LOCUS       sequence-431813-        9171 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 9171)
  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 9171)
  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: GRM5-DuET
FEATURES             Location/Qualifiers
     source          1..9171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        96..475
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        476..679
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     630..650
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     724..743
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        724..742
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4623..4640)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4629..4853
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4899..5327
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4986..5005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5196..5217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5336..5356)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5341..5670
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5521..5656
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5583..5602
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5737..6531
                     /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(5791..5810)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6401..6420
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6705..6826
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6742..6761)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6796..6815
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6875..6891)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6875..6891)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6888..6910)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6899..6915
                     /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(6923..6929,6930..6947,6948..6953))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6968..6989
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7106..7123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7277..7865)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7357..7376)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8036..8827,8828..8896))
                     /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     8659..8678
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8897..9001)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9076..9095)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
       61 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      121 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      181 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      241 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      301 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      361 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      421 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      481 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      541 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      601 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      661 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
      721 aattaatacg actcactata gggagaccca agctggctag cgccaccatg aagacgatca
      781 tcgccctgag ctacatcttc tgcctggtat tcgccgacta caaggacgat gatgacgcca
      841 gcatcgatca gtccagcgag cgacgggtgg tggctcatat gccaggagac atcataatcg
      901 gggcattgtt cagcgtccat caccagccaa ctgtcgacaa ggtccatgag cgaaaatgcg
      961 gcgcggtacg cgagcagtac ggcattcaaa gggtggaggc catgttgcac acactggaga
     1021 gaattaacag cgatcctacc ttgctgccta acattacatt gggatgcgaa atccgcgatt
     1081 cctgctggca cagcgcagtg gctctggagc aaagcattga atttatccgg gatagtttga
     1141 tcagcagcga agaggaggag ggactggtac gatgcgttga tggtagcagc agctccttca
     1201 ggtctaagaa acccatcgta ggggttatcg ggcctggatc ctcctccgtt gcgatccaag
     1261 ttcagaacct gctgcaactg tttaatattc cccagattgc ttatagtgca acttccatgg
     1321 acttgtccga taagaccctt tttaagtatt tcatgcgggt agttccaagc gacgctcagc
     1381 aggcaagggc catggtggat atcgtgaagc gctacaactg gacttatgtc agtgctgtgc
     1441 atactgaggg caattatggg gagtccggaa tggaagcatt caaggacatg tctgctaagg
     1501 agggaatctg catcgcccac agctacaaga tctatagtaa cgctggggag cagtcctttg
     1561 ataagttgct gaaaaagctc actagccacc ttcccaaggc ccgagttgtc gcctgcttct
     1621 gcgagggaat gacagttcgg ggcttgctca tggctatgcg gaggctgggg ttggcaggag
     1681 aattcctgct gctcggatca gatggttggg ccgatagata cgacgtcaca gatggctacc
     1741 aaagggaagc agttggaggc attaccatca agctgcagtc tcctgatgtg aagtggttcg
     1801 acgactacta cttgaagctg aggcccgaga caaatcacag aaacccatgg ttccaagagt
     1861 tttggcaaca cagatttcaa tgcaggctgg aaggcttccc acaggaaaat tccaagtaca
     1921 ataagacctg taactcatca ctgacgttga aaactcacca tgtacaggat tcaaagatgg
     1981 gattcgtgat taatgctatc tactctatgg cctatggcct ccacaatatg caaatgagct
     2041 tgtgtcccgg ctacgcaggc ctctgtgacg caatgaaacc catagatggg cgaaagttgc
     2101 tcgaaagtct gatgaagact aacttcaccg gagtgtccgg cgatacaatc ctgtttgacg
     2161 aaaacggtga ttctcctgga cgctacgaga tcatgaactt caaagagatg ggcaaggatt
     2221 attttgatta tatcaacgtt gggagttggg ataacggcga attgaaaatg gacgatgatg
     2281 aggtgtggag caaaaagtcc aacatcatcc ggagcgtgtg ctccgaacca tgcgagaagg
     2341 ggcagattaa ggtgataagg aagggagagg tgagttgctg ttggacttgc accccttgca
     2401 aagagaacga gtacgtcttt gacgaatata cctgcaaggc ttgccagctg gggtcctggc
     2461 ctacggatga tttgaccgga tgcgatctga tccccgtgca gtatctgaga tggggcgacc
     2521 cagagcccat tgcagcggtg gttttcgctt gtctcggcct gcttgcaact ctgttcgtga
     2581 ctgttgtatt catcatttac cgagataccc ccgtggttaa gagcagttct agagaactgt
     2641 gctacattat cctggccggc atatgccttg gttatctgtg tacgttctgc ctcattgcca
     2701 aaccaaaaca gatctattgc tatctgcagc gcattgggat cggcctgtct cccgccatgt
     2761 cctattcagc gctcgtgacc aaaaccaatc gaatcgcgag gatcctggca gggtctaaga
     2821 aaaaaatatg taccaagaaa ccaagattta tgtccgcctg tgcccagctc gtgatcgcct
     2881 tcatcctgat ctgtatccag cttggcatta tagttgccct ttttataatg gagcccccag
     2941 acatcatgca tgactaccct tccatcaggg aagtgtacct catctgcaat actaccaatc
     3001 tgggggtggt aaccccactg ggatataatg gcctcctgat cctgagctgt accttttacg
     3061 cgtttaagac gcgcaatgtg cccgcgaatt ttaatgaggc caaatatatt gcttttacca
     3121 tgtatacgac ttgtataatc tggctggcct tcgtgccaat ctattttgga tccaattata
     3181 agataattac catgtgcttt agcgtttcct tgagcgccac cgtggcactc ggatgcatgt
