LOCUS       sequence-430832-        6893 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 6893)
  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 6893)
  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: GPR61-DuET
FEATURES             Location/Qualifiers
     source          1..6893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      52..640
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     541..560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     794..811
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    928..949
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(964..969,970..987,988..994)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1002..1018
                     /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     1007..1029
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1026..1042
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1026..1042
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1091..1212
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1102..1121)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1156..1175
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1386..2180)
                     /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(1497..1516)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2107..2126
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2247..2576)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2261..2396
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2315..2334)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2561..2581
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2590..3018)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2700..2721)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2912..2931
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3064..3288
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3277..3294
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4731..4742)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(4896..4915)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4897..4915)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4960..5163)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4989..5009)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5164..5543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5715..5734
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5809..5913
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5914..5982,5983..6774)
                     /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(6132..6151)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
       61 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      121 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      181 gcagatacca aataccgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      241 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      301 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      361 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      421 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      481 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      541 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
      601 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      661 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      721 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      781 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
      841 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      901 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      961 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     1021 tcacacagga aacagctatg accatgatta cgccaagctc tagctagagg tcgacggtat
     1081 acagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     1141 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     1201 aataaacaag ttggggtggg cgaagaactc cagcatgaga tccccgcgct ggaggatcat
     1261 ccagccggcg tcccggaaaa cgattccgaa gcccaacctt tcatagaagg cggcggtgga
     1321 atcgaaatct cgtgatggca ggttgggcgt cgcttggtcg gtcatttcga accccagagt
     1381 cccgctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg
     1441 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata
     1501 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg
     1561 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
     1621 gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct
     1681 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
     1741 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
     1801 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
     1861 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
     1921 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
     1981 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
     2041 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
     2101 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
     2161 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatccgaaa
     2221 atggatatac aagctcccgg gagctttttg caaaagccta ggcctccaaa aaagcctcct
     2281 cactacttct ggaatagctc agaggcagag gcggcctcgg cctctgcata aataaaaaaa
     2341 attagtcagc catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc
     2401 ggagttaggg gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg
     2461 cctgctgggg agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt
     2521 tgcatacttc tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc
     2581 cacagaatta attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     2641 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     2701 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     2761 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     2821 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     2881 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     2941 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     3001 cgccgctaca gggcgcgtgg ggataccccc tagagcccca gctggttctt tccgcctcag
     3061 aagccataga gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct
     3121 tgctgtcctg ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg
     3181 caatttcctc attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag
     3241 gcacggggga ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc
     3301 agcgggttta aacgggccct ctagactcga gttaggcagg ggccacctga gaagtctcgg
     3361 tcttagacac ggtggtgctg gcctcatcga attcaccggt gcgtccaccg gtatcgatgc
     3421 tctccagccg ggggctagcg gcgggtctca ggtagctgtc agacatgatg atatcactgg
     3481 taagttgctg ttccaagaat tcggatgttt cctcggcgat ctgtccaggt atccggaagt
     3541 cgacggctgg tggttcctgc ttaggggatg gcagaggccg actgacccag ctttcgctag
     3601 ggcagccagt tccctgcaga aactggagga agttctcctc gatggacccc tctctactag
     3661 gcagtcgcaa ttcttcctcc ggagcgggtt tgaagaagca cacgaactgc tttgaaagct
     3721 cgccgcggat ctgcctgttc aggcatccgt aaaagaatgg attagatgta aaacagaagt
     3781 agccaatcca tgtcacgacg ctttccacct gcccggtgga gatgggttgg gcggagagag
     3841 caacatagag gtgaaaggag aagtagggca accagcacag caggaattga cctcccactg
     3901 ccagcaatac aactgcggcc ttgccaccgc caaaggtcct gtgtggagtg gtctggggcg
     3961 cgccagagga tgtcaccata gtggagcgac ttgacaggct ctcagatctt tgcctgggcg
     4021 tctccatcca cgtaggcaaa ggcccgtgct gcatcgcggc cacgcgtgcg accctgaaca
     4081 ttgaacagta cacgaccagg atcagcagga ggggcagcag aaagtaaagg acggcgaaca
     4141 cgaccacgaa cagctggcaa taagctgagt ggctccattg caatgagcag cccgggggca
     4201 cggagggggc tccttcttcc caggacactc ttccgaggac aggcactgag gccatggcca
     4261 gtgccttcac ccaaaccccg accaatacgg aagcgaccag gcccagggtc attctgactt
     4321 cataccgcat ggggtgtaca acataataat agcgctccac gtttattgct gacacagaca
     4381 gtatggccag ggacacaaag caaacggaaa ggaacaagta aagccggcat gcaacctcgc
     4441 cgaagagggc gtggtcaaaa agcgctgagc tgctgagcat ggccagaggc atgagtgtca
     4501 gtgcggcgag caaatccacc aggcacagat gaaacacaaa gacaaatttc cgcagtgccg
     4561 gtgtcttagc gataacggcc ataacggcgg cgttgccggc aacggctgtc aagtcgagaa
     4621 gcaacatgaa gaacaaagcg acggattcgg aggccacgtc ccggaggccc acttcaggta
     4681 cgccgctagc tgtggatggt ccgggagtct gtggcacgcg tcccaacgta ctggagttcc
     4741 cactggactg cggtatggga cttgattcca tatcgatgct ggcgtcatca tcgtccttgt
     4801 agtcggcgaa taccaggcag aagatgtagc tcagggcgat gatcgtcttc atggtggcgc
     4861 taagcttaag tttaaacgct agccagcttg ggtctcccta tagtgagtcg tattaatttc
     4921 gataagccag taagcagtgg gttctctagt tagccagaga gctctgctta tatagacctc
     4981 ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg
     5041 aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact
     5101 tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat
     5161 caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa
     5221 ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg
     5281 cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc
     5341 cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat
     5401 tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta
     5461 tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg attactatta
     5521 ataactagtc aataatcaat gtcaacgcgt atatctggcc cgtacatcgc gaagcagcgc
     5581 aaaacgccta accctaagca gattcttcat gcaaatgtcg gtcaagcctt gccttgttgt
     5641 agcttaaatt ttgctcgcgc actactcagc gacctccaac acacaagcag ggagcagata
     5701 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata ggggatcggg
     5761 agatctcccg atccgtcgac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5821 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5881 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5941 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6001 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6061 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6121 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6181 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6241 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6301 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6361 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6421 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6481 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6541 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6601 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6661 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6721 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6781 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6841 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tga
//
