LOCUS       sequence-430806-        6665 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 6665)
  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 6665)
  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: GPER-DuET
FEATURES             Location/Qualifiers
     source          1..6665
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     418..437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(656..760)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(835..854)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1026..1405
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1406..1609
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1560..1580
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1654..1673
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1654..1672
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3047..3064)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3053..3277
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3323..3751
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3410..3429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3620..3641
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3760..3780)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3765..4094
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3945..4080
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4007..4026
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4161..4955
                     /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(4215..4234)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4825..4844
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5129..5250
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5166..5185)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5220..5239
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5299..5315)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5299..5315)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5312..5334)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5323..5339
                     /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(5347..5353,5354..5371,5372..5377))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5392..5413
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5530..5547)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5701..6289)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5781..5800)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6460..6665,1..586,587..655))
                     /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"
ORIGIN
        1 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
       61 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
      121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
      181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
      241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
      301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
      361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
      421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
      481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
      541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
      601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
      661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
      721 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
      781 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
      841 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
      901 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acatttgcat
      961 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     1021 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     1081 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     1141 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     1201 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     1261 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     1321 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     1381 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     1441 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     1501 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     1561 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
     1621 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca
     1681 agctggctag cgtttaaact taagcttagc gccaccatga agacgatcat cgccctgagc
     1741 tacatcttct gcctggtatt cgccgactac aaggacgatg atgacgccag catcgatatg
     1801 gacgtaacat cccaagcaag aggagtgggt cttgagatgt acccggggac tgcgcagccc
     1861 gcagctccta acactacttc acccgagctg aacctgtcac accctctcct gggcactgcg
     1921 ctcgcgaacg gaaccgggga gctgtctgag caccagcagt acgtcatagg cctgttcctg
     1981 tcctgtttgt ataccatttt tctcttcccc attgggtttg tggggaacat cctgattctt
     2041 gttgttaaca tcagcttcag ggagaaaatg acgattcctg acttgtactt tatcaacctg
     2101 gccgtcgccg acttgatttt ggtggcggat tctctgattg aggtttttaa cctccacgag
     2161 agatattacg acatcgctgt gctgtgtacc tttatgagcc tctttctgca ggttaacatg
     2221 tattcctcag tgttttttct gacctggatg tcattcgaca gatacatcgc cctcgccagg
     2281 gctatgaggt gctctctgtt ccgcacgaaa caccacgccc ggcttagttg cgggctcatt
     2341 tggatggcct cagtgagcgc aacactggtg ccgtttactg ccgtacacct ccagcataca
     2401 gacgaagcct gtttttgctt cgctgatgtt agggaggtcc aatggctgga agttactctg
     2461 ggtttcatag ttccatttgc catcataggg ctttgctata gcttgatcgt tcgagtactg
     2521 gtgagagcac acagacaccg cggactcagg cctcgccgcc agaaggcact gaggatgatc
     2581 ctcgccgtcg tgctggtatt ttttgtgtgc tggctgccag agaatgtctt cattagcgtt
     2641 catttgctgc agaggactca acctggtgcc gccccctgta agcagagctt caggcacgcc
     2701 caccccctca caggccatat cgtgaatttg gccgcttttt ctaattcctg tctgaaccct
     2761 ctgatctaca gtttcctggg ggaaaccttt agggacaaac tgcgattgta catagaacaa
     2821 aagaccaacc tgccggccct gaatcggttc tgccacgcgg ccctcaaagc tgtgattccg
     2881 gactcaactg agcagagtga tgtccgattt agctctgcgg tcatcgatac cggtggacgc
     2941 accggtgaat tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc tcaggtggcc
     3001 cctgcctaac tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     3061 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3121 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3181 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3241 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3301 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3361 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3421 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3481 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3541 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3601 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3661 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3721 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     3781 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3841 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3901 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3961 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4021 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4081 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     4141 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4201 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4261 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4321 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4381 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4441 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4501 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4561 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4621 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4681 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4741 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4801 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4861 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4921 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4981 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     5041 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5101 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5161 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5221 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5281 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5341 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5401 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5461 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5521 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5581 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5641 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5701 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5761 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5821 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5881 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     5941 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6001 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6061 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6121 taactacggc tacactagaa gaacggtatt tggtatctgc gctctgctga agccagttac
     6181 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6241 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6301 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6361 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6421 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6481 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6541 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6601 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6661 gcgca
//
