LOCUS       sequence-430756-        6986 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 6986)
  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 6986)
  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: C3AR1-DuET
FEATURES             Location/Qualifiers
     source          1..6986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(599..618)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(600..618)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(663..866)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(692..712)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        867..1246
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1418..1437
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1512..1616
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1617..1685,1686..2477)
                     /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(1835..1854)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2648..3236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3137..3156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3390..3407
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3524..3545
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3560..3565,3566..3583,3584..3590)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3598..3614
                     /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     3603..3625
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3622..3638
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3622..3638
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3687..3808
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3698..3717)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3752..3771
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3982..4776)
                     /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(4093..4112)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4703..4722
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4843..5172)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4857..4992
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4911..4930)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5157..5177
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5186..5614)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5296..5317)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5508..5527
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5660..5884
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5873..5890
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 aaaaacaggt atgcacatca caaaagcgac gacccagatg cagccgcaaa tgctgcaggc
       61 catcccaaca tttctgtggt tctgacacca aataggcttg aacaccacga gacatctatc
      121 cagacttatt gcggtaagca agaagacgga tgcaaacata ttaagcacta tgatggaggg
      181 tatcagcttg cagaggaacc gcccataagg ccattggccc tgcaaagcca ggtgcgccaa
      241 agagaagggc agggacaggc agcacaggag gtcggcgagt gtaaggtgca ggaaccaaat
      301 tgtgttcact gtcctctgca ttttcaagcc ggccacccac aacacgagcc cattgcctgg
      361 caggcccagg agaaaagtca gactgagtat caccatagac aggatcacag ggggctcgtt
      421 ccaaggctga ctgagcaagt ccgttgagtt ggtctctgcg gaaaagctgg ccatatcgat
      481 gctggcgtca tcatcgtcct tgtagtcggc gaataccagg cagaagatgt agctcagggc
      541 gatgatcgtc ttcatggtgg cgctaagctt aagtttaaac gctagccagc ttgggtctcc
      601 ctatagtgag tcgtattaat ttcgataagc cagtaagcag tgggttctct agttagccag
      661 agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg
      721 gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca
      781 ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca
      841 ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac
      901 tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta
      961 ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag
     1021 tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt
     1081 actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca
     1141 ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat
     1201 gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcaacg cgtatatctg
     1261 gcccgtacat cgcgaagcag cgcaaaacgc ctaaccctaa gcagattctt catgcaaatg
     1321 tcggtcaagc cttgccttgt tgtagcttaa attttgctcg cgcactactc agcgacctcc
     1381 aacacacaag cagggagcag atactggctt aactatgcgg catcagagca gattgtactg
     1441 agagtgcacc ataggggatc gggagatctc ccgatccgtc gacgtcaggt ggcacttttc
     1501 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1561 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1621 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1681 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1741 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1801 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1861 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1921 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1981 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     2041 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     2101 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2161 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2221 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2281 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2341 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2401 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2461 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2521 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2581 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2641 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2701 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2761 ctggcttcag cagagcgcag ataccaaata ccgttcttct agtgtagccg tagttaggcc
     2821 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2881 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2941 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3001 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3061 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3121 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3181 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3241 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3301 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3361 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3421 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     3481 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     3541 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     3601 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctctagc
     3661 tagaggtcga cggtatacag acatgataag atacattgat gagtttggac aaaccacaac
     3721 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3781 aaccattata agctgcaata aacaagttgg ggtgggcgaa gaactccagc atgagatccc
     3841 cgcgctggag gatcatccag ccggcgtccc ggaaaacgat tccgaagccc aacctttcat
     3901 agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca
     3961 tttcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg
     4021 ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc
     4081 aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc
     4141 cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa
     4201 gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct
     4261 ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac
     4321 aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa
     4381 tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac
     4441 tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag
     4501 cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt
     4561 cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag
     4621 gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc
     4681 agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc
     4741 cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc
     4801 ttgatcagat ccgaaaatgg atatacaagc tcccgggagc tttttgcaaa agcctaggcc
     4861 tccaaaaaag cctcctcact acttctggaa tagctcagag gcagaggcgg cctcggcctc
     4921 tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt
     4981 taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat
     5041 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta
     5101 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc
     5161 taactgacac acattccaca gaattaattc gcgttaaatt tttgttaaat cagctcattt
     5221 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5281 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5341 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5401 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5461 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5521 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5581 cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat accccctaga gccccagctg
     5641 gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct
     5701 tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta
     5761 ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct
     5821 tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc
     5881 acagtcgagg ctgatcagcg ggtttaaacg ggccctctag actcgagtta ggcaggggcc
     5941 acctgagaag tctcggtctt agacacggtg gtgctggcct catcgaattc accggtgcgt
     6001 ccaccggtat cgattacggt tgtagagttc cgctcggaga ttacattatt gcttgggcag
     6061 tgggtgctgc gggtgagttc ttcagagaag gcggcctcca ggatgccctg gatgctctgt
     6121 cttgcctttt tgcgaaaatc tttccccagg agggcgtaca ggaaaggatt aaagcaagaa
     6181 ttggcgctgg caagggcgat gcacacgtga tcccaagaca taagggtctt cccgagaggg
     6241 gtttcgggat cggtcagcag ggacagcact ccaaagatgt gataaggagt ccagcacacc
     6301 aagaacaccg ccacgacgac cactgcgacc cgaaaggtct tgctttggct cttagcgaac
     6361 cgccctcgct gcatccggaa cacaataaaa gaatagcaag caatcatgat aacacttggc
     6421 aacagaaagc ctacaaccag ccgtgtgatt gtgatagcca ccaagggggt aggcacttga
     6481 tcgtcgtctg tgaactggcc aaggttgtaa tagtcttgaa aaccctgagg cagctcactc
     6541 tcgtaaaagg agttactgct tgcggaagga aacagcttca gatgagtgct cagaaatgca
     6601 tcggaattat cgaggggact ggtctcgtgg tcctcaattg ggaacccact gggaatttta
     6661 ggggagacga cgtctgcagg cttaaacacg ttactgtaca gattctggga agtaagccta
     6721 gcactgcctc tggggagact gtccgcggat ggtcgctgaa aggtttgtgg ctgaaaaacg
     6781 gttggcactg tccaagggtg gtcgtttgtc tggaaggagg aaggatcaag cctgtcgttc
     6841 atctctccgg gaggctgcac gatgttttcc aagctgcggt tctccagagg gtcgccgtag
     6901 aaatcggggt aatccagaga acttgacagc ccaaatttgt acccgcaccg attatgattg
     6961 tccgtggtga aaatctctct atagac
//
