcacgcgtag accacggggt gaaggaattc gcctgggtat
     4561 ctcggaaggg agagcacggt tttcagatcc atatcgatgc tggcgtcatc atcgtccttg
     4621 tagtcggcga ataccaggca gaagatgtag ctcagggcga tgatcgtctt catggtggcg
     4681 ctaagcttaa gtttaaacgc tagccagctt gggtctccct atagtgagtc gtattaattt
     4741 cgataagcca gtaagcagtg ggttctctag ttagccagag agctctgctt atatagacct
     4801 cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg
     4861 gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac
     4921 ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca
     4981 tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
     5041 aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg
     5101 gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact
     5161 ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta
     5221 ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt
     5281 atgtaacgcg gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt
     5341 aataactagt caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg
     5401 caaaacgcct aaccctaagc agattcttca tgcaaatgtc ggtcaagcct tgccttgttg
     5461 tagcttaaat tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat
     5521 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat aggggatcgg
     5581 gagatctccc gatccgtcga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5641 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5701 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5761 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5821 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5881 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5941 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6001 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     6061 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     6121 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     6181 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6241 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6301 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6361 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6421 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6481 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6541 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6601 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6661 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6721 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6781 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6841 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6901 accaaatacc gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6961 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7021 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     7081 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     7141 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     7201 gtatccggta agcggcaggg
//
