LOCUS       sequence-431747-        6581 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 6581)
  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 6581)
  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: ADORA3-DuET
FEATURES             Location/Qualifiers
     source          1..6581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(79..183)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(258..277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        449..828
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        829..1032
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     983..1003
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1077..1096
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1077..1095
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2386..2403)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2392..2616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2662..3090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2749..2768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2959..2980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3099..3119)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3104..3433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3284..3419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3346..3365
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3500..4294
                     /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(3554..3573)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4164..4183
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4468..4589
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4505..4524)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4559..4578
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4638..4654)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4638..4654)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4651..4673)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4662..4678
                     /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(4686..4692,4693..4710,4711..4716))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4731..4752
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4869..4886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5040..5628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5120..5139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5799..6581,1..9,10..78))
                     /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     6422..6441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
       61 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
      121 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
      181 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
      241 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
      301 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
      361 caaggcttga ccgacatttg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
      421 cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa
      481 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
      541 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
      601 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
      661 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
      721 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
      781 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
      841 tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
      901 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
      961 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1021 ataagcagag ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat
     1081 acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt agcggccgcg
     1141 ccaccatgaa gacgatcatc gccctgagct acatcttctg cctggtattc gccgactaca
     1201 aggacgatga tgacgccagc atcgatatgc caaataacag cactgcactg tcccttgcta
     1261 atgtcacata catcactatg gagatcttca tcggcctgtg cgctattgtg gggaatgtcc
     1321 ttgtgatctg tgtagtgaaa ctgaacccca gcctgcaaac caccaccttt tactttatcg
     1381 tgagcctcgc actcgctgac atcgctgtgg gagtgctggt gatgcccctg gccatcgtgg
     1441 tatctctggg gatcactatc catttttatt catgcttgtt tatgacttgt ctgcttctga
     1501 ttttcacaca cgcctctatc atgtctctgc tggctatcgc agtggacaga tatcttcggg
     1561 tgaagctcac cgtcaggttc cgcatccctg gcttgccagg gtgcatcctc tccttccagc
     1621 tgaaagtgtg tttcttgcca gtgatgtggc tgttcatcct cctgagtctc gcactgatct
     1681 ctgatgctat ggtaatggac gaaaaagtta agcgctcctt cgtactggat acggcctcag
     1741 ccatttgtaa ctacaacgcc cattataaaa atcacccaaa atactggtgc cgaggatatt
     1801 tcagggacta ctgtaacatt atcgctttct cccccaactc aaccaaccac gtggcgctgc
     1861 gagataccgg aaatcagctc atcgttacca tgagttgcct taccaaggag gatacagggt
     1921 ggtattggtg cggcattcag cgcgacttcg cacgggatga tatggacttt accgaactga
     1981 ttgtaactga tgacaaaggc acacttgcaa atgatttctg gtctggtaag gacctgagcg
     2041 gcaacaagac cagatcttgt aaggcgccca aggtcgtgcg aaaggccgat cggtcccgca
     2101 ccagtatcct gatcatttgc attctgataa ccggactggg aatcatttcc gtaatctctc
     2161 acctcacgaa aagacggaga tcccagagga acagacgagt tggtaataca cttaagccat
     2221 ttagtcgcgt gctgacacca aaggagatgg ctcctacgga gcagatgatc gataccggtg
     2281 gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg
     2341 tggcccctgc ctaatctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     2401 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     2461 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2521 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2581 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     2641 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     2701 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     2761 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2821 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2881 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     2941 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3001 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     3061 agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg
     3121 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3181 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3241 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3301 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3361 cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3421 aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac
     3481 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3541 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3601 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     3661 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     3721 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     3781 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     3841 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     3901 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     3961 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4021 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4081 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4141 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4201 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4261 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4321 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4381 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4441 ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa
     4501 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4561 ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag
     4621 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4681 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4741 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4801 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4861 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4921 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4981 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5041 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5101 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5161 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5221 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5281 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5341 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5401 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5461 aactacggct acactagaag aacggtattt ggtatctgcg ctctgctgaa gccagttacc
     5521 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5581 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5641 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5701 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5761 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5821 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5881 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5941 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6001 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6061 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6121 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6181 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6241 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6301 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6361 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6421 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6481 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6541 gcacccaact gatcttcagc atcttttact ttcaccagcg t
//
