LOCUS       sequence-431820-        6572 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 6572)
  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 6572)
  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: LPAR6-DuET
FEATURES             Location/Qualifiers
     source          1..6572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(266..370)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(445..464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        636..1015
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1016..1219
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1170..1190
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1264..1283
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1264..1282
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2564..2581)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2570..2794
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2840..3268
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2927..2946)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3137..3158
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3277..3297)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3282..3611
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3462..3597
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3524..3543
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3678..4472
                     /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(3732..3751)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4342..4361
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4646..4767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4683..4702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4737..4756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4816..4832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4816..4832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4829..4851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4840..4856
                     /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(4864..4870,4871..4888,4889..4894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4909..4930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5047..5064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5218..5806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5298..5317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5977..6572,1..196,197..265))
                     /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 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
       61 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
      121 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
      181 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
      241 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
      301 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
      361 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
      421 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
      481 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
      541 aacaaggcaa ggcttgaccg acatttgcat gaagaatctg cttagggtta ggcgttttgc
      601 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
      661 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      721 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      781 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      841 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      901 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      961 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     1021 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     1081 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     1141 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1201 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     1261 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttagc
     1321 gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt cgccgactac
     1381 aaggacgatg atgacgccag catcgatatg gtgtcagtga atagctctca ctgcttctac
     1441 aacgatagct tcaagtatac gctttacggg tgcatgttta gtatggtgtt cgtgttgggc
     1501 cttatctcca attgtgttgc aatctacatc tttatctgcg tgttgaaggt gcgcaatgaa
     1561 acaaccacat atatgattaa tctcgctatg tctgatcttc tgtttgtctt tacactgcca
     1621 tttcggatct tctattttac caccagaaac tggccattcg gggacctgtt gtgtaagata
     1681 tccgtcatgc tgttttacac taatatgtat gggagtattc tttttctgac atgtattagc
     1741 gtggacagat tcctcgcgat tgtgtaccct tttaaaagta agactctcag aaccaagaga
     1801 aacgcaaaga tcgtgtgcac tggcgtgtgg ctcaccgtga ttggtggcag cgcccctgct
     1861 gtttttgtgc agagcaccca ttcccagggc aataacgcaa gcgaagcctg cttcgagaat
     1921 ttccccgagg ctacatggaa gacatatctg agccgcattg ttatatttat tgaaattgta
     1981 ggtttcttca tcccactgat tcttaatgtg acttgctcta gtatggtctt gaagaccctc
     2041 acgaagccgg tgaccctgtc ccgcagtaaa atcaataaaa ccaaagtgtt gaagatgatt
     2101 tttgtgcacc tgatcatatt ctgtttttgc tttgtgcctt acaacattaa tttgatactc
     2161 tactcactgg tgcggaccca gacctttgtg aactgtagcg tggtagcagc ggtgcgcacc
     2221 atgtacccta taacactgtg cattgccgtg tcaaattgtt gttttgaccc catagtctac
     2281 tacttcacct cagataccat ccagaatagc attaagatga aaaactggtc agttaggagg
     2341 agtgatttta gattctcaga agtgcacggt gccgaaaact ttattcaaca taacctgcag
     2401 acactgaaga gcaaaatttt tgataatgag tcagctgcta tcgataccgg tggacgcacc
     2461 ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct
     2521 gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2581 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2641 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2701 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2761 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2821 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2881 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2941 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3001 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3061 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3121 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3181 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3241 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3301 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3361 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3421 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3481 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3541 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3601 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     3661 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3721 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3781 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3841 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3901 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3961 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4021 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4081 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4141 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4201 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4261 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4321 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4381 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4441 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4501 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     4561 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4621 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4681 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4741 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4801 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4861 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4921 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4981 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5041 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5101 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5161 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5221 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5281 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5341 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5401 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5461 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5521 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5581 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5641 ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc cagttacctt
     5701 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5761 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5821 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5881 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5941 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6001 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6061 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6121 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6181 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6241 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6301 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6361 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6421 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6481 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6541 gtcattctga gaatagtgta tgcggcgacc ga
//
