LOCUS       sequence-430841-        6899 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 6899)
  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 6899)
  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: GPRC5C-DuET
FEATURES             Location/Qualifiers
     source          1..6899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      125..713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     614..633
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     867..884
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1001..1022
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1037..1042,1043..1060,1061..1067)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1075..1091
                     /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     1080..1102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1099..1115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1099..1115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1164..1285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1175..1194)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1229..1248
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1459..2253)
                     /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(1570..1589)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2180..2199
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2320..2649)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2334..2469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2388..2407)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2634..2654
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2663..3091)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2773..2794)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2985..3004
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3137..3361
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3350..3367
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4975..4994)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4976..4994)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5039..5242)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5068..5088)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5243..5622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5794..5813
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5888..5992
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5993..6061,6062..6853)
                     /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(6211..6230)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
       61 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
      121 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
      181 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      241 gcttcagcag agcgcagata ccaaataccg ttcttctagt gtagccgtag ttaggccacc
      301 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      361 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
      421 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
      481 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
      541 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      601 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      661 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      721 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      781 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
      841 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
      901 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      961 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     1021 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     1081 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag
     1141 aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1201 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1261 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     1321 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     1381 aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt
     1441 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     1501 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     1561 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     1621 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     1681 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
     1741 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     1801 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     1861 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     1921 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     1981 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     2041 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     2101 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     2161 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     2221 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     2281 atcagatccg aaaatggata tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc
     2341 aaaaaagcct cctcactact tctggaatag ctcagaggca gaggcggcct cggcctctgc
     2401 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     2461 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     2521 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     2581 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     2641 ctgacacaca ttccacagaa ttaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2701 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     2761 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     2821 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     2881 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     2941 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3001 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3061 gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt
     3121 ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc
     3181 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     3241 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     3301 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     3361 gtcgaggctg atcagcgggt ttaaacgggc cctctagact cgagttaggc aggggccacc
     3421 tgagaagtct cggtcttaga cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca
     3481 ccggtatcga tatcccacac atagggattc ctgaacacct gagagttctt tccatctttt
     3541 gggggagtag cggcttggtg agactgagcg gagtacatgt cttctgcccg aagcgttgag
     3601 tttgcactgc ccataacctg gctgttggcg gttgctctgg ggaggataat gtcgtaagct
     3661 ccctcagaag gcaccttatg catcaatgcc atttcggtgg gttgatacac ggacgtcaac
     3721 agttgcccgt tgtaccctga gtaaggtgac actggtcgct tcgcggctac tggttcgtcc
     3781 attgaaaagg ctttattttc cacaaacata gactgtccct tctgctcctt cagaatagtt
     3841 tcgtagccga ctccccttgt aggatacata tcaccctgat aagactgttc aggggaactt
     3901 ttagttacct gtgagacctc gggtatcaca taaaacagca cgaaagccca tgcgttggcg
     3961 gccaaggcaa tggccagggt agggtcgtcc cacgtagggc tgttatgctg cttattgcca
     4021 taggtgtaca tcacaatcca gactacccat atagccacac tagttgccgt ggtgagcaac
     4081 acgaagacgc catgtttccg ccacctcttg tatctgccac acagggccgg ccaagcaccg
     4141 aggaaggcgc ccagcaacag caacatgaca taaatcaagg ccatcacaaa gtccatattg
     4201 gcgatggcgc atggtgatgc cactgcccac cccgcgcttg aatttccctg tggtccgccc
     4261 tctccggaac ctctcaccag tgtaatgata agccattctg tattaattat gacttcaacc
     4321 aaagtgagca gcagagccac ggtgaaaatg acccatcccc ttggaccatg gtttttgcgc
     4381 gccagaaaat tcagtgcaaa aacgtgtgca gccaggcaac tgaagcagat ggcaaagagc
     4441 acgccaaaga ggaaccgtcg actggcgcaa gtagagaagt cgggcttcac gacgcatgcg
     4501 aaaaccagac aaaacagccc aagggtcccg aggagaaaaa atacctgagt accaagcaat
     4561 gaccgtttct tggtgtcctg tacgaaggga agagaggcca ccaagatgat ggtcaggacg
     4621 aaagtagtga cgatccccgc gccggcaacc gcttcgagga cgattcccca cgctccggat
     4681 ctgtcgcaca gattgtagta gagggggttc agtccctgag aacagccagg ggggacatga
     4741 ccttgggccc aagctccggg aaacagaaac aagggcagcc cgaggcacat taccagagcc
     4801 ttatgaattg ccattcgggc accgagtccg ggctctggct gtgtccccat atcgatgctg
     4861 gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct cagggcgatg
     4921 atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg gtctccctat
     4981 agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag
     5041 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg
     5101 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga
     5161 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga
     5221 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc
     5281 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt
     5341 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     5401 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     5461 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
     5521 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc
     5581 ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc
     5641 gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg caaatgtcgg
     5701 tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca
     5761 cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag
     5821 tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg
     5881 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5941 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6001 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6061 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6121 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6181 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6241 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6301 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6361 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6421 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6481 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6541 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6601 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6661 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6721 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6781 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6841 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaac
//
