LOCUS       sequence-430743-        6536 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 6536)
  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 6536)
  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: ADORA2B-DuET
FEATURES             Location/Qualifiers
     source          1..6536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(31..822,823..891))
                     /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     654..673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(892..996)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1071..1090)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1262..1641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1642..1845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1796..1816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1890..1909
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1890..1908
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3154..3171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3160..3384
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3430..3858
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3517..3536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3727..3748
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3867..3887)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3872..4201
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4052..4187
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4114..4133
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4268..5062
                     /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(4322..4341)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4932..4951
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5236..5357
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5273..5292)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5327..5346
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5406..5422)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5406..5422)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5419..5441)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5430..5446
                     /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(5454..5460,5461..5478,5479..5484))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5499..5520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5637..5654)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5808..6396)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5888..5907)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
       61 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
      121 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
      181 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
      241 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
      301 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
      361 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
      421 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
      481 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
      541 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
      601 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
      661 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
      721 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
      781 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
      841 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
      901 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
      961 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     1021 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     1081 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     1141 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacat ttgcatgaag
     1201 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     1261 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     1321 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     1381 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     1441 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     1501 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     1561 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     1621 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     1681 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     1741 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     1801 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
     1861 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
     1921 ggctagcgtt taaacttaag cttagcgcca ccatgaagac gatcatcgcc ctgagctaca
     1981 tcttctgcct ggtattcgcc gactacaagg acgatgatga cgccagcatc gatatgttgc
     2041 ttgagaccca ggatgccctt tatgtggccc tggaactggt catcgcggct ctcagcgtgg
     2101 ctggaaatgt gctggtgtgc gctgccgttg gaacagcgaa cacccttcag actccaacta
     2161 attattttct cgttagcctg gcagccgccg acgtggctgt aggtctgttc gccattccat
     2221 tcgccatcac tatcagcctc gggttttgca cggacttcta cggatgcctt ttcttggctt
     2281 gcttcgtgct cgttcttact caaagtagca tatttagcct gctggcagtg gccgttgacc
     2341 gatacctggc tatctgcgtg ccactgaggt ataagagcct ggtaaccgga accagagcac
     2401 gcggcgttat cgctgtgctt tgggttctcg ccttcgggat cggcttgact ccttttctcg
     2461 ggtggaactc aaaagatagc gccaccaaca actgcaccga gccttgggat ggaactacca
     2521 atgaaagctg ctgcctcgtg aagtgcctgt tcgagaacgt ggtgcccatg tcctacatgg
     2581 tgtacttcaa cttcttcggc tgcgttttgc cccccttgct gataatgttg gtgatttata
     2641 ttaagatttt tctcgtggcc tgtcgacagc tccagaggac cgagctgatg gaccacagcc
     2701 gaactacgct gcaacgagaa attcacgccg ccaagagcct ggccatgatt gtgggtatct
     2761 ttgcactgtg ttggttgcca gtgcacgcgg tgaattgtgt tactctgttc caaccagctc
     2821 aggggaaaaa caaacccaaa tgggcgatga atatggcaat cctcctgtct catgccaata
     2881 gcgttgtcaa tccaatcgtg tatgcctata gaaaccgcga cttcagatat acctttcaca
     2941 aaatcatatc caggtatctt ctctgtcagg cggatgtgaa gtccggcaat ggccaggctg
     3001 gcgtacagcc tgccttggga gtggggctga tcgataccgg tggacgcacc ggtgaattcg
     3061 atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct gcctaactcg
     3121 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     3181 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3241 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3301 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3361 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     3421 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3481 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3541 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     3601 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     3661 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     3721 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     3781 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     3841 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3901 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3961 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4021 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4081 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4141 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4201 aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc
     4261 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     4321 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     4381 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4441 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4501 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4561 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4621 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4681 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4741 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     4801 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4861 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4921 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4981 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     5041 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     5101 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5161 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5221 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5281 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5341 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     5401 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     5461 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     5521 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5581 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5641 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5701 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5761 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5821 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5881 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5941 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     6001 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     6061 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     6121 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6181 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6241 actagaagaa cggtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6301 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6361 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6421 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6481 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatcta
//
