LOCUS       sequence-430742-        6776 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 6776)
  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 6776)
  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: ADORA2A-DuET
FEATURES             Location/Qualifiers
     source          1..6776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    141..262
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(178..197)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     232..251
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(311..327)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(311..327)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(324..346)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    335..351
                     /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(359..365,366..383,384..389))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    404..425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(542..559)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(713..1301)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(793..812)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1472..2263,2264..2332))
                     /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     2095..2114
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2333..2437)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2512..2531)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2703..3082
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3083..3286
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3237..3257
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3331..3350
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3331..3349
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4835..4852)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4841..5065
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5111..5539
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5198..5217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5408..5429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5548..5568)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5553..5882
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5733..5868
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5795..5814
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5949..6743
                     /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(6003..6022)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6613..6632
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
       61 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
      121 tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca
      181 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
      241 ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg
      301 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
      361 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
      421 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
      481 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
      541 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
      601 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
      661 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
      721 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
      781 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
      841 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
      901 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
      961 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     1021 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     1081 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     1141 gctacactag aagaacggta tttggtatct gcgctctgct gaagccagtt accttcggaa
     1201 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     1261 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     1321 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     1381 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     1441 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     1501 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     1561 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     1621 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     1681 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     1741 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     1801 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     1861 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     1921 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     1981 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     2041 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     2101 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     2161 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     2221 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     2281 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     2341 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     2401 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     2461 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
     2521 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
     2581 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa
     2641 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
     2701 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
     2761 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     2821 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     2881 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     2941 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     3001 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     3061 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     3121 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3181 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     3241 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     3301 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     3361 tggctagcgt ttaaacttaa gcttagcgcc accatgaaga cgatcatcgc cctgagctac
     3421 atcttctgcc tggtattcgc cgactacaag gacgatgatg acgccagcat cgatatgcct
     3481 atcatgggtt ctagcgtgta tatcactgta gagcttgcaa tcgcggtctt ggccatcctc
     3541 gggaacgtcc tcgtgtgttg ggccgtatgg ctgaatagca acctccagaa cgtgaccaat
     3601 tactttgtgg tgtctcttgc cgcggctgac attgccgtag gggtgctcgc gatcccgttc
     3661 gccattacaa tctccaccgg cttttgcgct gcatgtcatg ggtgtttgtt tatagcctgt
     3721 ttcgtcctcg ttctgacaca gtctagcatc ttcagtttgc tggccatcgc cattgatagg
     3781 tatatcgcca tccggatccc acttcgctat aacgggcttg tcacagggac acgcgccaaa
     3841 ggaatcattg caatctgctg ggtgctgtca tttgccatcg ggctgacacc gatgttgggc
     3901 tggaataatt gcggtcagcc gaaggagggc aaaaaccatt ctcagggctg tggggagggt
     3961 caggtcgcct gccttttcga ggacgtagtc cccatgaact acatggtcta ttttaatttt
     4021 ttcgcatgtg tgcttgtgcc gttgctgttg atgctggggg tgtatctcag gatttttttg
     4081 gccgctcgcc gccagctcaa gcagatggaa tctcagccct tgcctggcga gcgcgcccgg
     4141 tccactctcc agaaggaagt gcatgctgca aaaagtctgg ccattattgt aggccttttc
     4201 gccttgtgtt ggctgcccct gcatatcatt aactgcttta cattcttttg cccagactgc
     4261 tcccatgcac ccctgtggct tatgtacctg gcaatcgtgt tgtcccatac gaactccgtg
     4321 gttaacccat tcatatatgc ctataggatt cgggaattta gacaaacatt ccgcaaaatc
     4381 atacgctccc acgttctgag gcaacaggag ccttttaagg ccgcaggtac cagcgcccgc
     4441 gtgctggccg cccacggtag cgacggtgaa caagttagtc tgaggctgaa tggtcatcct
     4501 ccgggcgtgt gggctaacgg cagtgcccct caccctgaaa ggaggcctaa cggttacgct
     4561 ctcggattgg tttctggtgg cagcgcgcag gagagccagg gcaatacagg gctccccgac
     4621 gtggagctcc tgagtcacga gctgaaaggc gtctgccctg agcctcccgg gctggatgac
     4681 cctctggcgc aggatggtgc tggggtttct atcgataccg gtggacgcac cggtgaattc
     4741 gatgaggcca gcaccaccgt gtctaagacc gagacttctc aggtggcccc tgcctaactc
     4801 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     4861 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     4921 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     4981 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     5041 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     5101 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5161 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5221 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5281 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5341 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5401 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5461 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5521 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     5581 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     5641 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     5701 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     5761 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     5821 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     5881 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     5941 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     6001 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     6061 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     6121 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     6181 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     6241 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     6301 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     6361 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     6421 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     6481 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6541 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     6601 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     6661 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     6721 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaag
//
