LOCUS       sequence-430792-        6821 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 6821)
  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 6821)
  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: EDNRA-DuET
FEATURES             Location/Qualifiers
     source          1..6821
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     499..518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(737..841)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(916..935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1107..1486
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1487..1690
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1641..1661
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1735..1754
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1735..1753
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3284..3301)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3290..3514
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3560..3988
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3647..3666)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3857..3878
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3997..4017)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4002..4331
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4182..4317
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4244..4263
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4398..5192
                     /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(4452..4471)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5062..5081
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5366..5487
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5403..5422)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5457..5476
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5536..5552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5536..5552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5549..5571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5560..5576
                     /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(5584..5590,5591..5608,5609..5614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5629..5650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5767..5784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5938..6526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6018..6037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6697..6821,1..667,668..736))
                     /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 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
       61 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      121 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
      181 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
      241 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
      301 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
      361 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
      421 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
      481 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
      541 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
      601 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
      661 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
      721 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
      781 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
      841 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
      961 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     1021 aggcttgacc gacatttgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     1081 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
     1141 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     1201 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     1261 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     1321 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1381 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1441 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     1501 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     1561 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     1621 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     1681 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
     1741 gactcactat agggagaccc aagctggcta gcgtttaaac ttaagcttag cgccaccatg
     1801 aagacgatca tcgccctgag ctacatcttc tgcctggtat tcgccgacta caaggacgat
     1861 gatgacgcca gcatcgatat ggagacactg tgtctcaggg cctccttctg gctcgcactg
     1921 gtagggtgtg tcatatctga taaccctgaa cggtacagta ccaatctgag taatcatgtc
     1981 gatgacttta caacgtttcg gggcactgag ctgagctttc tcgtgactac gcaccagccc
     2041 accaatctcg tcctgcctag caacgggagt atgcacaact actgccccca gcagacgaaa
     2101 ataacaagcg ccttcaagta tatcaacacc gtgatatcct gcactatctt catagttggc
     2161 atggtgggga acgcgaccct gctccgcatt atttatcaga acaagtgtat gagaaacgga
     2221 ccgaatgcac ttatcgcaag tctggcactc ggagacctga tctacgtcgt cattgatctg
     2281 ccaataaacg tgtttaagct cctggccggc cgctggccct tcgatcacaa cgattttggg
     2341 gtgtttcttt gcaagctctt tccttttttg cagaaatcat ccgtgggcat caccgttctc
     2401 aatctgtgtg cgctgtccgt tgaccgatat cgcgccgtcg caagctggtc ccgggtccag
     2461 ggaatcggaa tcccactggt gaccgctatc gagatcgtgt caatctggat actgtccttt
     2521 atcctcgcga ttccggaggc aatcgggttt gtgatggtcc ctttcgagta tcgaggcgag
     2581 cagcataaaa cctgcatgtt gaatgctaca tcaaagttca tggagtttta tcaggatgtg
     2641 aaagattggt ggcttttcgg attctatttt tgcatgccct tggtttgtac cgcaattttt
     2701 tatactctga tgacatgcga gatgctcaat aggagaaacg gatctctgag gattgcactg
     2761 tccgagcacc tgaaacagcg gcgggaggtt gcgaagactg tcttctgtct ggtcgtgatc
     2821 ttcgctctgt gctggttccc cctgcatctg agccgcattc tcaagaaaac cgtctacaac
     2881 gagatggaca aaaatagatg cgaattgctc tcctttttgc ttttgatgga ctatattggg
     2941 attaaccttg ccaccatgaa ttcctgtatc aacccaattg ctctctactt tgtgtcaaag
     3001 aaattcaaga actgtttcca aagctgtctc tgttgctgtt gctaccagtc taagagtctg
     3061 atgacctcag tgcccatgaa tgggacaagt atccagtgga agaaccatga ccagaataac
     3121 cacaacacag accgctcatc ccataaggat agcatgaaca tcgataccgg tggacgcacc
     3181 ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct
     3241 gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     3301 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3361 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3421 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3481 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3541 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3601 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3661 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3721 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3781 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3841 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3901 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3961 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     4021 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4081 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4141 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4201 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4261 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4321 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     4381 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4441 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4501 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4561 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4621 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4681 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4741 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4801 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4861 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4921 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4981 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     5041 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     5101 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     5161 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     5221 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5281 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5341 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     5401 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5461 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5521 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5581 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5641 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5701 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5761 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5821 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5881 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5941 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6001 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6061 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6121 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6181 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6241 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6301 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6361 ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc cagttacctt
     6421 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6481 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6541 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6601 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6661 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6721 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6781 gataactacg atacgggagg gcttaccatc tggccccagt g
//
