LOCUS       sequence-430865-        6650 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 6650)
  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 6650)
  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: LPAR4-DuET
FEATURES             Location/Qualifiers
     source          1..6650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1198..1217)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1199..1217)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1262..1465)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1291..1311)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1466..1845
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2017..2036
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2111..2215
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2216..2284,2285..3076)
                     /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(2434..2453)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3247..3835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3736..3755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3989..4006
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4123..4144
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4159..4164,4165..4182,4183..4189)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4197..4213
                     /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     4202..4224
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4221..4237
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4221..4237
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4286..4407
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4297..4316)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4351..4370
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4581..5375)
                     /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(4692..4711)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5302..5321
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5442..5771)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5456..5591
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5510..5529)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5756..5776
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5785..6213)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5895..5916)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6107..6126
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6259..6483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6472..6489
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 gttgtctggt cggacacttc ttcctgaatc gcaggcagag agggtttagt tgtcagtggg
       61 gtttctgtct taaacagtga ttccattcgg atgtgcgcgt taatgtagaa gctcttctga
      121 aaactctcca gtgtgaaata atagatgaaa gggtcgaagc agcagttcag ggtagcgagg
      181 cacaatgtaa taggatacat aatcttagcg aacctttcca ggaagcagtt ggtgatggcc
      241 tgactccgca ccagtgcata caggaaaagc acagaattat acggcacgaa gcacacaacg
      301 aacaccgcca tatggactgt gatcatcttg agcactttct tcttatttgt gccgatctgg
      361 gagagcgtcg ctggtttccg caaagtcctc aagaccacgg atgaacaact cacattcaga
      421 atcagtggga tgataaaccc taccacttct ataaaaattg taatctttga caggtacgtc
      481 ttccagactc gctttgaaaa accctcgaaa catgtagtgg ttgcgttatt cacattcgtg
      541 gtgctaaaca gagaagcaga aataccccca gagagcacga gaatccacac tcccgcacac
      601 actatagcgg agttccttct ggtgcggatc gttcgtgacc gaaatggata gactatggcc
      661 aggaacctgt ccacgctgat acatgtcagg aacagcatgg acccgtagat attggtcaga
      721 aaggccgttc cactgatttt gcacagggta tccccaaatg gccagtgccg gttgaaattg
      781 tagaagatct tgaagggcaa ggtacaaacg aacagcaaat ctgagacggc aaggttggta
      841 ataaaaatgg cggtttcgga ccgcattttc atcctaaagc agaacacgaa caaggacacg
      901 gaatttgtaa taagcccgag gatgaaaacc acggaataaa cggcgccgtt cagattgtac
      961 ttaaaggaat catcgactat acaagtgttg ttagctgtgg cattgcccaa tcgaggccgc
     1021 agggagctat tggaatcctg gaattgaaag tcaatgaatc gccggtcacc catatcgatg
     1081 ctggcgtcat catcgtcctt gtagtcggcg aataccaggc agaagatgta gctcagggcg
     1141 atgatcgtct tcatggtggc gctaagctta agtttaaacg ctagccagct tgggtctccc
     1201 tatagtgagt cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga
     1261 gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg
     1321 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat
     1381 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat
     1441 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact
     1501 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
     1561 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
     1621 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
     1681 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
     1741 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
     1801 accccgtaat tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg
     1861 cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt
     1921 cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca
     1981 acacacaagc agggagcaga tactggctta actatgcggc atcagagcag attgtactga
     2041 gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg
     2101 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2161 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2221 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2281 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2341 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2401 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2461 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2521 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2581 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2641 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2701 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2761 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2821 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2881 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2941 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3001 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3061 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3121 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3181 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3241 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3301 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3361 tggcttcagc agagcgcaga taccaaatac cgttcttcta gtgtagccgt agttaggcca
     3421 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3481 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3541 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3601 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3661 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3721 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3781 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3841 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3901 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3961 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4021 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4081 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     4141 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     4201 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctctagct
     4261 agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact
     4321 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     4381 accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc
     4441 gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata
     4501 gaaggcggcg gtggaatcga aatctcgtga tggcaggttg ggcgtcgctt ggtcggtcat
     4561 ttcgaacccc agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     4621 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     4681 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     4741 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     4801 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     4861 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     4921 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     4981 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     5041 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     5101 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     5161 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     5221 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     5281 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     5341 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     5401 tgatcagatc cgaaaatgga tatacaagct cccgggagct ttttgcaaaa gcctaggcct
     5461 ccaaaaaagc ctcctcacta cttctggaat agctcagagg cagaggcggc ctcggcctct
     5521 gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt
     5581 aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg
     5641 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa
     5701 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct
     5761 aactgacaca cattccacag aattaattcg cgttaaattt ttgttaaatc agctcatttt
     5821 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5881 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5941 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6001 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6061 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6121 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6181 ccgccgcgct taatgcgccg ctacagggcg cgtggggata ccccctagag ccccagctgg
     6241 ttctttccgc ctcagaagcc atagagccca ccgcatcccc agcatgcctg ctattgtctt
     6301 cccaatcctc ccccttgctg tcctgcccca ccccaccccc cagaatagaa tgacacctac
     6361 tcagacaatg cgatgcaatt tcctcatttt attaggaaag gacagtggga gtggcacctt
     6421 ccagggtcaa ggaaggcacg ggggaggggc aaacaacaga tggctggcaa ctagaaggca
     6481 cagtcgaggc tgatcagcgg gtttaaacgg gccctctaga ctcgagttag gcaggggcca
     6541 cctgagaagt ctcggtctta gacacggtgg tgctggcctc atcgaattca ccggtgcgtc
     6601 caccggtatc gataaaggta ctttccagca tcagttcacc gccattgttt
//
