LOCUS       sequence-430903-        6539 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 6539)
  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 6539)
  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: GPR146-DuET
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     621..640
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(859..963)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1038..1057)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1229..1608
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1609..1812
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1763..1783
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1857..1876
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1857..1875
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3124..3141)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3130..3354
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3400..3828
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3487..3506)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3697..3718
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3837..3857)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3842..4171
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4022..4157
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4084..4103
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4238..5032
                     /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(4292..4311)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4902..4921
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5206..5327
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5243..5262)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5297..5316
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5376..5392)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5376..5392)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5389..5411)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5400..5416
                     /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)."
     promoter        complement(join(5424..5430,5431..5448,5449..5454))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5469..5490
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5607..5624)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5778..6366)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5858..5877)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6537..6539,1..789,790..858))
                     /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"
ORIGIN
        1 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
       61 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
      121 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
      181 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
      241 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
      301 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
      361 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
      421 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
      481 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
      541 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
      601 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
      661 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
      721 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
      781 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
      841 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      901 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      961 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     1021 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     1081 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     1141 caaggcttga ccgacatttg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     1201 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
     1261 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1321 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1381 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1441 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     1501 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     1561 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1621 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1681 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1741 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1801 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
     1861 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt agcggccgcg
     1921 ccaccatgaa gacgatcatc gccctgagct acatcttctg cctggtattc gccgactaca
     1981 aggacgatga tgacgccagc atcgatatgt ggtcctgtag ctggttcaac ggtaccgggt
     2041 tggtcgaaga gcttccggcg tgccaggacc tgcagctggg acttagcctc ctcagcctgc
     2101 tgggtctggt ggtcggtgtg cctgtgggct tgtgctataa tgcgttgctg gtgttggcca
     2161 acctgcactc aaaggcttct atgaccatgc ctgacgttta ctttgttaat atggccgttg
     2221 cagggcttgt gttgagtgcc ttggcacctg tccatctgct cgggcctccc agcagcaggt
     2281 gggctctgtg gagcgtgggc ggcgaagtgc atgtcgccct ccagattcca tttaatgtat
     2341 cctccctggt tgctatgtac agtacagctc ttctgtcact ggatcattac attgagcggg
     2401 ctctcccaag aacgtatatg gcatccgttt acaacacacg ccacgtatgc ggttttgtgt
     2461 gggggggagc gctgttgact tcatttagtt ctctgttgtt ctacatttgc agtcacgtga
     2521 gcacccgggc cctcgaatgt gctaaaatgc agaacgccga ggcagcagat gctaccctgg
     2581 tgtttatcgg atacgtggtg cctgccctgg ccaccctgta tgcattggtg cttctgagtc
     2641 gcgtccgaag ggaagacact ccgcttgata gggacactgg acggctggaa ccaagcgccc
     2701 acaggctgtt ggtcgcgaca gtgtgcaccc aattcggcct gtggacaccc cactacctga
     2761 tcttgctcgg gcacaccgtg attattagca ggggtaaacc agtggacgct cattatcttg
     2821 ggctgctcca tttcgtaaaa gacttctcaa aactgctggc cttctcttcc agctttgtta
     2881 cgcccctcct gtacaggtac atgaatcaat cttttccatc aaagctccag aggcttatga
     2941 aaaagttgcc ctgcggcgat cggcattgta gtcctgacca catgggtgtg cagcaggtct
     3001 tggccatcga taccggtgga cgcaccggtg aattcgatga ggccagcacc accgtgtcta
     3061 agaccgagac ttctcaggtg gcccctgcct aatctagagg gcccgtttaa acccgctgat
     3121 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3181 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3241 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3301 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3361 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3421 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3481 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3541 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3601 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3661 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3721 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3781 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     3841 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3901 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3961 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     4021 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     4081 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     4141 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     4201 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4261 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4321 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4381 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     4441 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4501 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4561 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4621 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4681 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4741 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4801 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4861 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4921 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4981 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     5041 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5101 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5161 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     5221 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5281 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     5341 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5401 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5461 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5521 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5581 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5641 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5701 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5761 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5821 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5881 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5941 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6001 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6061 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6121 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6181 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cggtatttgg tatctgcgct
     6241 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6301 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6361 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6421 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6481 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagtta
//
