LOCUS       sequence-430771-        7310 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 7310)
  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 7310)
  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: CHRM3-DuET
FEATURES             Location/Qualifiers
     source          1..7310
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    144..265
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(181..200)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     235..254
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(314..330)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(314..330)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(327..349)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    338..354
                     /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(362..368,369..386,387..392))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    407..428
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(545..562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(716..1304)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(796..815)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1475..2266,2267..2335))
                     /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     2098..2117
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2336..2440)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2515..2534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2706..3085
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3086..3289
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3240..3260
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3334..3353
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3334..3352
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5372..5389)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5378..5602
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5648..6076
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5735..5754)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5945..5966
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6085..6105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6090..6419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6270..6405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6332..6351
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6486..7280
                     /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(6540..6559)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7150..7169
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
       61 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
      121 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
      181 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
      241 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
      301 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      361 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      421 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
      481 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
      541 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      601 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
      661 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      721 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      781 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
      841 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
      901 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
      961 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1021 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1081 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1141 acggctacac tagaagaacg gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1201 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1261 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1321 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1381 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     1441 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1501 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1561 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1621 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     1681 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     1741 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     1801 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     1861 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     1921 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     1981 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2041 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     2101 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2161 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2221 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     2281 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     2341 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     2401 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     2461 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     2521 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     2581 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acatttgcat
     2641 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     2701 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     2761 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     2821 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     2881 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     2941 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     3001 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     3061 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     3121 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     3181 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     3241 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
     3301 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca
     3361 agctggctag cgtttaaact taagcttagc gccaccatga agacgatcat cgccctgagc
     3421 tacatcttct gcctggtatt cgccgactac aaggacgatg atgacgccag catcgatatg
     3481 acgctgcata ataattcaac cacaagccct ctgttcccta acatcagtag tagctggata
     3541 cactctccta gcgacgccgg acttcctccc ggaaccgtga cccactttgg gtcatataac
     3601 gtctctcggg ccgcaggcaa tttttcttcc ccagatggga caaccgacga tccgctcggc
     3661 ggccacactg tatggcaggt ggttttcata gcctttctga ccggcattct ggctctggtc
     3721 acaatcatcg gcaatattct ggtaattgtg tccttcaagg taaacaaaca actgaagact
     3781 gtcaataact actttctgct tagtttggca tgcgccgatc tgattatcgg agtgataagt
     3841 atgaatctct tcactactta catcattatg aacagatggg ctctgggtaa cttggcttgc
     3901 gacctgtggc tcgcaattga ctatgtcgca agcaacgcca gtgtgatgaa ccttctggtt
     3961 attagcttcg accgctattt tagcattaca cgccctctga cctacagagc gaaacgcact
     4021 acaaagcggg caggagttat gatcggcctg gcctgggtca tttccttcgt gctctgggca
     4081 ccagccatct tgttctggca gtatttcgtc ggtaagagga cagtcccacc aggcgagtgc
     4141 ttcatccaat ttttgtccga gccaaccata acattcggca cagccatcgc ggcattttac
     4201 atgccagtga ctatcatgac cattctgtac tggagaatct acaaagaaac cgaaaagcgg
     4261 acaaaggagt tggcagggct tcaggcatca ggcacagagg ctgaaacaga aaacttcgtg
     4321 cacccaacag ggagttcacg cagttgcagc tcctacgagc tgcagcagca gagtatgaag
     4381 cgctcaaaca gaagaaagta cggcaggtgc cacttttggt ttactacaaa gagctggaag
     4441 ccaagctctg aacagatgga tcaagatcat agctcatcag actcatggaa taataatgac
     4501 gccgccgcta gtctggagaa cagtgccagt agcgatgagg aggacatcgg tagtgaaacc
     4561 agggcgatat atagtatcgt gcttaagctg ccaggtcatt caactatact gaattctacc
     4621 aaacttccca gtagcgataa tcttcaggtg cctgaggagg agcttggcat ggtcgacttg
     4681 gagcgcaagg cggataaact gcaggctcag aagtcagttg acgatggagg ctctttccct
     4741 aagtcatttt ccaaactccc aatccagttg gagagcgccg tcgacaccgc aaaaacgagc
     4801 gatgtgaatt catccgttgg caaaagcaca gcaactttgc ctctttcctt caaagaggcc
     4861 acactcgcta agcgattcgc actcaaaacc cggtcacaga taaccaagcg caagcgaatg
     4921 tcactcgtga aagaaaaaaa agcagcacag acattgagcg ccatcttgct cgcttttatc
     4981 ataacatgga caccctacaa tatcatggtc ctcgtgaaca ctttctgcga ctcctgtatt
     5041 cctaagactt tctggaacct cggctactgg ctgtgctata tcaacagtac agttaaccct
     5101 gtgtgctacg ctctctgtaa caagaccttt aggaccacgt tcaagatgct cctcctgtgc
     5161 cagtgtgaca aaaagaaacg acgaaagcag caatatcagc agcgacagag tgtgatcttt
     5221 cataaacggg cgccagaaca agcgctcatc gataccggtg gacgcaccgg tgaattcgat
     5281 gaggccagca ccaccgtgtc taagaccgag acttctcagg tggcccctgc ctaactcgag
     5341 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     5401 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5461 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     5521 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     5581 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     5641 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     5701 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5761 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5821 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5881 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5941 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     6001 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     6061 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     6121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     6181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     6241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     6301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     6361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     6421 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     6481 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     6541 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     6601 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     6661 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     6721 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     6781 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     6841 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     6901 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     6961 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     7021 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     7081 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     7141 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     7201 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     7261 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
//
