LOCUS       sequence-430906-        9068 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 9068)
  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 9068)
  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: GRM1-DuET
FEATURES             Location/Qualifiers
     source          1..9068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(2046..2063)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2052..2276
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2322..2750
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2409..2428)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2619..2640
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2759..2779)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2764..3093
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2944..3079
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3006..3025
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3160..3954
                     /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(3214..3233)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3824..3843
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4128..4249
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4165..4184)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4219..4238
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4298..4314)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4298..4314)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4311..4333)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4322..4338
                     /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(4346..4352,4353..4370,4371..4376))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4391..4412
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4529..4546)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4700..5288)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4780..4799)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5459..6250,6251..6319))
                     /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     6082..6101
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6320..6424)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6499..6518)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6690..7069
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7070..7273
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7224..7244
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     7318..7337
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7318..7336
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaatgagtac gtgcaagacg aatttacttg caaggcctgc gatctgggat ggtggcccaa
       61 tgcagatttg acaggttgcg agcccattcc cgtgcgatat ctggagtgga gcaatataga
      121 aagcattatc gcaatcgcct ttagctgtct ggggatactg gtgacactct ttgtgaccct
      181 tatatttgtc ctttaccggg acacccccgt ggtcaaaagt tcttccagag agctgtgcta
      241 catcatcctg gcaggcatct tcctggggta cgtttgtcct tttacgctga tagcaaagcc
      301 caccaccacc tcttgctatc tgcagagact ccttgtagga ctctccagcg caatgtgtta
      361 ttcagctctg gtgacgaaga ccaaccgaat cgccaggatt ctcgccggta gcaaaaagaa
      421 aatctgtacc cgcaaaccaa gattcatgtc agcttgggca caggtaataa ttgccagcat
      481 cctgatctcc gtccagctga ccctcgtagt cactctgatt attatggaac ccccaatgcc
      541 catcctctcc tatccatcaa tcaaggaagt atatctgatt tgtaacacat ccaatctcgg
      601 cgtcgttgct cctttgggtt ataatggcct gttgataatg tcctgtactt actatgcctt
      661 taagacgaga aatgtgccgg ctaatttcaa tgaggctaag tacatcgctt ttaccatgta
      721 tactacctgc attatctggc tggcgttcgt cccgatctac tttggctcca actataagat
      781 tatcactaca tgtttcgccg tgtctctgag tgtcacagtg gctctggggt gtatgtttac
      841 tcccaaaatg tatattataa tcgctaaacc cgagcgcaac gtgcggtctg cttttacaac
      901 cagcgatgta gtgcggatgc atgtcggaga tggcaaactg ccatgtagat caaatacatt
      961 tctgaacatt ttcaggagga agaaagctgg agccggtaac gccaattcta acggtaaatc
     1021 tgtgtcttgg tcagagcccg ggggcgggca agtccccaaa ggacagcata tgtggcatcg
     1081 gctctccgta catgtgaaaa ccaatgagac agcatgcaac cagaccgcgg ttatcaagcc
     1141 tctcacaaag agttaccagg gtagcgggaa gagcctcact tttagcgaca cctcaactaa
     1201 gactctgtat aacgtcgagg aggaagaaga cgcccaacct atcaggttca gcccccccgg
     1261 gtcaccgagt atggtggtgc atcgccgggt tcccagcgcc gctacaactc caccactccc
     1321 atcccatctg actgctgagg aaactccact cttccttgcc gaacctgcac tgcccaaagg
     1381 gttgccccct cctctccagc agcagcagca gcctcctccc cagcagaaga gtttgatgga
     1441 tcaactgcag ggggtggtgt ccaacttttc aaccgcgatc ccggattttc atgcagttct
     1501 cgccgggcca gggggacctg gaaatggtct tcggagcctt tatcccccac caccgccccc
     1561 ccagcacctg cagatgttgc ctttgcaact cagtacgttc ggcgaagagc ttgtgtcccc
     1621 ccccgcagat gacgacgatg actctgagag atttaagctg ctccaggagt acgtgtatga
     1681 gcatgaaaga gagggcaaca ctgaggaaga cgaacttgag gaagaagaag aggatctcca
     1741 ggcggcctct aaactcacgc ctgatgattc tcctgctttg actcccccca gtccgttccg
     1801 cgactccgtg gcctctggat ccagcgttcc ctcttccccc gttagtgaga gcgtgctttg
     1861 caccccgcca aacgtgtcct acgcttcagt aatcctcaga gactacaaac agagcagttc
     1921 tacactcatc gataccggtg gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc
     1981 taagaccgag acttctcagg tggcccctgc ctaatctaga gggcccgttt aaacccgctg
     2041 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2101 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2161 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2221 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2281 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2341 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2401 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2461 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2521 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     2581 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2641 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2701 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     2761 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2821 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2881 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2941 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3001 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3061 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3121 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3181 