LOCUS       sequence-431810-        8180 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 8180)
  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 8180)
  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: GRM4-DuET
FEATURES             Location/Qualifiers
     source          1..8180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1384..1403)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1385..1403)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1448..1651)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1477..1497)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1652..2031
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2203..2222
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2297..2401
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2402..2470,2471..3262)
                     /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(2620..2639)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3433..4021
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3922..3941
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4175..4192
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4309..4330
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4345..4350,4351..4368,4369..4375)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4383..4399
                     /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     4388..4410
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4407..4423
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4407..4423
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4472..4593
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4483..4502)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4537..4556
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4767..5561)
                     /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(4878..4897)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5488..5507
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5628..5957)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5642..5777
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5696..5715)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5942..5962
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5971..6399)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6081..6102)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6293..6312
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6445..6669
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6658..6675
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 atatatttaa ggagctgtgt accatccacg gggtccatcc gagggcacag ccccacgcga
       61 cccggacaca ggtctctatg catcgcatgg agggcgtgtc ccattgcgta taccgcgtcg
      121 ataacaaatt ggaccttccc ttcttgctca taggctgagt cttggccaat ccgctctcta
      181 tttgtacact tcttgacgtg tgatcctttt ttcagggcat gtctggagag cttgcagtga
      241 aaattatcct cccaaaactc agcaaaccat atattcctcc tgttgttgtc cagggtgcgg
      301 ctgctaaaat agcgatcgaa tcctcgcaca ctcatccgct tgggcaggat ggtaacggcc
      361 ccctctgcga cctcttccag gtgcaatacg ggggcgatct tgctccccca actgtctgat
      421 cccatccaaa aaaagtgccc cgtttggttc gctctgcggg cggcttccaa tactcgccgg
      481 atatcatctt cattggcgaa gattatcaca gctctcgcgt tggaggtttc aagcaggcgg
      541 cgaattatct tatcaaattc acctgctttt ggctcccttg gtattttaac ggactgagca
      601 atacaaaccc cgccatcctc tctgctcttc tgaataaaag cctcaactcc ggattcgcca
      661 tatgagcctt cactagccac ggttgacacg tagttccatt tcaaggcgcg cacgatgtcc
      721 accatggctt gtgcctgata agtgtcagat ggcactaccc gggagaagaa atcatagcga
      781 ctgttatcgg acaaatcggg cgctgtagag gcgtaagaga tctgtggaat tttgaagagc
      841 cgcaagatat tagcgaccat gattgacacg ctggatccgg atgccccgat cacgcctacg
      901 actcgctcag gcttggtaat aataggaggg cctccgctcc cacaccggac ctcagtacca
      961 tctttctcaa taagcgcctg gacgaaggta aggctttgct caagcgcatg cgtgtctcga
     1021 ctacacgtgt caagaatcct tgcgcccagt gtgatattgg gcagcagatc ggggtcgttg
     1081 tttatcctgt cgagagcaaa gagcattgcc tccagtctgt gaatgccttt ctctttcttc
     1141 agctcgccgc acggtttgcc ctctgaaccc ctaccatgca caggaaagag ccccccgagg
     1201 gtaatatcac cgtcaatccg aatgctgttc atgtgaggat gtccttttgg cttatcgatg
     1261 ctggcgtcat catcgtcctt gtagtcggcg aataccaggc agaagatgta gctcagggcg
     1321 atgatcgtct tcatggtggc gcggccgcta agcttaagtt taaacgctag ccagcttggg
     1381 tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt tctctagtta
     1441 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     1501 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     1561 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     1621 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     1681 tgtactgcca agtaggaaag tcccataagg tcatgtactg gacataatgc caggcgggcc
     1741 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     1801 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     1861 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     1921 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     1981 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     2041 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     2101 aaatgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     2161 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     2221 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     2281 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2341 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2401 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     2461 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     2521 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     2581 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     2641 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     2701 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     2761 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     2821 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     2881 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     2941 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3001 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3061 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     3121 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     3181 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     3241 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     3301 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     3361 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3421 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3481 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     3541 ggtaactggc ttcagcagag cgcagatacc aaataccgtt cttctagtgt agccgtagtt
     3601 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     3661 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     3721 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     3781 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     3841 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     3901 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3961 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4021 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4081 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     4141 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     4201 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     4261 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     4321 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     4381 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     4441 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     4501 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     4561 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     4621 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     4681 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     4741 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     4801 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     4861 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     4921 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     4981 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     5041 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     5101 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     5161 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     5221 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     5281 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     5341 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     5401 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     5461 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     5521 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     5581 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     5641 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     5701 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     5761 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     5821 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     5881 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     5941 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     6001 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6061 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6121 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6181 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6241 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6301 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6361 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     6421 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     6481 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     6541 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     6601 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     6661 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagattag gcaggggcca
     6721 cctgagaagt ctcggtctta gacacggtgg tgctggcctc atcgaattca ccggtgcgtc
     6781 caccggtatc gatgatcgca tggttcgtgt atgtgacata cgtctgcttg gttgcgagtg
     6841 cgggggcctc caggttttcg cacagctcgg attttgcctc gccattgggg cggaagtttc
     6901 ccttttgcgt gaatttatta gacatggtag cggctgtgac aacggccttc aggctgcgtt
     6961 ttcgtttggg cacgttttgt tcggggtgga agagaatgat atacacctta ggcatgtaca
     7021 gcatgccgag tgaaacagag gcggacaggc tcactgagac tgtcagggtt gtggtctgaa
     7081 tatacagctt gtccgcactt tgtgaggtcc caaaaaaaat tgggatgaat gcgagccaga
     7141 cgatgcaggt agtgtacatg gtaaacccga tgggtttggc ttcattaaat gtttcgggga
     7201 ctccgcgcgt cttgatggcg tagactgtac acgttaccat gagcagcatg gagtagccga
     7261 gcagacagat aaggctaagg tcagagatgt cacacttaag gacgcctctg gcgaagcgag
     7321 gatcaagtgt cctctggtcc tggaaatcca cgacagagtg ggaagggtca acaacgaacc
     7381 acacgcagat tcccagcagc tggaggctga tcaaagagaa ggtaatagcg agttgtgaag
     7441 ccggacttat gaagcgagga gcactcacac tgcgtttacc ttgttcgaat atcctataga
     7501 tcctattggt cttggtcagg agggccgcat aggagatgct catgccgagt cccagaaaga
     7561 tccgcctcaa agagcaggtc ccgaggtcgg gctcggcaat catcaggaag gtggtagcgt
     7621 agcagaggaa gatcccagcg agcaggacgt agctaagttc tctcccgctg gccttcacga
     7681 tgggagtgtc gttatatctc acaaatgtaa tgaccacgaa aagggtcgcg gcaatgccaa
     7741 ctacagcaag aaagaggggc aggaccgccc atgggctacc ccactccagc ttgatgattg
     7801 gaataggtcg gcagccggtc ctgttctcgg taggtcgcat gtcgtaaggg caagtcttgc
     7861 atgtatatcg gtcaacttgg tactggtatc cggtacaggg ttcacaatgc cagcagcagg
     7921 gcataccttt cactgtcttt ttccgttcgc cgggctggca cgggagtgag cagatgctcc
     7981 gagggagctg ctgtccgctt cctggccagt gcattcgctc gatccgcagg tgaagatggt
     8041 ctgtccaaga tccaattact ttgtactcgg cactgtcatt tctcagctgg tactggtata
     8101 tatcgtaccg tcctggagcg tcgccgtttt cattaaaagt gactggattg ccggcgattc
     8161 cggaaaaatt cacgttccta
//
