LOCUS       sequence-430914-        6785 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 6785)
  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 6785)
  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: NMUR2-DuET
FEATURES             Location/Qualifiers
     source          1..6785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      388..976
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     877..896
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1130..1147
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1264..1285
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1300..1305,1306..1323,1324..1330)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1338..1354
                     /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     1343..1365
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1362..1378
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1362..1378
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1427..1548
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1438..1457)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1492..1511
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1722..2516)
                     /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(1833..1852)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2443..2462
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2583..2912)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2597..2732
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2651..2670)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2897..2917
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2926..3354)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3036..3057)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3248..3267
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3400..3624
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3613..3630
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5124..5143)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5125..5143)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5188..5391)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5217..5237)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5392..5771
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5943..5962
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6037..6141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6142..6210,6211..6785,1..217)
                     /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(6360..6379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
       61 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
      121 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
      181 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
      241 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
      301 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
      361 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
      421 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
      481 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ccgttcttct
      541 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
      601 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
      661 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
      721 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
      781 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
      841 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
      901 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
      961 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     1021 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     1081 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     1141 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     1201 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     1261 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     1321 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     1381 tgattacgcc aagctctagc tagaggtcga cggtatacag acatgataag atacattgat
     1441 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1501 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg ggtgggcgaa
     1561 gaactccagc atgagatccc cgcgctggag gatcatccag ccggcgtccc ggaaaacgat
     1621 tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt
     1681 gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga
     1741 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     1801 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     1861 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     1921 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     1981 ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     2041 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     2101 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     2161 gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc
     2221 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     2281 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt
     2341 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     2401 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     2461 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga
     2521 aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg atatacaagc tcccgggagc
     2581 tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag
     2641 gcagaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa
     2701 tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt
     2761 gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt
     2821 ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag
     2881 cctggggact ttccacaccc taactgacac acattccaca gaattaattc gcgttaaatt
     2941 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     3001 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     3061 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     3121 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     3181 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     3241 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3301 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat
     3361 accccctaga gccccagctg gttctttccg cctcagaagc catagagccc accgcatccc
     3421 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     3481 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     3541 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     3601 atggctggca actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag
     3661 attaggcagg ggccacctga gaagtctcgg tcttagacac ggtggtgctg gcctcatcga
     3721 attcaccggt gcgtccaccg gtatcgatcg tcttattgaa gtggaaggac tgatagttag
     3781 tccgggacat ctgctcgcta ctcagggcgg caggcaggtg gctattgtgc attgaagact
     3841 gacagggaaa ctgcggcccg atatcttcgg tgagttccac gaagtggcac tccgtgagaa
     3901 agatatttct ctgagccggt gggagctggg ggtcgtgctg actatgccac tgtttgtgaa
     3961 atgatgagat tacattctgg aatgcagcct ggaatcttct gctaagcaga ttgtaaatta
     4021 taggattaac ggcggaggac agataaaaga atacgcctga aacgacgtgc accaggttga
     4081 acacggcagc aaggctttca gaccattcct cgacaaaaga aaaaaacagc cggtcaatgt
     4141 ggaatggggc ccagcaaata gcaaaaacca gtaccaacac aaaaagcatc ttgttcacag
     4201 atttgcgaca agggcgctgg atgttggcat tcccctcatc tgcctcaagg cttttgtctt
     4261 ttttcagccg cagggccatg aggtagtaca gtactgatat aactgtcatc ggcaacaggt
     4321 agaacaaaaa acttgtgact tggataatga agttgtagat ccacataggc ttaataacgg
     4381 tgcatgtggc agagccgggc acaagggacc cattaggaaa gtaatgaaat ttaattccat
     4441 gaatactagt atttggcagg gaaaacagaa ctgagaagcc ccacacgatg cccagaattc
     4501 tgagggcccg ccggcgggtg gattgcagct tagctcgaaa tgggtgcagg atggcgacat
     4561 agcgttccac agacactgtc gttatgctga ggatggaagc gaaacagacg gtctcaaaaa
     4621 gagcagtctt gaagtaacaa cccacggggc caaacagaaa aggataattt ctccacatct
     4681 cgtatacctc cagaggcatg cccaacagaa gcacgagcag atcagacact gccagactaa
     4741 agaggtaata atttgttgga gttttcatgg cctgatgctg cagaataacc aggcacacaa
     4801 gcacgttccc tatgacgccg accacaaaaa tgggcacgta cacgactgac acgggcaaaa
     4861 agaagtggga tcgtcttggg ccgcagagga aagccaggta ttcctctgta gagttcaggt
     4921 gcttctggaa aggatcctcc agcttctgtt ggtaaatcca actggcgttc tgaagttttt
     4981 ccatcccgga catatcgatg ctggcgtcat catcgtcctt gtagtcggcg aataccaggc
     5041 agaagatgta gctcagggcg atgatcgtct tcatggtggc gcggccgcta agcttaagtt
     5101 taaacgctag ccagcttggg tctccctata gtgagtcgta ttaatttcga taagccagta
     5161 agcagtgggt tctctagtta gccagagagc tctgcttata tagacctccc accgtacacg
     5221 cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg
     5281 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
     5341 tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat
     5401 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg
     5461 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
     5521 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     5581 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     5641 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     5701 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa
     5761 taatcaatgt caacgcgtat atctggcccg tacatcgcga agcagcgcaa aacgcctaac
     5821 cctaagcaga ttcttcatgc aaatgtcggt caagccttgc cttgttgtag cttaaatttt
     5881 gctcgcgcac tactcagcga cctccaacac acaagcaggg agcagatact ggcttaacta
     5941 tgcggcatca gagcagattg tactgagagt gcaccatagg ggatcgggag atctcccgat
     6001 ccgtcgacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6061 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6121 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6181 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6241 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6301 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6361 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6421 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6481 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6541 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6601 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6661 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6721 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6781 gttgc
//
