LOCUS       sequence-430735-        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: SSTR5-DuET
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1217..1236)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1218..1236)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1281..1484)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1310..1330)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1485..1864
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2036..2055
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        2130..2234
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2235..2303,2304..3095)
                     /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(2453..2472)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3266..3854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3755..3774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4008..4025
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4142..4163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4178..4183,4184..4201,4202..4208)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4216..4232
                     /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     4221..4243
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4240..4256
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4240..4256
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4305..4426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4316..4335)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4370..4389
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4600..5394)
                     /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(4711..4730)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5321..5340
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5461..5790)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5475..5610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5529..5548)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5775..5795
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5804..6232)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5914..5935)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6126..6145
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    6278..6502
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     6491..6508
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 cagtttgctg gtctgcatca acccgtttgc tgcggcccgg tgtgcgggtg gtgtggcctc
       61 ttgctgctgt cgaatcctgt ctggtcgagg ctcggttgca tctgcgtcct tcgctccgct
      121 cccctttcta aggcaaagta ctttctgaaa gctttgccgg aagttatcag agaggaaccc
      181 atagaggact gggttggcac aggagttggc gtaagacaat ataactacga agaagtagag
      241 tcctgctgat gctggttcct gcggcagtgc tacggcgaga ttgacgatat tcactgtgaa
      301 gaaaggcagc cagcagcccg cgaacaccag caccaccacc aacaccattc tggtcacctt
      361 gcgttcggac cggcgcctca cacagccaac tctgaccccg gcggcccgaa cttttacgac
      421 tatcagcaga tagcaaaggc aaatgaccag cagaggggcg aagaatccca gaacagcggt
      481 gtaaataata aacacagcgc cccaaagtcc tacgggctca ggccaggaag cattacatgt
      541 ccctccctct tgcacgtctg cgaacacgag cagagggagg ctcatacaga gggacagcac
      601 ccatgcagca gcacttgcca gtttcgccac cctaggcctc ctccatcggg cagatgagag
      661 cggatggacc acggcgaggt accgatcaac actcatcact gtcaggcaaa aaacggaggt
      721 aaactgattg acgccatcaa gggtcataac cagcctgcac agcacaggtc cgaaaggcca
      781 gaaagatgcc gcattctgag tggccaggaa cggcaatccg agcatataca gcacgtcggc
      841 tactgccaag ttgagaatat agatattcgt gacagttttc atctttgcaa accgcaacac
      901 tacgtaaatc accagtgtgt tgccgcccaa cccagcggcg cagaccagca ggtacaggac
      961 tgggaccagt acagcccggg ctccggctga aggagcgggg cccaccagcg ttctattgtc
     1021 gccaccaccg ctggctgcgc cgggagaaga agcgttccag ctaggagtgc tggcagggaa
     1081 gagtggttcc atatcgatgc tggcgtcatc atcgtccttg tagtcggcga ataccaggca
     1141 gaagatgtag ctcagggcga tgatcgtctt catggtggcg ctaagcttaa gtttaaacgc
     1201 tagccagctt gggtctccct atagtgagtc gtattaattt cgataagcca gtaagcagtg
     1261 ggttctctag ttagccagag agctctgctt atatagacct cccaccgtac acgcctaccg
     1321 cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg
     1381 tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca
     1441 aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg
     1501 actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa
     1561 tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac
     1621 acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg
     1681 aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg
     1741 tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata
     1801 tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt caataatcaa
     1861 tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc
     1921 agattcttca tgcaaatgtc ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg
     1981 cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa ctatgcggca
     2041 tcagagcaga ttgtactgag agtgcaccat aggggatcgg gagatctccc gatccgtcga
     2101 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2161 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2221 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     2281 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     2341 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     2401 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     2461 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     2521 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     2581 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2641 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2701 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2761 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2821 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2881 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2941 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3001 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3061 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3121 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3181 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3241 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3301 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3361 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatacc gttcttctag
     3421 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3481 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3541 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3601 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3661 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3721 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3781 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3841 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3901 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3961 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4021 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4081 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     4141 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     4201 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     4261 attacgccaa gctctagcta gaggtcgacg gtatacagac atgataagat acattgatga
     4321 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4381 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttgggg tgggcgaaga
     4441 actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg aaaacgattc
     4501 cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg
     4561 gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact cgtcaagaag
     4621 gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg
     4681 gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg
     4741 atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc
     4801 caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg
     4861 catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc
     4921 cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg
     4981 tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc
     5041 atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc
     5101 cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc
     5161 tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc
     5221 attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag
     5281 ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag
     5341 cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa
     5401 cgatcctcat cctgtctctt gatcagatcc gaaaatggat atacaagctc ccgggagctt
     5461 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     5521 agaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     5581 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     5641 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     5701 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     5761 tggggacttt ccacacccta actgacacac attccacaga attaattcgc gttaaatttt
     5821 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5881 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5941 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     6001 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     6061 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     6121 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     6181 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtggggatac
     6241 cccctagagc cccagctggt tctttccgcc tcagaagcca tagagcccac cgcatcccca
     6301 gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc
     6361 agaatagaat gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg
     6421 acagtgggag tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat
     6481 ggctggcaac tagaaggcac agtcgaggct gatcagcggg tttaaacggg ccctctagac
     6541 tcgagttagg caggggccac ctgagaagtc tcggtcttag acacggtggt gctggcctca
     6601 tcgaattcac cggtgcgtcc accggtatcg at
//
