LOCUS       sequence-430750-        6617 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 6617)
  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 6617)
  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: AGTR1-DuET
FEATURES             Location/Qualifiers
     source          1..6617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..29)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     64..83
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(143..159)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(143..159)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(156..178)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    167..183
                     /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(191..197,198..215,216..221))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    236..257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(374..391)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(545..1133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(625..644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1304..2095,2096..2164))
                     /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     1927..1946
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2165..2269)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2344..2363)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2535..2914
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2915..3118
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3069..3089
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3163..3182
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3163..3181
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4508..4525)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4514..4738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4784..5212
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4871..4890)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5081..5102
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5221..5241)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5226..5555
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5406..5541
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5468..5487
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5622..6416
                     /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(5676..5695)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6286..6305
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    join(6590..6617,1..94)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
       61 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
      121 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      181 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
      241 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
      301 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
      361 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      421 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      481 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      541 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      601 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      661 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      721 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      781 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      841 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      901 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      961 ggcctaacta cggctacact agaagaacgg tatttggtat ctgcgctctg ctgaagccag
     1021 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     1081 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1141 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1201 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     1261 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     1321 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     1381 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     1441 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     1501 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     1561 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     1621 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     1681 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     1741 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     1801 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     1861 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     1921 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     1981 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     2041 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     2101 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     2161 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     2221 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     2281 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     2341 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     2401 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     2461 catttgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     2521 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     2581 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     2641 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     2701 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     2761 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     2821 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     2881 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     2941 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     3001 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     3061 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     3121 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
     3181 ggagacccaa gctggctagc gtttaaactt aagcttagcg ccaccatgaa gacgatcatc
     3241 gccctgagct acatcttctg cctggtattc gccgactaca aggacgatga tgacgccagc
     3301 atcgatatga tcctcaactc ctcaactgaa gacggtataa aaagaatcca ggatgattgc
     3361 ccaaaagcag ggcggcataa ctacatattc gtgatgatcc ccactcttta tagtattatc
     3421 tttgtggtgg gcatcttcgg gaattcactc gttgtgatcg tgatttactt ttatatgaag
     3481 cttaagacgg tggcctccgt tttcctgctg aatctcgctt tggccgacct gtgctttctt
     3541 ctcaccttgc ccttgtgggc cgtgtacacc gcgatggagt accgatggcc tttcggtaac
     3601 tacctgtgta agatcgctag cgccagcgta tcttttaatc tttatgcctc tgttttcctc
     3661 ctcacatgcc tgtctattga tcggtatctg gccatcgtac atccgatgaa atcaagactt
     3721 aggaggacaa tgctggttgc taaggtaacc tgtattatta tttggctgct ggcaggtctg
     3781 gcttccctgc ctgcaattat tcatcgcaac gtctttttca tcgagaacac caacattacc
     3841 gtctgcgcct ttcactacga gagtcaaaat tccacgctcc caataggact gggcctgaca
     3901 aaaaacattc tcggttttct ctttccattc ctcatcatct tgacctcata taccttgatc
     3961 tggaaggcac tgaaaaaggc ctatgagatc cagaaaaaca aacccagaaa cgacgatatt
     4021 tttaaaatca tcatggctat tgtgctgttt tttttcttca gttggatccc acaccagatc
     4081 tttaccttcc ttgacgtctt gattcaactg ggcatcatca gagattgccg cattgccgac
     4141 atcgtggaca cggccatgcc cattacgatt tgcatcgcct actttaataa ctgccttaat
     4201 cccctgttct acgggttcct cggcaagaaa ttcaagcggt actttcttca gctgctcaaa
     4261 tacatcccgc ccaaggcaaa aagccactca aatctgtcaa ccaagatgag tacactgagt
     4321 tacagaccat ccgacaacgt gagcagttct accaagaaac ccgcaccatg tttcgaggtc
     4381 gaaatcgata ccggtggacg caccggtgaa ttcgatgagg ccagcaccac cgtgtctaag
     4441 accgagactt ctcaggtggc ccctgcctaa ctcgagtcta gagggcccgt ttaaacccgc
     4501 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4561 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4621 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4681 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     4741 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     4801 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4861 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4921 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4981 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5041 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5101 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     5161 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     5221 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5281 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     5341 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     5401 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5461 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     5521 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     5581 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     5641 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     5701 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     5761 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     5821 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     5881 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     5941 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     6001 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     6061 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     6121 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     6181 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     6241 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     6301 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     6361 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     6421 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     6481 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     6541 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     6601 tgcagcttat aatggtt
//
