LOCUS       sequence-430908-        8099 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 8099)
  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 8099)
  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: GRM6-DuET
FEATURES             Location/Qualifiers
     source          1..8099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      725..1313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1214..1233
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1467..1484
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1601..1622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1637..1642,1643..1660,1661..1667)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1675..1691
                     /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     1680..1702
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1699..1715
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1699..1715
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1764..1885
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1775..1794)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1829..1848
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2059..2853)
                     /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(2170..2189)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2780..2799
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2920..3249)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2934..3069
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2988..3007)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3234..3254
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3263..3691)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3373..3394)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3585..3604
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3737..3961
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3950..3967
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(6775..6794)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6776..6794)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(6839..7042)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(6868..6888)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        7043..7422
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     7594..7613
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7688..7792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7793..7861,7862..8099,1..554)
                     /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(8011..8030)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
       61 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
      121 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
      181 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
      241 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
      301 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
      361 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
      421 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
      481 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
      541 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
      601 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
      661 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
      721 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
      781 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      841 gcttcagcag agcgcagata ccaaataccg ttcttctagt gtagccgtag ttaggccacc
      901 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      961 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1021 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1081 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     1141 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     1201 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     1261 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     1321 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     1381 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     1441 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     1501 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     1561 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     1621 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     1681 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctctagctag
     1741 aggtcgacgg tatacagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1801 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1861 cattataagc tgcaataaac aagttggggt gggcgaagaa ctccagcatg agatccccgc
     1921 gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga
     1981 aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt
     2041 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     2101 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     2161 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     2221 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     2281 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc
     2341 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     2401 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     2461 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     2521 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     2581 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     2641 ggccagccac gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc
     2701 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     2761 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     2821 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     2881 atcagatccg aaaatggata tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc
     2941 aaaaaagcct cctcactact tctggaatag ctcagaggca gaggcggcct cggcctctgc
     3001 ataaataaaa aaaattagtc agccatgggg cggagaatgg gcggaactgg gcggagttag
     3061 gggcgggatg ggcggagtta ggggcgggac tatggttgct gactaattga gatgcatgct
     3121 ttgcatactt ctgcctgctg gggagcctgg ggactttcca cacctggttg ctgactaatt
