LOCUS       sequence-430863-        6632 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 6632)
  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 6632)
  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: LPAR1-DuET
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      222..810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     711..730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     964..981
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1098..1119
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1134..1139,1140..1157,1158..1164)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1172..1188
                     /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     1177..1199
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1196..1212
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1196..1212
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1261..1382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1272..1291)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1326..1345
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1556..2350)
                     /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(1667..1686)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2277..2296
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2417..2746)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2431..2566
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2485..2504)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2731..2751
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2760..3188)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2870..2891)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3082..3101
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3234..3458
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3447..3464
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4805..4824)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4806..4824)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4869..5072)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4898..4918)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5073..5452
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5624..5643
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5718..5822
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5823..5891,5892..6632,1..51)
                     /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(6041..6060)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
       61 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
      121 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
      181 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
      241 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      301 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
      361 aataccgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
      421 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
      481 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
      541 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
      601 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
      661 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
      721 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
      781 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
      841 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
      901 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
      961 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     1021 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1081 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     1141 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     1201 aacagctatg accatgatta cgccaagctc tagctagagg tcgacggtat acagacatga
     1261 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     1321 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
     1381 ttggggtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat ccagccggcg
     1441 tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga atcgaaatct
     1501 cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga accccagagt cccgctcaga
     1561 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     1621 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     1681 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     1741 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     1801 gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     1861 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     1921 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     1981 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     2041 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     2101 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     2161 cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     2221 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     2281 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     2341 gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatccgaaa atggatatac
     2401 aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     2461 ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     2521 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     2581 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     2641 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     2701 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagaatta
     2761 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     2821 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     2881 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     2941 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     3001 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     3061 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     3121 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     3181 gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga
     3241 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     3301 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     3361 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     3421 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta
     3481 aacgggccct ctagactcga gttaggcagg ggccacctga gaagtctcgg tcttagacac
     3541 ggtggtgctg gcctcatcga attcaccggt gcgtccaccg gtatcgatga ccacggaatg
     3601 atcgttggag tgtacaccgg ccaggattgt gtggttgagg gatgaagctg accgatctga
     3661 accttcagtg gggcctgtag gattctcgct cctctggcag cagaggatct gccgaaaagt
     3721 tgcggacatc tctttatctc gataagagta aataatagga ttcattgcgg agttgaattc
     3781 ggccaacaaa agaaagaact tctcataggc aagaacgtca cactggggac aacacacgtc
     3841 gagcagaagc agtacgagcc cgggggtcca acaaatgata aatgcgccca acacgattac
     3901 gacggttttg aggagggaca tcattgtgtc ccgattccgt ctgggtccgc tgctgtgtcg
     3961 agacatccgc attgtgcgtt gtctcacgta tccgaagatg tgagcataca gtaccaccat
     4021 cacgacaaat gtcacgagat taaagatggc ccagaaaacc agataactgt cagaatacag
     4081 aggtgccata ttggaacagt tctcgatgtc gcaaatgcaa ttccaaccca cgctaggaat
     4141 ggcgcccata acgatagcca tggtccagat caccacgatc accaccacca ccctccggtt
     4201 tgacatccgt gtatgaagtt gcattcggaa caccgttatg tgtctctcaa ttgcgatggc
     4261 caacaggttt gccacagagg cagtcaggga agtgtcgatg agtccctggc gcaacagcca
     4321 agttgagaca gtaagtctgc gggtgttggg cccggtatta aacatcaagt agaaatatgc
     4381 caaccccgcg aagaagtcag ctgcggcaag atttgccatc aagtagtaaa ttggaaaatg
     4441 gaatctgcgg ttcacgtaga tagccaccat caccagcaag tttgcaagca tgataaagat
     4501 gcaaacggta ataccaaggc ccatgaccaa tttagacacc gtattccatt cggtggccag
     4561 atgcttcccg gatctgttat agaaaaaggc gatgctttcg ttgtaaaagc actgaggctc
     4621 attcatcgct gtaaactggg gctgagagat aactgggata ctggtgctaa tagcggccat
     4681 atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct
     4741 cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg
     4801 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
     4861 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     4921 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     4981 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     5041 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     5101 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     5161 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     5221 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     5281 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     5341 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     5401 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     5461 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     5521 caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     5581 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     5641 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     5701 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5761 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5821 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5881 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5941 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6001 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6061 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6121 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6181 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6241 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6301 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6361 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6421 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6481 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6541 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6601 acacgacggg gagtcaggca actatggatg aa
//
