LOCUS       sequence-430834-        6797 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 6797)
  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 6797)
  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: GPR63-DuET
FEATURES             Location/Qualifiers
     source          1..6797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      184..772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     673..692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     926..943
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1060..1081
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1096..1101,1102..1119,1120..1126)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1134..1150
                     /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     1139..1161
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1158..1174
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1158..1174
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1223..1344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1234..1253)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1288..1307
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1518..2312)
                     /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(1629..1648)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2239..2258
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2379..2708)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2393..2528
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2447..2466)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2693..2713
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2722..3150)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2832..2853)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3044..3063
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3196..3420
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3409..3426
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4932..4951)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4933..4951)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4996..5199)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5025..5045)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5200..5579
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5751..5770
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5845..5949
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5950..6018,6019..6797,1..13)
                     /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(6168..6187)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
       61 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
      121 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      181 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      241 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      301 cttcagcaga gcgcagatac caaataccgt tcttctagtg tagccgtagt taggccacca
      361 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
      421 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
      481 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
      541 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
      601 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      661 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      721 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      781 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      841 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      901 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      961 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     1021 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     1081 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     1141 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc tctagctaga
     1201 ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac cacaactaga
     1261 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1321 attataagct gcaataaaca agttggggtg ggcgaagaac tccagcatga gatccccgcg
     1381 ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa
     1441 ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc
     1501 gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     1561 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     1621 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     1681 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     1741 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
     1801 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     1861 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     1921 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     1981 tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     2041 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     2101 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
     2161 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     2221 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     2281 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     2341 tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc taggcctcca
     2401 aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc ggcctctgca
     2461 taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg cggagttagg
     2521 ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag atgcatgctt
     2581 tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc tgactaattg
     2641 agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc acaccctaac
     2701 tgacacacat tccacagaat taattcgcgt taaatttttg ttaaatcagc tcatttttta
     2761 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2821 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2881 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2941 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     3001 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     3061 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     3121 ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc cctagagccc cagctggttc
     3181 tttccgcctc agaagccata gagcccaccg catccccagc atgcctgcta ttgtcttccc
     3241 aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca
     3301 gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca
     3361 gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag
     3421 tcgaggctga tcagcgggtt taaacgggcc ctctagactc gagttaggca ggggccacct
     3481 gagaagtctc ggtcttagac acggtggtgc tggcctcatc gaattcaccg gtgcgtccac
     3541 cggtatcgat caccactgtg cgatgctccc cacacacata aacggcgctc ggtctgatcc
     3601 gccgcttagt atgcccgggc agttgaggca ggaatttgaa gcttttcggc atcatatcaa
     3661 ggcacgcgtc gtggaacttt ttgatccgcc aatagtatat aagagggtta agagcgctct
     3721 tcaagtaaca gagccacagc agccaagtgc tgatctcaaa gaagttatgc tggtagtaaa
     3781 aatgcttaga aaaagtggct acaaggctat aggttgtgaa gggggcccaa catacgatga
     3841 agacggcaaa aaggatcaga attgtagtga aggctctcgt tttaaatccc atgtcgatac
     3901 tcatttggaa tggccgctga agactcatga gcccaagctt actggcctgg gacaggcaga
     3961 taccttcagg gtaactgtgg atgcggaggg cgttgtgcct caaagtattg aggatgccca
     4021 taaaactgta gagtataaca agaaagggaa tgaagaagct gatcaagctg atgagaataa
     4081 catacgcctg gtagcctggg tttgtcgtgt agccaaacac gcattgggga gcgcgactgg
     4141 ggatctgcag gtctggattc cccacggcca gagggaacgc aacgcagaaa cttgtagccc
     4201 agctgacagc tatcagcacc ttagctctgt aggggttcag cttatcttgc cgctgaacga
     4261 tgatcaggaa gcggtcgata gaaatgataa ggaggatagc taccccttcg atcacgaaca
     4321 gccaaaaaaa catagcagac actctacaaa agaattttcc aaaaatccac cgagtagtga
     4381 gaatggtgac caaggcaaag ggcatgttca ggactgcaag cagcatatca gcaaaggcaa
     4441 gagaggccag gagaatgttg atggcgctgc gcatggcggc tttctggtac accatcaggc
     4501 acactacaag gtttcccaaa aaactgacaa acaggatgaa gatcatgatg gcactcagag
     4561 ttatctgcag gggcaggttc agactcttaa aggcagcggg agtagtaggg acagctgtag
     4621 agttcactgt cagtgaggac aggccggtgg gtgccatagt ctcaaaggaa tatctcagca
     4681 gggggctgag gtcgggatgc tgaaaaggcg gggggagagt gatgttcatg taagtgttct
     4741 cgtacaccac gaatgtggta ttagaggtgc cggtgtgaaa agcagtaagc accgcactga
     4801 aaaccatatc gatgctggcg tcatcatcgt ccttgtagtc ggcgaatacc aggcagaaga
     4861 tgtagctcag ggcgatgatc gtcttcatgg tggcgctaag cttaagttta aacgctagcc
     4921 agcttgggtc tccctatagt gagtcgtatt aatttcgata agccagtaag cagtgggttc
     4981 tctagttagc cagagagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     5041 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     5101 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     5161 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     5221 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     5281 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     5341 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     5401 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     5461 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     5521 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     5581 acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt
     5641 cttcatgcaa atgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta
     5701 ctcagcgacc tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga
     5761 gcagattgta ctgagagtgc accatagggg atcgggagat ctcccgatcc gtcgacgtca
     5821 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5881 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5941 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     6001 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     6061 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     6121 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     6181 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     6241 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     6301 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     6361 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     6421 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     6481 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     6541 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6601 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6661 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6721 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6781 ataggtgcct cactgat
//