     3181 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacaccctaa
     3241 ctgacacaca ttccacagaa ttaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3301 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3361 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3421 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3481 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3541 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3601 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3661 gccgcgctta atgcgccgct acagggcgcg tggggatacc ccctagagcc ccagctggtt
     3721 ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc
     3781 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     3841 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     3901 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     3961 gtcgaggctg atcagcgggt ttaaacgggc cctctagatt aggcaggggc cacctgagaa
     4021 gtctcggtct tagacacggt ggtgctggcc tcatcgaatt caccggtgcg tccaccggta
     4081 tcgattttgt gggcctcggc atcttctcct ttaggaggcg cggcgaccgt tgaggtggct
     4141 ttcagggagc gttttctctt ctgcacgttc tgctctgggt ggaacaaaat aacatacgtc
     4201 ttgggcacat agagcatccc aagtgatacg gaggcactga gggacaagct cacggtgaga
     4261 gtggtagtct ggatgtagat cttctcggcg gactgggcgg tcccaaaaaa gattggaacg
     4321 aatgccagcc aaataatgca tgtggtatac atggtgaagc caatgggctt ggcttcattg
     4381 aatgtctctg gaacgcccct agccttgatg gcatagacgg tgcaagtgac catcaagagc
     4441 aggctatagc cgaggcaacc tatgagagaa aggtcgctca tgtcacactt caggaccccc
     4501 cgcgcctgct cgggatccac tgttcgttgc tcttcatagt caatgacgct gtgcggtggt
     4561 ctagcaccca gccaagcaat cattccgacc acttgcaatg aggtcaaact aaaagtaatg
     4621 accagctggc tagtggggct gataaagggt ggtggtgtta ctgagcgctt tccctgttcg
     4681 aagattctgt atattctgtt agtcttggtg agcagggctg agtagctcag tgtggttccc
     4741 aagccgagaa acagtcgtct agcggcacac acggcggctc ctggttcggc caccatcaga
     4801 aaggtaatag cgtagatcaa gaatattcca gtaagcaaaa cgtatgacag ttcgcggccg
     4861 gaggctctca cgatcggtgt gttgttatag cggacgaaag ttgcaaccac cgtcgtcgtg
     4921 gccacaatcc ccaataccgc aagcagaagt ggaggagctg cccaggggga actccatgac
     4981 agtctaacca caggcgtagg ccgacagccg gtgtggtttg gagtcgggcg catgtcgcct
     5041 ggacatgctt cgcatgtaaa ctcgtccact tggaatctgt acccatcaca agcttcgcag
     5101 tgccagcagc aagggacccc ttttaccatc tttttgcgtt cgcctggccc acaaggcaat
     5161 gaacacaagc tgctaggcac ctcgtgggga tcgccactcc actgcaatgc ctccacatcg
     5221 aggcggagcg tctcggccca ttgccccacc gcctgatatc ccccgctgga ggcggatcca
     5281 ttggtggcct gatactgaaa gatatcgtac cttccagggg catccccatt ttcgttgaac
     5341 atcactgggg ttcccgcgga gccgttgaat cgcacagctc taatatactg caggagcatc
     5401 cgcccatcag tgggctccat ggctggacaa aggccggtgt gtccggggca cagtgcctga
     5461 tgcatgctgt gcagggcgtg agctatagcg tacacggcgt cgatcacgaa ctggaccttg
     5521 ccctcttgct cgtaggtaga atctcgtcca atccgttcct cgccggtgca ttttctggta
     5581 ctgtcatcag attgggtgcc gctactggtc agcttgcagt taaaattttc ttcccagaac
     5641 tctgcgaacc aaatattgcg gcgattattt tcaagggacc tggtcatgaa gtactgatca
     5701 aacccatcaa tacttgctcg cttcgggagg attgtaattg ccccgacggc tacatcctcc
     5761 aaggacaaga ttggacttgt ttttgccccc cagctatcgg atccaaccca caggaaatgg
     5821 ccggtcaggt tcgcttggcg agctgcttcc agcacccttc ggatatcgtc ttcgttggca
     5881 aagatgatga tgccccgagc attaggggtt tccatcagcc gccgaatcac tttgctgaac
     5941 tctcctggtt tgggctctcg tggtatcttg attgactggg cgatgcacac tcccccggcc
     6001 tcccgtgaaa tttgtacgaa ggcctctacc cctgactcac cgtagttgcc ctcagaggcc
     6061 agagtactca cgtagttcca ccccaatgcc ctgacaatat ccaccattgc ctgtgcctgg
     6121 tagctatcag ggggtaccac tcgactgaag aagtcgtacc tagtagaatc agacaattcg
     6181 ggggccgtgg atgcgtagga aatttggggg attgcaaaca ggcgaagtac atttgccacc
     6241 ataatggaga cagaggaagc ggaagcgccc accacggcca ccaccctctc ggggggagca
     6301 ggtctaagtg ggggcacccc gccggggcat ctaactccca cctcgtctcc atcacctctg
     6361 cctctgatca gggcctgcac gaagctaagg gcttgctcca gtgcgtaagt atctcggctg
     6421 caggtatcca gcagtcgggc ccccagccgc accccgggca gcagttcggg gtcggcattc
     6481 accctgtcga gagcatacag catcgcctcc aatctatgca ctccctgttc ctttttcagc
     6541 tgtccgcagg ccctcccagc tgcgcctctt gcatgcacgg ggaacagtcc tccgagggtc
     6601 aagcctccag ccagcctgac gcttccagcg gccctggcga gtccatcgat gctggcgtca
     6661 tcatcgtcct tgtagtcggc gaataccagg cagaagatgt agctcagggc gatgatcgtc
     6721 ttcatggtgg cgcggccgct aagcttaagt ttaaacgcta gccagcttgg gtctccctat
     6781 agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt agccagagag
     6841 ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc aatggggcgg
     6901 agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa ctcccattga
     6961 cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca cgcccattga
     7021 tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag atgtactgcc
     7081 aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc catttaccgt
     7141 cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact gccaagtggg
     7201 cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt ggcgttacta
     7261 tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt cagccaggcg
     7321 ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga actaatgacc
     7381 ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta tatctggccc
     7441 gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg caaatgtcgg
     7501 tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg acctccaaca
     7561 cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt gtactgagag
     7621 tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca cttttcgggg
     7681 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     7741 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     7801 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     7861 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     7921 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     7981 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     8041 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagt
//
