LOCUS       sequence-430843-        8318 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 8318)
  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 8318)
  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: GPRC6A-DuET
FEATURES             Location/Qualifiers
     source          1..8318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(87..416)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      101..236
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(155..174)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     401..421
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(430..858)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(540..561)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     752..771
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    904..1128
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1117..1134
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4161..4180)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4162..4180)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4225..4428)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4254..4274)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4429..4808
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4980..4999
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5074..5178
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5179..5247,5248..6039)
                     /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(5397..5416)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6210..6798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6699..6718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6952..6969
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7086..7107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7122..7127,7128..7145,7146..7152)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7160..7176
                     /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     7165..7187
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7184..7200
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7184..7200
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    7249..7370
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7260..7279)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7314..7333
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(7544..8318,1..20))
                     /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(7655..7674)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     8265..8284
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatccgaaa
       61 atggatatac aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct
      121 cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa
      181 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
      241 ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg
      301 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt
      361 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc
      421 cacagaatta attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
      481 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
      541 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      601 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
      661 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      721 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      781 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      841 cgccgctaca gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag
      901 aagccataga gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct
      961 tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg
     1021 caatttcctc attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag
     1081 gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc
     1141 agcgggttta aacgggccct ctagactcga gttaggcagg ggccacctga gaagtctcgg
     1201 tcttagacac ggtggtgctg gcctcatcga attcaccggt gcgtccaccg gtatcgataa
     1261 tggagctcat cctcttcctc gggagtgttt tactcactga tgtagcgttc tcccggcaga
     1321 tgtgggcaaa ggcctgggcc tgcaggtcct tggatttctg ccaagttgcg gacttgcctg
     1381 aagaactggg gtttgtcata gtaacgttac cggacatact gtccagactg gctggtgaca
     1441 gagcgatact actcacacta tgagaggaat agctgtaaat catcttcaga aaggcggatt
     1501 tagtgtttat ctcttgtttg caaataatta catagcattt cggaatgaag gtgcagtaca
     1561 ggatgccgta attagaaata aggatcacaa taatctctac tgcaggaacg tatttcccga
     1621 aggtggtggc atagattgga atgaaggtga tccatgctat aaagtaaatc agcataccaa
     1681 aagttatgaa cttggcctcg ttataattct cgtacttccc cttaaatgcg aagatgaaac
     1741 agataaaggc caggatggcg atatagccca acattgtacc aaatgcaagg atgctaccct
     1801 cctcgcactc caagatgata actcgcggca gagaaacgtt cacctcaacg gtaggtgcgg
     1861 cgaagatcaa ccacagggtg caaataacaa cctggatgcc tgtacaggtg aatatgatca
     1921 ggattgggcg atagagacat ttcaagaact tctgcaactt agggtcaaat gaaaatgcca
     1981 ggaggatctt gaggctcttt gtcaggatac aagatataca cagggtaaag gagacgccaa
     2041 acatagtctg tctcgtcttg caagtgaaat cctggggctc tccgataaag aaagatgtgc
     2101 tagcaaagtt caagaagtgg caaagaagaa tcacataaca aaccctcaga cccccactag
     2161 acttcacgac gggtgtatta agattcctgg tgaatataat ccccacaacg aggacgaaaa
     2221 taatgcccag aagagacagg ataagcagga ggatggccag agaatcgttc caattcagat
     2281 actccacctc cttctcgaag cacatagtgg accgcacagg agcccaatgg gttttattgt
     2341 tgcacaacag acagtgtggc atatctgtct ggtttgtata atggttctct gggcagtttt
     2401 gacactcgta acagcatatg tgctgggacc gggttgtctt tttcatctgg ccaggagagc
     2461 attctttaga gcatttactt tgtatttgct tcaggttgcg gaactcattt ttggtttcct
     2521 gatcggggat gatgaagacg tcgttttgca agtcgtactc agccattttt gtcactgtca
     2581 tgtgcccgtt gatttctttc cacaggacaa cgtcataccc agtattgaga tcaccgtgtg
     2641 cgtcaaaatg aaatgaattc cacccatcgg tgaaggttac gttcttcagc acccccagca
     2701 gctcccaagg ctgaaaagca ttgggatttt ggcagtctct ggcctggcac aggtcgcgaa
     2761 ttgcgtagcc gagggcgaaa actgccagct gaatggaatg aatcaagcca ggctcggcgt
     2821 agtcccacag aaaatcattc ctcattacaa aattcctctc gatagctttg ttggctttgt
     2881 aagccagggt ccgttggctg tggttgaaga tgcattgact gagatcagtg tccttcacgt
