LOCUS       sequence-430740-        6854 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 6854)
  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 6854)
  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: VIPR2-DuET
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(690..709)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(691..709)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(754..957)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(783..803)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        958..1337
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1509..1528
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1603..1707
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1708..1776,1777..2568)
                     /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     complement(1926..1945)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2739..3327
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3228..3247
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3481..3498
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3615..3636
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3651..3656,3657..3674,3675..3681)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3689..3705
                     /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)."
     primer_bind     3694..3716
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3713..3729
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3713..3729
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3778..3899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3789..3808)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3843..3862
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4073..4867)
                     /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(4184..4203)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4794..4813
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4934..5263)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4948..5083
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5002..5021)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5248..5268
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5277..5705)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5387..5408)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5599..5618
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5751..5975
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5964..5981
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 ttccggaact agaatacaac acgtcatcct tcaccaacac gcttattgcc ctcaggataa
       61 atgacaaaaa gagattaaga tggatgtagt tcctggtgca gtgcagtttc cgaaagaggc
      121 acagaatgat actgcctgtg gccagactca tcagactaac gctgtatccg agggtgtaga
      181 tggctttaac aaggatataa aatgtgattt tgctctcatc ctctggatcg ctgtacccgc
      241 atgcatccac aaagtctgga aatgtctcgc tccaaccatc agatgtgcag ttcttagaga
      301 tattcccggc tttagagtaa aagttagaaa aaacttttgg acatggcacg gtaactgtct
      361 ccccgacgtt ggctggtctc caacaggtga tgttatccca cactccgctg caggccttgt
      421 gcttctcggt ctggcttcta aggagttcgg cgcacttagt ttcctcctcc tggatctcca
      481 ggtggaagcg gcactcgggg tggattgaat ttacaggcgc caagagccag catgtcaaca
      541 aggcaggtgg caggagcgtc cgcatatcga tgctggcgtc atcatcgtcc ttgtagtcgg
      601 cgaataccag gcagaagatg tagctcaggg cgatgatcgt cttcatggtg gcgctaagct
      661 taagtttaaa cgctagccag cttgggtctc cctatagtga gtcgtattaa tttcgataag
      721 ccagtaagca gtgggttctc tagttagcca gagagctctg cttatataga cctcccaccg
      781 tacacgccta ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc
      841 ccgttgattt tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa
      901 tccccgtgag tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat
      961 ggtaatagcg atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat
     1021 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
     1081 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
     1141 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
     1201 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
     1261 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
     1321 agtcaataat caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg
     1381 cctaacccta agcagattct tcatgcaaat gtcggtcaag ccttgccttg ttgtagctta
     1441 aattttgctc gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct
     1501 taactatgcg gcatcagagc agattgtact gagagtgcac cataggggat cgggagatct
     1561 cccgatccgt cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1621 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1681 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1741 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1801 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1861 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1921 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1981 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     2041 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2101 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2161 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2221 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     2281 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2341 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2401 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2461 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2521 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2581 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2641 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2701 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2761 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2821 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2881 accgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2941 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3001 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3061 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3121 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3181 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3241 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3301 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3361 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3421 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3481 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     3541 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     3601 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     3661 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     3721 agctatgacc atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa
     3781 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3841 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg
     3901 gggtgggcga agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc
     3961 cggaaaacga ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt
     4021 gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga
     4081 actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
     4141 gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
     4201 acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
     4261 agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
     4321 cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct
     4381 gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
     4441 gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
     4501 gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
     4561 ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
     4621 cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct
     4681 cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
     4741 cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
     4801 catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
     4861 caatcatgcg aaacgatcct catcctgtct cttgatcaga tccgaaaatg gatatacaag
     4921 ctcccgggag ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga
     4981 atagctcaga ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat
     5041 ggggcggaga atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg
     5101 ggactatggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     5161 ctggggactt tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc
     5221 ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt
     5281 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     5341 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     5401 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     5461 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     5521 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     5581 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     5641 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     5701 cgcgtgggga taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc
     5761 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
     5821 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
     5881 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     5941 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac
     6001 gggccctcta gactcgagtt aggcaggggc cacctgagaa gtctcggtct tagacacggt
     6061 ggtgctggcc tcatcgaatt caccggtgcg tccaccggta tcgatgatga ctgatgtctc
     6121 agtttgaagg aaggactggg ctcgggaacc cctgtgaaac tgcagggcgc cctcggatcc
     6181 attgcgagag aaagaagaac cacagaccct ataatctctt gaggcactgg gggtaggaca
     6241 cctggagcgc cacttcctct tcaactcgca ctgcacctcg ctgttcagga agcagtaaag
     6301 cacggcgact accagaccct gaaatgaccc cagacacagt tcgaacagaa tttggtattt
     6361 acttgaaata ctaataggga atactgcaaa caccatgtag tggacgccaa aaagaggtat
     6421 caacaggagg gtgctcttag ccaaccgttt gtattggctt tggtcgttac cgcccacatc
     6481 agggctagta agcttctgga gcaagatccg aatgatacta ataaacagga caaaattcac
     6541 aatgatgcta atcaggattg gtatccgaat aacccaccat ggcacggaat ggtcattagt
     6601 gtcccaacag ccggtgtctt ccagatagag ccgggccgca gtccatgccc caatgcagac
     6661 ggtaggaagg ccccatccaa tgagcaggta ggcaagaaag caccttcgtg gtggcagcat
     6721 agccaccaac aaagtgtgga ggtagagccc ctccaccaac agccagaaga aattggccat
     6781 aatacaatat tgcaggaaca ccagactgag cttgcaccct acccaactgg atggctgatc
     6841 gggacagtgc aggg
//
