LOCUS       sequence-430884-        6662 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 6662)
  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 6662)
  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: P2RY11-DuET
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..110
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     344..361
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    478..499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(514..519,520..537,538..544)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    552..568
                     /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     557..579
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     576..592
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     576..592
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    641..762
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(652..671)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     706..725
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(936..1730)
                     /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(1047..1066)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1657..1676
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1797..2126)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1811..1946
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1865..1884)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2111..2131
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2140..2568)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2250..2271)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2462..2481
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2614..2838
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2827..2844
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4215..4234)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4216..4234)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4279..4482)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4308..4328)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4483..4862
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5034..5053
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5128..5232
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5233..5301,5302..6093)
                     /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(5451..5470)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6264..6662,1..190)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
       61 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      121 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      181 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      241 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      301 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      361 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      421 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      481 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      541 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
      601 cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt
      661 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
      721 attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc
      781 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
      841 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca ggttgggcgt
      901 cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc aagaaggcga
      961 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     1021 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     1081 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     1141 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     1201 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     1261 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     1321 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     1381 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     1441 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     1501 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     1561 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     1621 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     1681 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     1741 cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg gagctttttg
     1801 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag
     1861 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     1921 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     1981 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     2041 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     2101 gactttccac accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt
     2161 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2221 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2281 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2341 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2401 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2461 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2521 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc
     2581 tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat
     2641 gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa
     2701 tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag
     2761 tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct
     2821 ggcaactaga aggcacagtc gaggctgatc agcgggttta aacgggccct ctagactcga
     2881 gttaggcagg ggccacctga gaagtctcgg tcttagacac ggtggtgctg gcctcatcga
     2941 attcaccggt gcgtccaccg gtatcgatct gagacagttc cctagactga ggctcggaag
     3001 gcttgggagc ggcggttgca ttcagaggga gggcctggcc agtagatttg gcatcctctg
     3061 ggttccaact atctctgtag cctggacaat gccggcagca gcaccccagg ctaggaacgg
     3121 cggccatgta cagcagggga tggacacaga aggcaagtgg catcaggccg cgcatcactt
     3181 gatatcccac atacgggccc agctcaaggg cggctgtagc ctgtgcgatg tctgcgaagc
     3241 tggggcaccg tgtggaccac cgccggcgtg catcaacatt cagcacgcgc attatatggt
     3301 atggcacata tgaagaggca tagagggcca cgccagaagc aaccagggcg gcgactctga
     3361 gtttctcagc aaccgtcatt ccgggggagc gcaaaacggc tctgccgagg gccccataag
     3421 cagcgagggt gagcaaaagg gggagcccgc atccgaggcc cgcaagcacc agactataag
     3481 cccggtacgc tgcgagtccg tggtcggcgg ttcccaggca cttgatacat gcttcagggc
     3541 gggccacaga gcagttcccg gcgccttgtt ggggcctctt aagatgggaa aatgacagcg
     3601 tgggcatggc aagcagggcc gcaagtaccc atccggctgc ggatacagcc caggcgtgtt
     3661 taggcctgag atgactccgt gcgaagaagg gatggacgat cccaagatac ctattgaggg
     3721 agatacaagt aatgaaaatg acactcccga gcaggttaca ggtgaacaga aacctttcca
     3781 gtctacaggc ggcctccccg tacctccagt gtttgggggg gtacagatac gctgccagtg
     3841 gtggcaatgt cagagcgcag agaagatcgg acacagccag ttgcacggag aacacaacag
     3901 caggatgcca tggcctttgc tttcgaatgc tgaacctgta cagtgccaga ccattgctag
     3961 caactgccac cagaaattcg acgacgagga tgggccagag gaagtccccc tggaagccgc
     4021 tgagcttgtc gtcggcggca gcgaggaagt tggcagggca agactttgcg ccgctcacat
     4081 ttgctgccat atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga
     4141 agatgtagct cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta
     4201 gccagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg
     4261 ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc
     4321 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
     4381 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
     4441 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
     4501 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     4561 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     4621 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     4681 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     4741 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     4801 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
     4861 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
     4921 attcttcatg caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca
     4981 ctactcagcg acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc
     5041 agagcagatt gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg
     5101 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     5161 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     5221 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     5281 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     5341 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     5401 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     5461 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5521 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5581 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5641 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5701 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5761 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5821 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5881 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5941 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     6001 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     6061 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     6121 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6181 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6241 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6301 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6361 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaataccgt tcttctagtg
     6421 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6481 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6541 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6601 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6661 ga
//
