LOCUS       sequence-430830-        6497 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 6497)
  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 6497)
  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: GPR55-DuET
FEATURES             Location/Qualifiers
     source          1..6497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     514..533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(752..856)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(931..950)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1122..1501
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1502..1705
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1656..1676
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1750..1769
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1750..1768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2975..2992)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2981..3205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3251..3679
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3338..3357)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3548..3569
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3688..3708)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3693..4022
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3873..4008
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3935..3954
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4089..4883
                     /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(4143..4162)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4753..4772
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5057..5178
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5094..5113)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5148..5167
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5227..5243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5227..5243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5240..5262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5251..5267
                     /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(5275..5281,5282..5299,5300..5305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5320..5341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5458..5475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5629..6217)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5709..5728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6388..6497,1..682,683..751))
                     /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 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
       61 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
      121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
      181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
      241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
      301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
      361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
      421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
      481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
      541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
      601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
      661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
      721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
      781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
      841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
      901 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      961 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     1021 agctacaaca aggcaaggct tgaccgacat ttgcatgaag aatctgctta gggttaggcg
     1081 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
     1141 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     1201 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     1261 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     1321 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1381 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1441 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     1501 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     1561 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     1621 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     1681 gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg cttactggct
     1741 tatcgaaatt aatacgactc actataggga gacccaagct ggctagcgtt taaacttaag
     1801 cttagcgcca ccatgaagac gatcatcgcc ctgagctaca tcttctgcct ggtattcgcc
     1861 gactacaagg acgatgatga cgccagcatc gatatgtcac agcaaaatac aagtggggac
     1921 tgtctcttcg acggggtcaa cgagctcatg aagacactcc agtttgccgt ccacattccg
     1981 accttcgtgc tgggcctgct gctgaacttg cttgccatcc acgggttctc tacattcctg
     2041 aagaatcggt ggcccgacta cgctgcaacc tcaatctata tgatcaacct tgccgttttc
     2101 gacctgctgc tggttctgtc cctgccattt aaaatggtgc tctctcaggt acaaagcccc
     2161 ttcccatcac tgtgcacact tgtcgagtgc ctgtactttg tctcaatgta tggttccgtg
     2221 tttactatct gtttcatttc catggaccgc ttccttgcaa ttcggtaccc attgcttgtt
     2281 tcacacctga ggtcacctcg caagatcttt gggatatgtt gcacaatctg ggtgctcgta
     2341 tggacaggga gcatccctat atacagcttc cacggtaagg tagagaaata tatgtgtttc
     2401 cacaacatgt ccgatgacac ttggagtgcg aaagtattct ttcctcttga ggtgtttgga
     2461 tttttgctcc ctatgggcat tatgggcttt tgctgtagtc ggtcaattca cattcttctc
     2521 ggtcgaagag accacacaca ggactgggtg caacaaaagg cctgcatata cagcattgcc
     2581 gcctcactgg cggtatttgt ggtgagcttt ctccccgtcc acctggggtt tttcctgcag
     2641 ttcctcgtga gaaactcttt tatcgtggag tgccgggcta aacagtctat atccttcttt
     2701 ttgcaacttt caatgtgctt ttcaaacgtg aactgctgcc tcgatgtctt ttgctattat
     2761 ttcgttatta aagaatttcg catgaacatc cgagcacatc ggcccagccg cgtgcagttg
     2821 gtgctgcagg acactaccat atctcggggt atcgataccg gtggacgcac cggtgaattc
     2881 gatgaggcca gcaccaccgt gtctaagacc gagacttctc aggtggcccc tgcctaactc
     2941 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     3001 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3061 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3121 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3181 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3241 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3301 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3361 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3421 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3481 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3541 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3601 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3661 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3721 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3781 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3841 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3901 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3961 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4021 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     4081 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4141 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4201 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4261 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4321 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4381 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4441 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4501 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4561 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4621 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4681 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4741 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4801 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4861 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4921 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4981 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5041 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5101 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5161 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5221 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5281 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5341 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5401 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5461 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5521 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5581 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5641 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5701 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5761 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5821 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5881 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5941 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6001 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6061 cactagaaga acggtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6121 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6181 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6241 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6301 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6361 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6421 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6481 gatacgggag ggcttac
//
