LOCUS       sequence-430755-        6713 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 6713)
  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 6713)
  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: BDKBR2-DuET
FEATURES             Location/Qualifiers
     source          1..6713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      32..620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     521..540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     774..791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    908..929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(944..949,950..967,968..974)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    982..998
                     /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     987..1009
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1006..1022
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1006..1022
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1071..1192
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1082..1101)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1136..1155
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1366..2160)
                     /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(1477..1496)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2087..2106
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2227..2556)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2241..2376
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2295..2314)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2541..2561
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2570..2998)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2680..2701)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2892..2911
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3044..3268
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3257..3274
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4696..4715)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4697..4715)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4760..4963)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4789..4809)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4964..5343
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5515..5534
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5609..5713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5714..5782,5783..6574)
                     /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(5932..5951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
       61 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      121 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aataccgttc
      181 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      241 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      301 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      361 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
      421 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
      481 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
      541 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
      601 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
      661 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
      721 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
      781 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
      841 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      901 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      961 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     1021 accatgatta cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat
     1081 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1141 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg
     1201 cgaagaactc cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa
     1261 cgattccgaa gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca
     1321 ggttgggcgt cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc
     1381 aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag
     1441 gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat
     1501 gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc
     1561 attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc
     1621 gtcgggcatg cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc
     1681 ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat
     1741 gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg
     1801 cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc
     1861 ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag
     1921 cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg
     1981 cagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc
     2041 tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc
     2101 gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat
     2161 gcgaaacgat cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg
     2221 gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
     2281 agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
     2341 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat
     2401 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     2461 ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
     2521 ggagcctggg gactttccac accctaactg acacacattc cacagaatta attcgcgtta
     2581 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2641 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2701 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2761 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2821 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2881 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2941 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg
     3001 ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca
     3061 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     3121 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     3181 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     3241 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta aacgggccct
     3301 ctagactcga gttaggcagg ggccacctga gaagtctcgg tcttagacac ggtggtgctg
     3361 gcctcatcga attcaccggt gcgtccaccg gtatcgatct gtctagagcc agcccagtcc
     3421 tggagcttgt ggatctgtcg ctccacgcta atggaggttc tcagggtacc catgctattt
     3481 tccatttgga ttggctctga tcggcagcct cccttttggc acaccccttg gtacacttcc
     3541 cagctcttct tcctaaaccg ttttcccaca ataacataca ccaatgggtt caagcagcta
     3601 ttggagtatg ccatgaagga cgcgatttga gtgataacgt cgatgatcct ttcgtcctga
     3661 caactactca ggattccgag tctgtgcaac gtgtccagga aagtgctgat ctggaaaggc
     3721 agccaacaga taatgaacaa cagcaaaacg accaagacca gcactgtggc ccttcgctcg
     3781 gtctgtatct ccttaaattt ttgcatttcg ttgtttcgca aaacttgcat gatctgcatt
     3841 gtacagaagg taatcacaga aagggggagc aaaaaaccca ccacgttcag cagcatgttt
     3901 gtgaagacct cccaaatcaa agatgggtaa ctgataacgc atgcggtaac attgtggccc
     3961 tcgtcagagt actccttcat cgttcggaag accagcatag gtgaactgag gaggagggtg
     4021 caaccccaaa tcaccaggct atagagctta gcccagcgta cgcccctcat gcggcccatg
     4081 ctcatagtct ttacaagggc gaggtacctg tcaatgctga cgagcataag gaagcagatt
     4141 gagctgtaca gattcatact gataattgca ttcaccacgc gacacaaggt ctcgccaaac
     4201 aaccaatcaa aattattgct gatagtaata gcccagaaag gcaggccaca ggccaaaatg
     4261 agatccgctg cggccaaatt acccaggtaa atttcggcca ccgtacagga gctcttatgg
     4321 aggcaaaaca cagagagaac aaagatgttt tccagcgtgg ccagaacaaa aagaacccaa
     4381 aggaaagggg gctggatagt gttcaaccag ccgagccatt ccacttgagg acatttagac
     4441 tgagcaaatg tcccgttgag agtggggccc tgcagggtca cgttaagcat gtcggcgcta
     4501 aaactcgcag ttgtgggcac gctgtcctcg cgcacagaaa ggaacataga aatcttccag
     4561 ggtgagaaca tatcgatgct ggcgtcatca tcgtccttgt agtcggcgaa taccaggcag
     4621 aagatgtagc tcagggcgat gatcgtcttc atggtggcgc taagcttaag tttaaacgct
     4681 agccagcttg ggtctcccta tagtgagtcg tattaatttc gataagccag taagcagtgg
     4741 gttctctagt tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc
     4801 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
     4861 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
     4921 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
     4981 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     5041 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     5101 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     5161 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     5221 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     5281 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     5341 gtcaacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca
     5401 gattcttcat gcaaatgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc
     5461 actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat
     5521 cagagcagat tgtactgaga gtgcaccata ggggatcggg agatctcccg atccgtcgac
     5581 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5641 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5701 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5761 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5821 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5881 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5941 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6001 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6061 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6121 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6181 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6241 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6301 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6361 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6421 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6481 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6541 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6601 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6661 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgt
//
