LOCUS       sequence-430746-        6926 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 6926)
  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 6926)
  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: ADRA2C-DuET
FEATURES             Location/Qualifiers
     source          1..6926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     318..337
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(556..660)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(735..754)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        926..1305
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1306..1509
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1460..1480
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1554..1573
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1554..1572
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2488..2499
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(3208..3225)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3214..3438
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3484..3912
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3571..3590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3781..3802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3921..3941)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3926..4255
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4106..4241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4168..4187
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4322..5116
                     /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(4376..4395)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4986..5005
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5290..5411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5327..5346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5381..5400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5460..5476)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5460..5476)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5473..5495)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5484..5500
                     /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(5508..5514,5515..5532,5533..5538))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5553..5574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5691..5708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5862..6450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5942..5961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6621..6926,1..486,487..555))
                     /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 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
       61 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
      121 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
      181 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
      241 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
      301 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
      361 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
      421 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
      481 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
      541 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
      601 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
      661 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta
      721 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
      781 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
      841 ggcttgaccg acatttgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
      901 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
      961 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1021 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1081 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1141 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1201 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1261 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1321 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1381 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1441 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1501 agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg
     1561 actcactata gggagaccca agctggctag cgtttaaact taagcttagc gccaccatga
     1621 agacgatcat cgccctgagc tacatcttct gcctggtatt cgccgactac aaggacgatg
     1681 atgacgccag catcgatatg gcctcacccg cacttgctgc cgcccttgcc gtggccgccg
     1741 cagctggccc gaatgcgtct ggagccggag agcgagggtc cggtggagtg gcaaacgcat
     1801 caggcgcctc atggggccct ccgcgcggcc agtatagcgc aggcgccgta gcagggctgg
     1861 cagctgtggt aggatttctg attgtattta ccgtggttgg taacgtcctg gtggtaatag
     1921 ccgttcttac ctcccgcgca ctgcgggcac cacaaaacct gtttctcgtg tcacttgcgt
     1981 cagccgatat actggtggca accctcgtga tgccgtttag ccttgccaac gaactgatgg
     2041 catactggta cttcgggcag gtgtggtgtg gagtctatct cgcacttgat gtgctctttt
     2101 gcacgtcttc catcgttcac ctgtgtgcaa tatcactgga cagatattgg tctgtcacac
     2161 aggccgtaga atacaatctg aagcgcaccc ctcggcgcgt caaggccaca attgtggctg
     2221 tgtggctcat tagcgctgtg atctcatttc cgcccctggt gagtctttac cgacaaccag
     2281 atggggcagc ctatccacag tgcggactta atgacgaaac gtggtacata cttagtagct
     2341 gtattgggtc cttttttgcc ccatgcttga tcatgggcct tgtgtatgcg agaatctaca
     2401 gggtggcaaa acttcgcacc agaacactct ccgaaaagag agccccggtc gggcccgatg
     2461 gcgcttcccc gaccacagag aatgggctgg gagctgcagc cggggctggg gaaaacggcc
     2521 actgcgcccc ccctcccgca gacgtcgagc cggatgagag cagtgctgcg gccgaaaggc
     2581 ggagaagaag gggcgctctc agaagagggg ggagacggag ggccggtgca gaaggaggcg
     2641 ctggcggtgc cgacgggcag ggcgctggcc caggagctgc cgagtcaggg gctcttaccg
     2701 cttcaagatc cccggggccc ggaggtcggc tgtctagggc aagcagtcgc tcagtggagt
     2761 tctttctgag taggcggaga cgggcccgga gcagtgtttg ccggcgcaag gtcgcacagg
     2821 cgcgggagaa gcgctttaca ttcgtgctgg cagtggtaat gggagttttc gtgctgtgtt
     2881 ggttcccatt cttcttttca tactcactgt acggcatttg ccgagaggct tgccaagtcc
     2941 ctggccctct gttcaagttc tttttctgga tcggctactg taatagcagt ctcaatccgg
     3001 ttatttacac cgtttttaac caggatttta ggcggtcatt taaacatatc ctgttcaggc
     3061 gcaggcgaag gggcttccga cagatcgata ccggtggacg caccggtgaa ttcgatgagg
     3121 ccagcaccac cgtgtctaag accgagactt ctcaggtggc ccctgcctaa ctcgagtcta
     3181 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3241 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3301 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3361 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3421 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3481 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3541 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3601 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3661 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3721 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3781 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3841 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3901 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3961 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4021 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     4081 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4141 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     4201 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4261 cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     4321 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4381 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4441 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4501 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4561 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4621 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4681 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4741 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4801 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4861 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4921 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4981 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5041 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5101 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5161 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5221 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5281 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     5341 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5401 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     5461 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5521 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5581 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5641 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5701 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5761 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5821 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5881 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5941 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     6001 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     6061 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     6121 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     6181 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     6241 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6301 agaacggtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6361 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6421 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6481 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6541 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6601 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6661 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6721 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6781 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6841 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6901 ccagttaata gtttgcgcaa cgttgt
//
