LOCUS       sequence-430808-        7124 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 7124)
  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 7124)
  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: GPR114-DuET
FEATURES             Location/Qualifiers
     source          1..7124
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     200..219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(438..542)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(617..636)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        808..1187
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1188..1391
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1342..1362
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1436..1455
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1436..1454
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3288..3305)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3294..3518
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3564..3992
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3651..3670)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3861..3882
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4001..4021)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4006..4335
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4186..4321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4248..4267
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4402..5196
                     /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(4456..4475)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5066..5085
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5370..5491
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5407..5426)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5461..5480
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5540..5556)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5540..5556)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5553..5575)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5564..5580
                     /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(5588..5594,5595..5612,5613..5618))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5633..5654
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5771..5788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5942..6530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6022..6041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6701..7124,1..368,369..437))
                     /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 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
       61 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
      121 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
      181 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
      241 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
      301 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
      361 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
      421 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
      481 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
      541 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
      601 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
      661 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
      721 aaggcttgac cgacatttgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      781 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      841 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      901 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      961 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1021 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1081 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1141 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1201 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1261 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1321 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1381 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata
     1441 cgactcacta tagggagacc caagctggct agcgtttaaa cttaagctta gcgccaccat
     1501 gaagacgatc atcgccctga gctacatctt ctgcctggta ttcgccgact acaaggacga
     1561 tgatgacgcc agcatcgata tggatcattg cggagcgctg ttcttgtgtc tgtgtctcct
     1621 cactctgcag aatgccacga ccgagacctg ggaggaactc ctcagctaca tggagaatat
     1681 gcaggttagc agaggcaggt cctctgtgtt cagctctaga cagctgcacc agcttgagca
     1741 gatgctcctt aataccagct tccctggcta caatctcacc ctgcagactc caacgattca
     1801 gtcactggca tttaagctga gctgcgactt ttccggcctg agcctgactt ccgcgacact
     1861 caaacgagtc ccgcaggctg gaggccagca cgcacgcggt cagcatgcca tgcagttccc
     1921 cgcggaactt actcgcgacg cgtgtaagac acgacccagg gaactgaggc tgatctgcat
     1981 ttatttttcc aatacacatt tttttaaaga cgaaaataat tctagcttgc tgaacaatta
     2041 cgtgctggga gcccagctca gtcatggcca cgtgaataac cttcgagatc ccgttaacat
     2101 atccttttgg cataaccaga gtctggaagg gtacacactg acctgcgtgt tctggaagga
     2161 gggggctagg aaacaaccat ggggcggatg gtcccctgag ggttgccgca cagagcagcc
     2221 aagtcactct caggtgctgt gccgctgcaa ccatctcaca tacttcgccg tgctcatgca
     2281 gctttcaccc gctctcgtcc ctgccgaact tctcgccccc ctgacgtata tttcactcgt
     2341 gggctgtagc atcagcatag tcgcctctct gatcaccgtg ctgctgcact tccattttag
     2401 aaagcagagc gactctctga cccgcatcca catgaatctc cacgcaagcg tcctgctgct
     2461 caatattgct ttcttgctgt cacccgcttt cgctatgtcc cctgtgccag ggagtgcttg
     2521 cactgccctc gcggcagcgc tccactatgc cctcctgtct tgtctgactt ggatggcaat
     2581 cgagggattt aacctctacc tcctgctggg gagagtatac aatatctaca tccggagata
     2641 cgtgttcaag ctgggcgttc ttggctgggg cgcccctgcg ctgctcgtgc tgttgtcact
     2701 gtccgtgaaa tcatctgtat atggcccatg tactattcct gttttcgaca gctgggagaa
     2761 cggaactggg ttccagaata tgtccatctg ctgggttcgc agcccagtgg ttcactcagt
     2821 gctggttatg ggatacggag gactgacctc tctcttcaac ttggtggtgc tggcatgggc
     2881 cctgtggacc cttcggcgac tgcgggagag agccgacgcc cccagcgtaa gagcttgcca
     2941 cgacaccgtg acagtcctcg gactgacggt tctgctcggt acaacgtggg cactcgcttt
     3001 tttcagcttc ggcgtgtttc tgcttccaca gctgtttctt ttcactatcc tgaattctct
     3061 ctacggcttt ttcctctttc tgtggttttg ctctcagcgc tgccgctccg aggcagaggc
     3121 gaaggcccaa atcgaggctt tctccagtag ccagactact caaatcgata ccggtggacg
     3181 caccggtgaa ttcgatgagg ccagcaccac cgtgtctaag accgagactt ctcaggtggc
     3241 ccctgcctaa ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     3301 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3361 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3421 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3481 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3541 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3601 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     3661 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     3721 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     3781 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3841 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3901 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3961 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     4021 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4081 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4141 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4201 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4261 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4321 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     4381 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     4441 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     4501 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     4561 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     4621 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     4681 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     4741 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     4801 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     4861 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     4921 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     4981 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5041 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5101 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5161 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5221 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     5281 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5341 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5401 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5461 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     5521 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5581 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5641 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5701 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5761 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5821 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5881 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5941 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6001 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6061 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6121 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6181 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6241 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6301 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6361 ctaactacgg ctacactaga agaacggtat ttggtatctg cgctctgctg aagccagtta
     6421 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6481 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6541 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6601 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6661 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6721 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6781 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6841 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6901 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6961 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7021 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7081 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggtt
//
