LOCUS       sequence-430749-        6764 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 6764)
  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 6764)
  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: ADRB3-DuET
FEATURES             Location/Qualifiers
     source          1..6764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(181..972,973..1041))
                     /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     804..823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1042..1146)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1221..1240)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1412..1791
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1792..1995
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1946..1966
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2040..2059
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2040..2058
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3538..3762
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3808..4236
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3895..3914)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4105..4126
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4245..4265)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4250..4579
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4430..4565
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4492..4511
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4646..5440
                     /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(4700..4719)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5310..5329
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5614..5735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5651..5670)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5705..5724
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5784..5800)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5784..5800)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5797..5819)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5808..5824
                     /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(5832..5838,5839..5856,5857..5862))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5877..5898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6015..6032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6186..6764,1..10))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6266..6285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
       61 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
      121 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
      181 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
      241 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
      301 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
      361 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
      421 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
      481 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
      541 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
      601 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
      661 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
      721 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
      781 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
      841 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
      901 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
      961 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     1021 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     1081 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     1141 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     1201 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     1261 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     1321 aggcaaggct tgaccgacat ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     1381 cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     1441 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     1501 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     1561 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     1621 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     1681 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     1741 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     1801 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     1861 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     1921 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     1981 tatataagca gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt
     2041 aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag cttagcgcca
     2101 ccatgaagac gatcatcgcc ctgagctaca tcttctgcct ggtattcgcc gactacaagg
     2161 acgatgatga cgccagcatc gatatggccc cgtggcccca tgagaattcc agccttgcac
     2221 cttggcctga ccttccaact ctggcaccaa ataccgccaa cacatccgga ctccctggcg
     2281 tcccctggga agctgctctg gctggggctc tgttggccct tgccgtgctg gctaccgtgg
     2341 ggggcaattt gttggtgatt gttgctatcg cctggacacc acggctccag acaatgacaa
     2401 atgtctttgt gacctctctg gctgctgctg atctggtgat gggtctgctc gtggtccccc
     2461 cagctgcgac cctggcactt accggccatt ggcctcttgg tgcgacaggg tgcgagctct
     2521 ggactagcgt ggatgtcctc tgtgtcacag cctccatcga aactctgtgt gccctcgctg
     2581 tcgacagata tctggccgtg actaatcccc tgaggtatgg agccctggtg acaaagaggt
     2641 gtgccagaac cgctgtggtg ctcgtctggg tggtcagcgc cgccgtgtca ttcgctccca
     2701 taatgtccca gtggtggcgg gtgggagccg acgcggaggc ccagcgctgc cacagcaacc
     2761 ctcgatgctg cgcgttcgct agcaatatgc catacgtgtt gctctctagc tcagtgagtt
     2821 tctatctgcc actcctggtg atgctgttcg tctatgcccg agtcttcgtt gtggcaacaa
     2881 ggcagttgag gctgctcagg ggggagttgg ggaggttccc cccagaggag tccccacctg
     2941 ctccgtctag gtctcttgca cccgctccag ttggtacctg cgcccctcca gaaggtgtgc
     3001 cagcatgcgg taggaggccc gcccgactcc tgcctctgag ggagcacaga gctctgtgta
     3061 ctcttggttt gatcatgggt acttttactc tgtgctggct gcccttcttt ctggcaaatg
     3121 tgttgagagc cttgggaggt ccctctctcg tccctggccc agcgttcctg gccctcaact
     3181 ggctcgggta cgccaatagt gcattcaatc ccttgattta ttgtcgaagt cccgatttcc
     3241 gaagcgcttt ccggagactt ttgtgtagat gcgggagacg actccccccg gaaccatgcg
     3301 ccgctgcacg accagctttg ttcccttcag gcgttcccgc cgcaaggtct tcccctgcac
     3361 agcctcgact gtgtcagagg ttggacggag cttcctgggg cgtcagcatc gataccggtg
     3421 gacgcaccgg tgaattcgat gaggccagca ccaccgtgtc taagaccgag acttctcagg
     3481 tggcccctgc ctaactcgag tctagagggc ccgtttaaac ccgctgatca gcctcgactg
     3541 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3601 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3661 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3721 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3781 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3841 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3901 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3961 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4021 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4081 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4141 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4201 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     4261 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4321 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4381 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4441 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4501 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4561 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4621 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4681 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4741 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4801 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4861 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4921 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4981 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5041 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5101 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5161 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5221 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5281 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5341 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5401 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5461 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5521 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5581 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     5641 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5701 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5761 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5821 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5881 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5941 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6001 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6061 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6121 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6181 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6241 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6301 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6361 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6421 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6481 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6541 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6601 tggcctaact acggctacac tagaagaacg gtatttggta tctgcgctct gctgaagcca
     6661 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6721 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaa
//
