LOCUS       sequence-430741-        6518 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 6518)
  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 6518)
  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: ADORA1-DuET
FEATURES             Location/Qualifiers
     source          1..6518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     522..541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(760..864)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(939..958)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1130..1509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1510..1713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1664..1684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1758..1777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1758..1776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3004..3021)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3010..3234
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3280..3708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3367..3386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3577..3598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3717..3737)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3722..4051
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3902..4037
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3964..3983
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4118..4912
                     /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(4172..4191)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4782..4801
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5086..5207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5123..5142)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5177..5196
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5256..5272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5256..5272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5269..5291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5280..5296
                     /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(5304..5310,5311..5328,5329..5334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5349..5370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5487..5504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5658..6246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5738..5757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6417..6518,1..690,691..759))
                     /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 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
       61 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
      121 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      181 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
      241 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
      301 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
      361 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
      421 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
      481 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
      541 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
      601 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
      661 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
      721 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
      781 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
      841 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
      901 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
      961 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
     1021 aaaatttaag ctacaacaag gcaaggcttg accgacattt gcatgaagaa tctgcttagg
     1081 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
     1141 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
     1201 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     1261 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     1321 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     1381 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     1441 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     1501 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     1561 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     1621 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     1681 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
     1741 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta
     1801 aacttaagct tagcgccacc atgaagacga tcatcgccct gagctacatc ttctgcctgg
     1861 tattcgccga ctacaaggac gatgatgacg ccagcatcga tatgcctccc agtatatccg
     1921 cgttccaagc tgcatacatt gggatcgaag tgcttatcgc acttgtgtct gtacccggga
     1981 atgtgctggt gatttgggcc gtgaaggtta atcaggcact gagagatgct acattttgct
     2041 tcatcgtctc tttggcagtg gccgacgtcg ccgtgggcgc tctcgtcatt cccctcgcca
     2101 tcctgattaa catcggacct cagacctact ttcacacctg tctgatggtc gcatgcccag
     2161 tgcttattct tacccagagc agcatcctgg cacttctggc aatcgccgtt gaccgatacc
     2221 tgagagtaaa gatcccactt cggtacaaaa tggtggtgac cccccggcga gctgctgtag
     2281 ctatagcagg atgttggatc ttgagctttg tagttggact gacaccaatg tttggatgga
     2341 acaatctctc agctgtggag agagcctggg cagccaacgg gtccatgggc gagcccgtca
     2401 ttaagtgtga atttgagaaa gtcatctcta tggagtatat ggtgtacttc aattttttcg
     2461 tgtgggtgct cccgcccctc ctgcttatgg tcctgatcta tctggaggtg ttttatttga
     2521 tccggaaaca actgaataag aaagtttccg ccagttccgg cgatccgcaa aaatactatg
     2581 gtaaagagct gaaaatcgct aagagtctcg ctctcatcct cttcttgttc gctcttagct
     2641 ggcttcccct ccatattctc aactgcataa ctctgttctg cccctcttgt cacaaaccat
     2701 ccatcctgac ttacattgcc atctttctca cccacggaaa ctctgctatg aaccctatcg
     2761 tgtacgcttt tagaattcag aagtttaggg tgactttttt gaagatctgg aacgaccact
     2821 tccggtgtca gcccgcccca cccattgacg aggacctgcc agaggaacgg cctgacgaca
     2881 tcgataccgg tggacgcacc ggtgaattcg atgaggccag caccaccgtg tctaagaccg
     2941 agacttctca ggtggcccct gcctaactcg agtctagagg gcccgtttaa acccgctgat
     3001 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3061 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3121 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3181 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3241 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3301 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3361 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3421 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3481 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3541 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3601 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3661 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     3721 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3781 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3841 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3901 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3961 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     4021 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     4081 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4141 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4201 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4261 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     4321 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4381 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4441 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4501 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4561 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4621 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4681 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4741 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4801 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4861 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4921 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4981 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5041 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     5101 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5161 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     5221 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5281 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5341 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5401 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5461 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5521 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5581 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5641 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5701 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5761 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5821 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5881 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5941 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6001 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6061 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cggtatttgg tatctgcgct
     6121 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6181 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6241 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6301 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6361 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6421 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6481 gcctgactcc ccgtcgtgta gataactacg atacggga
//
