LOCUS       sequence-430864-        6593 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 6593)
  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 6593)
  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: LPAR2-DuET
FEATURES             Location/Qualifiers
     source          1..6593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     444..463
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(584..913)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      598..733
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(652..671)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     898..918
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(927..1355)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1037..1058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1249..1268
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1401..1625
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1614..1631
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(2933..2952)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2934..2952)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2997..3200)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3026..3046)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3201..3580
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3752..3771
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3846..3950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3951..4019,4020..4811)
                     /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(4169..4188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4982..5570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5471..5490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5724..5741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5858..5879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5894..5899,5900..5917,5918..5924)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5932..5948
                     /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     5937..5959
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5956..5972
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5956..5972
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    6021..6142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6032..6051)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6086..6105
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(6316..6593,1..517))
                     /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(6427..6446)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
       61 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
      121 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
      181 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
      241 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
      301 tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
      361 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
      421 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
      481 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
      541 cttgatcaga tccgaaaatg gatatacaag ctcccgggag ctttttgcaa aagcctaggc
      601 ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggcagaggcg gcctcggcct
      661 ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa ctgggcggag
      721 ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca
      781 tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact
      841 aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc
      901 ctaactgaca cacattccac agaattaatt cgcgttaaat ttttgttaaa tcagctcatt
      961 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     1021 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     1081 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     1141 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1201 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1261 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1321 acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga taccccctag agccccagct
     1381 ggttctttcc gcctcagaag ccatagagcc caccgcatcc ccagcatgcc tgctattgtc
     1441 ttcccaatcc tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct
     1501 actcagacaa tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc
     1561 ttccagggtc aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg
     1621 cacagtcgag gctgatcagc gggtttaaac gggccctcta gactcgagtt aggcaggggc
     1681 cacctgagaa gtctcggtct tagacacggt ggtgctggcc tcatcgaatt caccggtgcg
     1741 tccaccggta tcgatgaggg tggaatccat cagcgggtgg ccattctcag ggagcatgat
     1801 tcgtgttgag gccccgccct gggcgctgga ggtgtaatgc actgattcgc gcgtagactg
     1861 tctcagacat gcgcaacaca gcagcctcct gaaggtcctc cgcatctcgg cgtctctaca
     1921 gctatagaca gcagcattaa ccaaggaatt agcttctgcg agcaacagga agtacttctc
     1981 aacagcgagg acattacagc tttcacagcc caacccgtcc aaaagcagaa cgacctgtcc
     2041 aggtgtccag cagacgacga aagcccccaa gattataaca acggttttca caagtgagag
     2101 agtcgtctcc ctatatcttg gatggcaaga cacgtgctcg gccatccgct gtacgcgtct
     2161 cctgacgtag aagaagattc tggtgtacac cgccaccatg aggagaaaca ccaggagaga
     2221 gctgagtgcc cacacagcca aataagatct ggacaggaga ggcgccatcc gagaacagcg
     2281 atcgagggcg caaaggcaat gccagctgtg agctggcaac aggcccaaac ccagggcagc
     2341 gacccatacg cccacgataa gcatcacgac tcttccgcga ggcagcctgc tgtggagctg
     2401 aacagccatt acgctccggt gtctttcaac agcaatagca agcagtgttg ccacagaggc
     2461 cgtgagagaa gtatccaaca atccctgtct aagaaaccat ccctccaggc tgagcctagc
     2521 agtcctaggg cctgtgtgga acatcagaaa cagataagct actccggcaa acagatctgc
     2581 ggcagccaga ttgcccagca gataataaat aggttgatga aaccggcggt tagaggcaat
     2641 tgctgcgatc accagcaggt tggtcagaag gaccagtaca ctaaccgtca ggcccagagc
     2701 cacaaccacg acatccttgg gtcgccagtg agaactcagc tcctttccag aattattgta
     2761 gaagaacccg atagtttcat tataataaca ttgacccatg atgaccatat cgatgctggc
     2821 gtcatcatcg tccttgtagt cggcgaatac caggcagaag atgtagctca gggcgatgat
     2881 cgtcttcatg gtggcgctaa gcttaagttt aaacgctagc cagcttgggt ctccctatag
     2941 tgagtcgtat taatttcgat aagccagtaa gcagtgggtt ctctagttag ccagagagct
     3001 ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag
     3061 ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg
     3121 tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg
     3181 tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa
     3241 gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca
     3301 ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca
     3361 gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg
     3421 ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg
     3481 ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc
     3541 gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt
     3601 acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca aatgtcggtc
     3661 aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca
     3721 caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     3781 caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa
     3841 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3901 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3961 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4021 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4081 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4141 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4201 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4261 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4321 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4381 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4441 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4501 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4561 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4621 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     4681 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4741 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4801 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4861 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4921 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4981 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5041 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5101 tcagcagagc gcagatacca aataccgttc ttctagtgta gccgtagtta ggccaccact
     5161 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5221 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5281 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5341 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5401 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5461 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5521 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5581 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5641 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5701 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     5761 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5821 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     5881 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     5941 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc tagctagagg
     6001 tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     6061 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     6121 tataagctgc aataaacaag ttggggtggg cgaagaactc cagcatgaga tccccgcgct
     6181 ggaggatcat ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg
     6241 cggcggtgga atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga
     6301 accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
     6361 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
     6421 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg
     6481 gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc
     6541 atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcc
//