     2941 aggcgcaagc gctcaagtgc atggcatact cgtgcaacag cttgtggctg tcggatggga
     3001 gcagatgcag attctgcaaa aatgagtgga agctggaaat gttgcctcgt ctgaatgcaa
     3061 aaccgaccac ctttccaatc ttcttaacgt taggtatagt ggtgatttta gtggctgtag
     3121 accagttatc agacgcaatc cacatcttat taatattcat ttcaattgcc ttattgaaga
     3181 ggtcgaaaac gtggaactgc ctaagaaaga ctacgatcac gttaacctgc gcttccagaa
     3241 ttattttctt cagtgttcgg ttgattctga cctcgatggt gttgtcactc agaaaggctg
     3301 gcaacacttc tttaaaagcg atacacacgt tattagcttc agcctggatg ataaatgtgt
     3361 tcaatgccag tcggccatag tcatcatctg tggtaataat accaatccaa ttccaaccgc
     3421 tcttttgaat caggtgagcc atagccttga tctggtggaa gtcactagga accgtcctca
     3481 agaaggatgg gaaccggatt ttgtcactga ggatttctgc tgtgctctcg tatcctactt
     3541 ggggcatgag ctgcaagttc aacatccggg acacagccat tgtaatttca gagtagccgc
     3601 ttccgatcac ggctttaacc ctgggcatgt aggaggagta gtcgcatttg aattcaactg
     3661 tctcgcgaga acaattgaac ttggacaaaa agcgaagtgt agcggccata gccactgtga
     3721 cttctgtgca ggtatcatag atctcgtacc ccagcttgac cccgggcaac agggttgaat
     3781 tattgatcat ttcaatacta tgaatcattg ccagggtctg cagaaataca ctgatctcga
     3841 agcccacgca ttcttggatc tgtggtcttc ttggagagtc ctcgctactc agcatttttt
     3901 cgtgaatagc aaacaggcca ccgataatga tgtgtccggg gcttgtcgct gccacaaaat
     3961 catcgggggt ttgacacggt tgggatgtgg ccagaataat cacaaagcaa gtaatcagga
     4021 ttatcagaaa tgccatatcg atgctggcgt catcatcgtc cttgtagtcg gcgaatacca
     4081 ggcagaagat gtagctcagg gcgatgatcg tcttcatggt ggcgctaagc ttaagtttaa
     4141 acgctagcca gcttgggtct ccctatagtg agtcgtatta atttcgataa gccagtaagc
     4201 agtgggttct ctagttagcc agagagctct gcttatatag acctcccacc gtacacgcct
     4261 accgcccatt tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt
     4321 ttggtgccaa aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga
     4381 gtcaaaccgc tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc
     4441 gatgactaat acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc
     4501 ataatgccag gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg
     4561 atacacttga tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca
     4621 atggaaagtc cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg
     4681 ggggtcgttg ggcggtcagc caggcgggcc atttaccgta agttatgtaa cgcggaactc
     4741 catatatggg ctatgaacta atgaccccgt aattgattac tattaataac tagtcaataa
     4801 tcaatgtcaa cgcgtatatc tggcccgtac atcgcgaagc agcgcaaaac gcctaaccct
     4861 aagcagattc ttcatgcaaa tgtcggtcaa gccttgcctt gttgtagctt aaattttgct
     4921 cgcgcactac tcagcgacct ccaacacaca agcagggagc agatactggc ttaactatgc
     4981 ggcatcagag cagattgtac tgagagtgca ccatagggga tcgggagatc tcccgatccg
     5041 tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5101 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5161 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5221 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5281 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5341 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5401 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5461 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5521 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5581 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5641 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5701 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5761 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5821 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5881 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5941 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6001 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6061 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6121 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6181 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6241 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6301 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa taccgttctt
     6361 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6421 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6481 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6541 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6601 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6661 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6721 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6781 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6841 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6901 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6961 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     7021 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     7081 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     7141 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     7201 catgattacg ccaagctcta gctagaggtc gacggtatac agacatgata agatacattg
     7261 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     7321 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt ggggtgggcg
     7381 aagaactcca gcatgagatc cccgcgctgg aggatcatcc agccggcgtc ccggaaaacg
     7441 attccgaagc ccaacctttc atagaaggcg gcggtggaat cgaaatctcg tgatggcagg
     7501 ttgggcgtcg cttggtcggt catttcgaac cccagagtcc cgctcagaag aactcgtcaa
     7561 gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga
     7621 agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt
     7681 cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat
     7741 tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt
     7801 cgggcatgcg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt
     7861 cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc
     7921 gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca
     7981 ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct
     8041 gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca
     8101 cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca
     8161 gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg
     8221 acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga
     8281 atagcctctc cacccaagcg gccggagaac ctgcgtgc
//