     3241 tcgttcctaa ggtatatatt attctggcta aaccagagcg caatgtccgc agcgctttca
     3301 cgacatctac cgtcgttagg atgcatgtgg gagatggaaa gtcaagcagt gcagccagta
     3361 ggagcagcag cctggtgaat ctgtggaaac ggcgagggag tagtggggaa accctgagat
     3421 acaaggacag aaggctcgct caacataaaa gcgagataga atgtttcacc cccaagggga
     3481 gcatgggtaa cggaggacgc gctactatgt catcttcaaa tggaaagtcc gtgacttggg
     3541 cccaaaatga aaaaagtagc cgcggacaac acctgtggca gcgcctgtca attcatatca
     3601 ataagaagga gaaccccaac cagaccgctg tgatcaaacc atttcccaag agtacagagt
     3661 cacgggggct gggagctggc gccggagccg gaggaagtgc gggcggcgtg ggggccacgg
     3721 gaggtgctgg atgcgccggg gccggaccag gggggcccga gtccccagat gcgggcccca
     3781 aagctctgta cgacgtggct gaggctgaag agcacttccc cgctcctgct cggccaagaa
     3841 gcccctcccc aatttcaacc ttgtcacatc gggccgggtc agcctcaaga accgatgatg
     3901 atgttcctag tcttcattcc gaacccgtgg ctcgaagctc ctcctctcag ggcagcctga
     3961 tggagcagat aagctccgtg gtgacaagat tcaccgccaa catttcagaa ttgaacagta
     4021 tgatgctgtc aaccgccgca cccagtcctg gtgttggcgc acctctctgc agtagctatc
     4081 tgatccccaa ggaaatccag ctgcctacta ctatgaccac cttcgctgag atccagcctc
     4141 tgccagccat tgaagtgact ggcggggcgc aaccagctgc aggcgctcag gctgctggcg
     4201 acgcggccag ggaaagtcca gccgctggac ctgaagccgc agcggctaag cctgatctcg
     4261 aagagttggt cgccctgacc ccgccaagcc cattccggga ctccgtggac tccggtagta
     4321 ctactcccaa ctccccagtc agtgagtccg cactttgtat tccaagctca cccaagtacg
     4381 acactctgat tattcgggat tacacgcagt cctcctccag cctgatcgat accggtggac
     4441 gcaccggtga attcgatgag gccagcacca ccgtgtctaa gaccgagact tctcaggtgg
     4501 cccctgccta aaagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
     4561 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     4621 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     4681 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4741 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4801 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     4861 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     4921 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     4981 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5041 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5101 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5161 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5221 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5281 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     5341 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5401 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     5461 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     5521 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     5581 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     5641 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     5701 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     5761 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     5821 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     5881 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     5941 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     6001 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     6061 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     6121 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     6181 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     6241 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     6301 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     6361 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     6421 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     6481 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     6541 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     6601 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     6661 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     6721 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     6781 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     6841 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     6901 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     6961 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     7021 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     7081 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     7141 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     7201 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     7261 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     7321 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     7381 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     7441 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     7501 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     7561 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     7621 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     7681 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     7741 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     7801 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     7861 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     7921 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     7981 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     8041 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     8101 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     8161 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     8221 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     8281 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     8341 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     8401 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     8461 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     8521 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     8581 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     8641 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     8701 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     8761 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     8821 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     8881 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     8941 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     9001 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     9061 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     9121 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta c
//
