LOCUS       sequence-430754-        6599 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 6599)
  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 6599)
  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: BDKBR1-DuET
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(257..1051)
                     /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(368..387)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     978..997
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1118..1447)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1132..1267
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1186..1205)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     1432..1452
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(1461..1889)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1571..1592)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1783..1802
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    1935..2159
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2148..2165
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(3473..3492)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3474..3492)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(3537..3740)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3566..3586)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3741..4120
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4292..4311
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4386..4490
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4491..4559,4560..5351)
                     /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(4709..4728)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5522..6110
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6011..6030
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6264..6281
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6398..6419
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6434..6439,6440..6457,6458..6464)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6472..6488
                     /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     6477..6499
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6496..6512
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6496..6512
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    join(6561..6599,1..83)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6572..6591)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
       61 ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag atccccgcgc
      121 tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag
      181 gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg
      241 aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg
      301 aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct
      361 cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc
      421 ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg
      481 catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga
      541 acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac
      601 cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc
      661 aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct
      721 cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc
      781 agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg
      841 ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg
      901 tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc
      961 agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag
     1021 aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat
     1081 cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct aggcctccaa
     1141 aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg gcctctgcat
     1201 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     1261 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     1321 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     1381 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     1441 gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct cattttttaa
     1501 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     1561 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     1621 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     1681 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     1741 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     1801 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     1861 cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc agctggttct
     1921 ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat tgtcttccca
     1981 atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac acctactcag
     2041 acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg caccttccag
     2101 ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag aaggcacagt
     2161 cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaggcag gggccacctg
     2221 agaagtctcg gtcttagaca cggtggtgct ggcctcatcg aattcaccgg tgcgtccacc
     2281 ggtatcgatg ttcctccaga acaactgaaa aatctccttt cgatgactag aggaaatggg
     2341 ggcaaggctc ttcggtgtgc attgcttgta cagctcccag accttggtcc gaaacagtcg
     2401 ccccacaaag acataaataa cgggattcag tgaggagtta gtgaaggcga aaaaattagc
     2461 caactggagt cccaggtcaa tgaagtcttc ccagaaacac cctcgcacgg cctgcacttg
     2521 aaacaggaac tccagaaagg cgaagaaatg gtagggagcc caacatacaa ggaaagccac
     2581 cacaagggtc agaatgagag ctgtggtttt gctatctttt ctcccgccac agcgtgtgcg
     2641 gctcacctct tccctcgtgc gcagtgacgc aaggatgtga tagttgaaga atacaatcgc
     2701 cgccaggggc aggagaaagc ccagaatatt gagttccaca atccttgcaa aatgccaggc
     2761 ctcgtgaggc aacagcagga tgcaggccgt tatgttgagg tcgggcacgg cttgaattga
     2821 gcgaagcaaa aaggttggaa tgctgagcaa acccccgacc acccagatca gaacgcatgt
     2881 cacccgtgcc tgccggcgcc tctgctgcct ccgagaggcc atagggtgaa ccagaactct
     2941 atacctgtct tgactgatag ccactaccaa aaagatgcta atgaacagat tcgccttgat
     3001 gactccgttg atcactctgc agaggagggc tccgaacggc caattaaact ggttccatat
     3061 gttttcagcc cagaatggca ggcccaacac aaagaccagg tcagaagcag cgaggttggc
     3121 caagtaaatt tctgcgacgt taagctgtcg gcggggcagc aaaaatacga ggagtacgaa
     3181 gaggtttccg agcaggccaa aaaagcagat tgagatgata aatgtcggca aaactctatg
     3241 cagcaaatcc cacgcctctg gggcattgtc gcaggcagta gcgttttgcg ggaacagctg
     3301 agattggttg gaagactgca gctccaaagg gggccaggag ctggccatat cgatgctggc
     3361 gtcatcatcg tccttgtagt cggcgaatac caggcagaag atgtagctca gggcgatgat
     3421 cgtcttcatg gtggcgctaa gcttaagttt aaacgctagc cagcttgggt ctccctatag
     3481 tgagtcgtat taatttcgat aagccagtaa gcagtgggtt ctctagttag ccagagagct
     3541 ctgcttatat agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag
     3601 ttgttacgac attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg
     3661 tcaatggggt ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg
     3721 tactgccaaa accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa
     3781 gtaggaaagt cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca
     3841 ttgacgtcaa tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca
     3901 gtttaccgta aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg
     3961 ggaacatacg tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg
     4021 ccatttaccg taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc
     4081 gtaattgatt actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt
     4141 acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca aatgtcggtc
     4201 aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca
     4261 caagcaggga gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4321 caccataggg gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa
     4381 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4441 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4501 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4561 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4621 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4681 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4741 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4801 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4861 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4921 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4981 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     5041 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     5101 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     5161 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     5221 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     5281 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     5341 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     5401 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     5461 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     5521 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5581 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5641 tcagcagagc gcagatacca aataccgttc ttctagtgta gccgtagtta ggccaccact
     5701 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5761 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5821 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5881 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5941 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     6001 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     6061 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     6121 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     6181 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     6241 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa
     6301 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     6361 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     6421 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     6481 gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc tagctagagg
     6541 tcgacggtat acagacatga taagatacat tgatgagttt ggacaaacca caactagaa
//
