LOCUS       sequence-430822-        6614 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 6614)
  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 6614)
  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: GPR20-DuET
FEATURES             Location/Qualifiers
     source          1..6614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(686..790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(865..884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1056..1435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1436..1639
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1590..1610
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1684..1703
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1684..1702
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3026..3043)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3032..3256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3302..3730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3389..3408)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3599..3620
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3739..3759)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3744..4073
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3924..4059
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3986..4005
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4140..4934
                     /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(4194..4213)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4804..4823
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5108..5229
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5145..5164)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5199..5218
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5278..5294)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5278..5294)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5291..5313)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5302..5318
                     /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(5326..5332,5333..5350,5351..5356))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5371..5392
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5509..5526)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5680..6268)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5760..5779)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6439..6614,1..616,617..685))
                     /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 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
       61 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
      121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
      181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
      241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
      301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
      361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
      421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
      481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
      541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
      601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
      661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
      721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
      781 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
      841 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
      901 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
      961 aacaaggcaa ggcttgaccg acatttgcat gaagaatctg cttagggtta ggcgttttgc
     1021 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     1081 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     1141 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     1201 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     1261 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     1321 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     1381 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
     1441 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
     1501 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
     1561 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
     1621 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga
     1681 aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttagc
     1741 gccaccatga agacgatcat cgccctgagc tacatcttct gcctggtatt cgccgactac
     1801 aaggacgatg atgacgccag catcgatatg cccagcgtga gtcctgctgg cccatctgct
     1861 ggggccgtgc ctaacgcaac tgccgtgacc actgtgcgca ccaatgcgtc aggccttgag
     1921 gtgccactgt tccacctctt cgcacgactc gacgaagagc ttcacggaac ctttccaggc
     1981 ctgtggcttg cgcttatggc ggtgcacggg gccatatttc tcgctgggct ggtgctcaat
     2041 ggcctggcgt tgtacgtatt ctgctgcaga acccgcgcca aaactccgag tgtgatctac
     2101 actatcaacc tggtcgtgac cgatcttctt gtggggctgt ccctccctac aagatttgcc
     2161 gtatattacg gggctcgcgg gtgtctgcgg tgcgcctttc cacacgtgct gggttacttt
     2221 ctcaatatgc attgtagtat cctttttctg acatgcatct gcgtggacag gtacttggct
     2281 atcgttagac cagaaggctc caggcggtgt cgacagccag cctgtgcccg cgcagtgtgt
     2341 gcttttgtat ggctcgcagc tggcgccgta acgctttccg tgcttggcgt gaccgggagc
     2401 agaccttgct gtcgggtctt tgccctcaca gtgctggagt ttctgttgcc tctccttgtg
     2461 atttccgtat tcactggtag gatcatgtgt gcactgtccc ggcccggact tctgcatcag
     2521 ggccggcagc ggagggtgcg ggcaatgcag ctgttgctca cagttctcat catttttctg
     2581 gtgtgcttca cccccttcca tgctagacag gtggctgtgg ctctctggcc agacatgcca
     2641 catcatacct ctctggtggt gtaccatgtg gccgttacac tctccagcct gaactcctgc
     2701 atggacccaa tagtctattg ttttgtgact tcaggatttc aggctacagt gcggggactg
     2761 tttggtcagc atggagaacg ggaaccctcc tcaggagacg tcgtgagcat gcaccgcagc
     2821 agtaagggtt ccggccggca tcacattctg tctgctggac ctcacgcact gactcaagcc
     2881 ctcgctaacg gacctgaagc gatcgatacc ggtggacgca ccggtgaatt cgatgaggcc
     2941 agcaccaccg tgtctaagac cgagacttct caggtggccc ctgcctaact cgagtctaga
     3001 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3061 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3121 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3181 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3241 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     3301 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3361 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3421 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3481 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3541 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3601 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3661 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3721 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3781 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     3841 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3901 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     3961 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     4021 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     4081 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     4141 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4201 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4261 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4321 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4381 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4441 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4501 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4561 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4621 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4681 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4741 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4801 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4861 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4921 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4981 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5041 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5101 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5161 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5221 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     5281 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5341 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5401 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5461 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5521 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5581 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5641 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5701 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5761 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5821 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5881 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5941 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6001 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6061 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6121 aacggtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6181 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6241 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6301 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6361 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6421 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6481 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6541 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6601 agatttatca gcaa
//