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3241 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3301 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3361 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3421 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3481 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3541 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3601 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3661 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3721 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3781 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3841 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3901 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3961 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4021 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4081 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4141 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4201 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4261 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4321 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4381 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4441 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4501 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4561 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4621 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4681 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4741 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4801 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4861 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4921 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4981 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5041 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5101 agttcttgaa gtggtggcct aactacggct acactagaag aacggtattt ggtatctgcg
     5161 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5221 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5281 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5341 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5401 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5461 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5521 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5581 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5641 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5701 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5761 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5821 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5881 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5941 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6001 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6061 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6121 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6181 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6241 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6301 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6361 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6421 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     6481 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     6541 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     6601 gcaaggcttg accgacattt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     6661 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     6721 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6781 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6841 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6901 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     6961 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     7021 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     7081 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     7141 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     7201 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     7261 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     7321 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tagcggccgc
     7381 gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt cgccgactac
     7441 aaggacgatg atgacgccag catcgatctg ccccggtcac ctggaaggaa agttctgctc
     7501 gccggagcta gtagccagcg cagcgtggca agaatggacg gagacgttat catcggcgca
     7561 ctgttcagtg tacaccacca accaccagct gaaaaggtgc cagagcggaa atgtggcgag
     7621 attagagagc aatacggaat tcagcgagtt gaggccatgt tccatacact cgacaaaata
     7681 aatgcagacc ctgtcctgct gcctaatatc actttgggaa gcgagatcag agacagctgc
     7741 tggcacagct cagttgcctt ggagcagtct atagagttca tcagggacag cctcatttcc
     7801 atccgcgacg agaaggacgg aataaaccga tgcctgccag acgggcagtc actgccaccc
     7861 ggtagaacta agaaacccat agccggggtt atagggccag gatcctctag cgtggccatc
     7921 caggtacaga atctgctgca actgtttgac atcccccaga tagcctactc cgccacttct
     7981 atagacttga gcgacaagac cctctacaag tacttcctta gggttgtgcc ttccgatacc
     8041 ctccaggcac gcgcgatgct ggatatcgtg aaacgctata attggaccta tgtctctgcc
     8101 gtgcacactg agggtaatta tggtgagagc ggaatggacg cttttaagga gctggctgca
     8161 caggaagggc tgtgtattgc acactccgat aaaatttact ccaatgccgg agaaaagtct
     8221 ttcgataggc tcctgaggaa gctgcgggag aggctgccta aagctagggt ggtggtgtgt
     8281 ttttgtgaag gaatgacagt tagaggactc ctgtcagcca tgcgcagact tggggtggtg
     8341 ggcgaattta gtctcattgg gagcgatggc tgggccgata gggacgaagt tatcgaaggt
     8401 tatgaggtcg aggcgaatgg aggtattact attaaacttc agagccccga ggtgagaagc
     8461 tttgatgact acttcctgaa attgcggctt gatacgaata ccaggaatcc ctggtttcca
     8521 gaattctggc agcatcggtt ccagtgtcgg ttgcccgggc acctgctcga aaaccctaat
     8581 tttaaaagaa tttgcaccgg caacgagagc ctcgaagaga actatgtgca agacagcaag
     8641 atgggttttg tgataaacgc tatatacgca atggctcatg gcctgcagaa tatgcaccac
     8701 gccctgtgcc cgggccacgt cggcctgtgc gatgcaatga aaccgattga tggttctaaa
     8761 cttctggatt tcctgattaa atcctccttc ataggagtga gcggcgaaga ggtgtggttc
     8821 gatgagaaag gagatgcacc tggccgctac gatatcatga atttgcaata cacggaggct
     8881 aacagatatg actacgtgca tgtcgggaca tggcacgaag gcgtgctcaa cattgacgat
     8941 tacaaaatcc agatgaacaa aagcggtgtt gtgagatctg tatgctctga gccatgcctc
     9001 aaaggccaga tcaaagtcat cagaaagggt gaggtgagct gttgttggat ctgcaccgcg
     9061 tgtaagga
//
