LOCUS       sequence-430844-        6692 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 6692)
  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 6692)
  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: GRPR-DuET
FEATURES             Location/Qualifiers
     source          1..6692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     305..324
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     558..575
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    692..713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(728..733,734..751,752..758)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    766..782
                     /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     771..793
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     790..806
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     790..806
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    855..976
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(866..885)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     920..939
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1150..1944)
                     /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(1261..1280)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1871..1890
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2011..2340)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2025..2160
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2079..2098)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2325..2345
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2354..2782)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2464..2485)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2676..2695
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2828..3052
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3041..3058
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4459..4478)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4460..4478)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4523..4726)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4552..4572)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4727..5106
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5278..5297
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5372..5476
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5477..5545,5546..6337)
                     /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(5695..5714)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6508..6692,1..404)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
       61 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
      121 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
      181 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
      241 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
      301 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
      361 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
      421 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
      481 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
      541 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
      601 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
      661 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      721 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      781 aatttcacac aggaaacagc tatgaccatg attacgccaa gctctagcta gaggtcgacg
      841 gtatacagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
      901 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
      961 ctgcaataaa caagttgggg tgggcgaaga actccagcat gagatccccg cgctggagga
     1021 tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag aaggcggcgg
     1081 tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca
     1141 gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     1201 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     1261 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     1321 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     1381 atgggtcacg acgagatcct cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc
     1441 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     1501 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     1561 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     1621 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     1681 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     1741 cgatagccgc gctgcctcgt cctgcagttc attcagggca ccggacaggt cggtcttgac
     1801 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     1861 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     1921 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatcc
     1981 gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc
     2041 tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa
     2101 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     2161 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     2221 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     2281 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     2341 attccacaga attaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     2401 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     2461 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     2521 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     2581 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     2641 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     2701 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     2761 aatgcgccgc tacagggcgc gtggggatac cccctagagc cccagctggt tctttccgcc
     2821 tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc
     2881 cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc
     2941 gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag
     3001 gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct
     3061 gatcagcggg tttaaacggg ccctctagac tcgagttagg caggggccac ctgagaagtc
     3121 tcggtcttag acacggtggt gctggcctca tcgaattcac cggtgcgtcc accggtatcg
     3181 attacgtatc gttcgtggca gatgttccca ttgataagtg agaaggtagc cactgatggg
     3241 tttgtacttt tcagggaagt catgcatgtt gtagaccgac ccgttgagtg actgcggata
     3301 atgagcccgg gctggcaaca caacagttga gtattgaatt gcttgcgaaa acttttactc
     3361 agcaaataca gtgcaaaggg attaacacaa gaattagtga aggccagcag cctggcacag
     3421 atggatgtca cgaaatgaag catggaggtg tcgacctcgg agtagtggta tgatcggtac
     3481 agatagatca catgattggg cagccaacag aatgcgaaaa ggcccacaaa gacgagaacg
     3541 gtctttgcaa gccgctttct gctttctatc tgtttcttaa cgtgtatatt gccttctact
     3601 gggaggttgt aagctgactg aatcaggttc ttcgctatga agtagtagta aacggagatt
     3661 atactcaagg ggatcacgta aaagacgagg aatgaggcca tagaatgaat cttagggtgc
     3721 agctcattgg agtgggggta tggggcgcag gaaataaaag tctggttcgt gctttcttcg
     3781 tgaaatgggt ggaggtcgga aaatactgct tcggggatgg ccaaaagcat gctgataatc
     3841 caaatgaacg ccgccttcaa gcagatcttc atgagggcat gggatgcctg gatatccatg
     3901 ggcctcacga tagctttgta gcgatccgca gaaagtgctg tgagggtgaa gacagaaact
     3961 cccactgagg tcagctgaat gaagggaatc agtttacaac ctattctccc aaacagccat
     4021 ctgtccgcca ggtatctaga ggcatcaaca ggggcacatg ttatcagcag aaggaggtca
     4081 cccaatgcca gagaacttat aaacaggtta gggacgttcc tcattgattt gacggtgcag
     4141 aagatcttga tcagcgtaat gttgccaatc agcccaatga gaattattac cccgtagacc
     4201 gcaggaatca cgtacagaat tcccggatga gaccagtcgt cattcacagg cagatcagca
     4261 gaatgggatg aaatattgca gtgcataaag tggtccactt ccagattcaa cagaaagcaa
     4321 tcgttgaggg ccatatcgat gctggcgtca tcatcgtcct tgtagtcggc gaataccagg
     4381 cagaagatgt agctcagggc gatgatcgtc ttcatggtgg cgctaagctt aagtttaaac
     4441 gctagccagc ttgggtctcc ctatagtgag tcgtattaat ttcgataagc cagtaagcag
     4501 tgggttctct agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac
     4561 cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt
     4621 ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt
     4681 caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga
     4741 tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat
     4801 aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat
     4861 acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat
     4921 ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
     4981 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca
     5041 tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc
     5101 aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa
     5161 gcagattctt catgcaaatg tcggtcaagc cttgccttgt tgtagcttaa attttgctcg
     5221 cgcactactc agcgacctcc aacacacaag cagggagcag atactggctt aactatgcgg
     5281 catcagagca gattgtactg agagtgcacc ataggggatc gggagatctc ccgatccgtc
     5341 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5401 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5461 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5521 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5581 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5641 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5701 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5761 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5821 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5881 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5941 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6001 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6061 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6121 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6181 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6241 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6301 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6361 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6421 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6481 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6541 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6601 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ccgttcttct
     6661 agtgtagccg tagttaggcc accacttcaa ga
//
