LOCUS       sequence-430909-        8183 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 8183)
  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 8183)
  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: GRM7-DuET
FEATURES             Location/Qualifiers
     source          1..8183
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(81..100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        272..651
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        652..855
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     806..826
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     900..919
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        900..918
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3811..3828)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3817..4041
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4087..4515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4174..4193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4384..4405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4524..4544)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4529..4858
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4709..4844
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4771..4790
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4925..5719
                     /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(4979..4998)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5589..5608
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5893..6014
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5930..5949)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5984..6003
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6063..6079)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6063..6079)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6076..6098)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6087..6103
                     /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(6111..6117,6118..6135,6136..6141))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6156..6177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6294..6311)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6465..7053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6545..6564)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7224..8015,8016..8084))
                     /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     7847..7866
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(8085..8183,1..6))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
       61 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
      121 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
      181 aggcaaggct tgaccgacat ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
      241 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
      301 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
      361 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
      421 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
      481 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
      541 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
      601 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
      661 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
      721 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
      781 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
      841 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
      901 aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag cttagcggcc
      961 gcgccaccat gaagacgatc atcgccctga gctacatctt ctgcctggta ttcgccgact
     1021 acaaggacga tgatgacgcc agcatcgatc aggagatgta tgccccccat tctatcagaa
     1081 ttgaagggga cgttactctc ggggggctgt ttccagtgca cgcaaagggc ccctctggtg
     1141 tgccctgcgg cgatataaag cgggaaaatg gaatccatcg gctggaggct atgctgtatg
     1201 cgttggatca gatcaacagt gaccccaacc tgctcccaaa cgtaacgttg ggcgcccgga
     1261 tccttgatac ttgtagcaga gacacatacg ctttggagca gtcactgaca ttcgtgcagg
     1321 ccctgattca aaaggacact agtgatgtcc ggtgtaccaa cggagagccc ccagtatttg
     1381 tgaagccaga aaaggtagtg ggtgtgatcg gagcgtctgg ctcctccgtt agcatcatgg
     1441 tggcaaacat tctcagactc ttccagatcc cacaaattag ctacgccagt accgctcccg
     1501 aattgtctga tgaccggaga tacgactttt tcagccgggt ggtgcccccg gatagcttcc
     1561 aggcccaggc tatggtcgat atcgtcaaag cccttggctg gaactacgtg tctacactgg
     1621 cttccgaagg ttcatacggc gagaagggcg tcgaaagctt cacccagatc agtaaggagg
     1681 caggggggct gtgcatcgcg caatccgtga gaatcccgca agagcgcaag gatagaacga
     1741 tagattttga ccggattatc aagcagttgc ttgatactcc caactcacgg gcagtggtga
     1801 tcttcgccaa cgatgaggac attaagcaga tacttgctgc cgctaagagg gcagaccagg
     1861 taggacactt tttgtgggtc ggcagtgata gctggggttc caagatcaat cctcttcacc
     1921 agcacgaaga tattgccgaa ggggcgataa caatccagcc gaaacgcgcc acagtggaag
     1981 gttttgacgc ctacttcacc agtcgaacct tggaaaacaa caggcgaaac gtgtggtttg
     2041 ccgagtactg ggaggagaat ttcaactgta agcttactat cagtggaagc aagaaggagg
     2101 atacagatcg gaaatgtaca ggacaggagc gcatcgggaa agactcaaac tacgagcaag
     2161 aggggaaggt ccagtttgtg attgatgcag tgtacgctat ggctcatgca ctgcaccata
     2221 tgaacaaaga cttgtgtgcc gattaccggg gagtttgtcc cgagatggag caggcaggtg
     2281 gcaagaagct cttgaaatac atccgcaacg tgaacttcaa cgggagcgct ggaacccccg
     2341 tgatgttcaa taaaaacggc gatgcaccag gccgatacga tattttccaa tatcaaacaa
     2401 ctaacacgag caaccctggg tataggctga tcggtcaatg gacagatgag cttcaactca
     2461 atattgagga catgcagtgg ggaaaaggcg tacgcgaaat accagcctct gtgtgcacac
     2521 tgccatgcaa accagggcag agaaaaaaaa cccagaaagg aaccccctgt tgttggactt
     2581 gcgaaccttg cgacggatat cagtatcagt ttgacgaaat gacgtgtcag cactgtccct
     2641 atgatcagcg gcctaatgaa aataggaccg ggtgtcaaga cattcccata atcaaactgg
     2701 agtggcactc cccatgggca gtaatcccag tgtttctggc tatgttgggg attatcgcaa
     2761 ccatttttgt tatggctaca ttcatccgat acaacgacac tccaatcgtc cgggcaagtg
     2821 gtagagagct gtcctatgtc ctgctcactg gaatcttcct gtgctatatc atcactttct
     2881 tgatgatcgc aaaacctgac gtcgctgttt gctccttcag gagggtgttc ctcggactgg
     2941 ggatgtgtat cagctacgcc gctcttctga caaagaccaa cagaatatat agaatctttg
     3001 aacaagggaa gaagagcgtg accgccccta gactgatctc tcctacatca caactggcca
     3061 tcaccagcag cctgatttca gtgcagctcc tcggagtctt tatctggttc ggggttgacc
     3121 cgcccaacat catcattgac tatgatgagc acaaaacgat gaatccggag caggccaggg
     3181 gtgtgctgaa gtgcgatatc accgacctcc agatcatttg tagcctcggg tacagtattc
     3241 ttttgatggt cacctgtacg gtttatgcaa tcaaaactcg gggggtacct gagaacttca
     3301 acgaggccaa gcctataggc ttcaccatgt ataccacatg tatcgtgtgg cttgctttca
     3361 tacctatctt tttcgggacc gcacaaagcg ccgaaaagct gtacattcaa actacaactt
     3421 tgaccatttc tatgaatctg tctgcgagcg tagctctggg tatgctttac atgcctaagg
     3481 tttacatcat cattttccac cccgaactta atgtacagaa gagaaagaga agttttaaag
     3541 ccgtcgtaac cgccgcaact atgagttcta gactcagcca taagccgtca gaccgaccta
     3601 acggagaggc aaagaccgaa ctctgcgaga acgtcgatcc aaactcccct gctgcaaaga
     3661 agaagtatgt cagctataat aacctcgtta ttatcgatac cggtggacgc accggtgaat
     3721 tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc tcaggtggcc cctgcctaat
     3781 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     3841 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     3901 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     3961 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4021 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     4081 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4141 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4201 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4261 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4321 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4381 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4441 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4501 aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     4561 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     4621 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4681 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4741 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc
     4801 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa
     4861 gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt
     4921 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
     4981 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct
     5041 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga
     5101 actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc
     5161 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg
     5221 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
     5281 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca
     5341 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga
     5401 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc
     5461 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga
     5521 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
     5581 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg
     5641 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct
     5701 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc
     5761 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
     5821 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
     5881 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     5941 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     6001 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
     6061 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6121 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6181 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     6241 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6301 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6361 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6421 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6481 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6541 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6601 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6661 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6721 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6781 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6841 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6901 agaagaacgg tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6961 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7021 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7081 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7141 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7201 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7261 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7321 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7381 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7441 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7501 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7561 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7621 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7681 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7741 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7801 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7861 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7921 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7981 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8041 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8101 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8161 tagaaaaata aacaaatagg ggt
//
