LOCUS       sequence-430728-        6617 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 6617)
  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 6617)
  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: PTGDR-DuET
FEATURES             Location/Qualifiers
     source          1..6617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      267..695
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(354..373)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     564..585
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(704..724)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        709..1038
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      889..1024
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1105..1899
                     /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(1159..1178)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1769..1788
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2073..2194
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2110..2129)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2164..2183
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2243..2259)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2243..2259)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2256..2278)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2267..2283
                     /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)."
     promoter        complement(join(2291..2297,2298..2315,2316..2321))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2336..2357
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2474..2491)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2645..3233)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2725..2744)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3404..4195,4196..4264))
                     /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     4027..4046
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4265..4369)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4444..4463)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4635..5014
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5015..5218
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5169..5189
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5263..5282
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5263..5281
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(join(6608..6617,1..8))
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6614..6617,1..221)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
       61 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
      121 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
      181 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
      241 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
      301 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
      361 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
      421 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
      481 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
      541 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
      601 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
      661 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
      721 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
      781 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
      841 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
      901 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
      961 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     1021 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     1081 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     1141 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     1201 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     1261 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     1321 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     1381 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     1441 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     1501 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     1561 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     1621 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     1681 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     1741 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     1801 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     1861 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     1921 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     1981 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     2041 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     2101 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2161 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     2221 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2281 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2341 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2401 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2461 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2521 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2581 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2641 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2701 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2761 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2821 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2881 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2941 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3001 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3061 ggcctaacta cggctacact agaagaacgg tatttggtat ctgcgctctg ctgaagccag
     3121 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3181 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3241 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3301 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3361 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3421 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3481 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3541 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3601 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3661 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3721 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3781 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3841 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3901 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3961 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4021 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4081 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4141 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4201 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4261 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4321 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4381 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     4441 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     4501 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     4561 catttgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     4621 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     4681 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     4741 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     4801 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     4861 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     4921 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     4981 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     5041 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     5101 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     5161 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     5221 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
     5281 ggagacccaa gctggctagc gtttaaactt aagcttagcg ccaccatgaa gacgatcatc
     5341 gccctgagct acatcttctg cctggtattc gccgactaca aggacgatga tgacgccagc
     5401 atcgatatga agtccccttt ctatcggtgc cagaacacca catctgtaga aaagggtaac
     5461 tctgccgtta tgggtggggt gcttttcagt actggcctgc tgggaaactt gctggcactt
     5521 ggcttgctcg cgaggagcgg actcggctgg tgtagcagaa ggccattgcg gcctctgccc
     5581 tctgtcttct acatgttggt gtgcggactg actgtgacag atctccttgg caagtgcctc
     5641 ctgtccccgg tcgtcctcgc ggcttacgca cagaaccgct ctttgcgggt gctcgccccc
     5701 gcacttgata acagcctgtg ccaggccttt gctttcttca tgagcttctt cgggctctct
     5761 tcaacactgc agttgctggc catggccctg gagtgctggt tgtctcttgg tcacccattc
     5821 ttctaccgac gccacatcac actcagactg ggtgccctgg tggcaccagt ggtaagtgcc
     5881 ttttcactgg ctttctgcgc cctgcccttt atgggctttg gcaagtttgt gcagtattgc
     5941 cctggcacct ggtgtttcat tcaaatggtg cacgaggaag gaagtctctc agtactcggc
     6001 tacagcgtgc tgtacagcag tctgatggcc ctcctggtcc tggcaaccgt gctctgtaat
     6061 ctgggggcta tgcggaatct ctatgccatg catagaaggc tccagcgcca cccccggtca
     6121 tgtactcggg attgcgcaga accacgcgcc gatgggcggg aagcttcccc acagcctctg
     6181 gaagaactgg atcatctcct gctgctggca ctgatgaccg tgctgttcac gatgtgttcc
     6241 cttcccgtga tttatagagc atactatgga gcgtttaagg acgtgaagga gaaaaacaga
     6301 acctccgagg aggccgagga tttgcgagcg ctccggttct tgtccgtcat ctctatcgtg
     6361 gatccatgga tcttcatcat cttccgctct ccagtattta ggatcttttt ccacaagatc
     6421 tttataaggc cactgaggta tagaagtaga tgttcaaaca gcaccaatat ggagtcctct
     6481 ctgatcgata ccggtggacg caccggtgaa ttcgatgagg ccagcaccac cgtgtctaag
     6541 accgagactt ctcaggtggc ccctgcctaa ctcgagtcta gagggcccgt ttaaacccgc
     6601 tgatcagcct cgactgt
//
