LOCUS       sequence-430882-        6650 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 6650)
  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 6650)
  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: OPRL1-DuET
FEATURES             Location/Qualifiers
     source          1..6650
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     430..449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(668..772)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(847..866)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1038..1417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1418..1621
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1572..1592
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1666..1685
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1666..1684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3044..3061)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3050..3274
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3320..3748
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3407..3426)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3617..3638
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3757..3777)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3762..4091
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3942..4077
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4004..4023
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4158..4952
                     /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(4212..4231)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4822..4841
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5126..5247
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5163..5182)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5217..5236
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5296..5312)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5296..5312)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5309..5331)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5320..5336
                     /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(5344..5350,5351..5368,5369..5374))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5389..5410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5527..5544)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5698..6286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5778..5797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6457..6650,1..598,599..667))
                     /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 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
       61 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
      121 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
      181 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
      241 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
      301 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
      361 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
      421 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
      481 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
      541 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
      601 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
      661 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
      721 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
      781 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
      841 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
      901 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
      961 cgacatttgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     1021 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     1081 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1141 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1201 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1261 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1321 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1381 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1441 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1501 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1561 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1621 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
     1681 tagggagacc caagctggct agcgtttaaa cttaagctta gcgccaccat gaagacgatc
     1741 atcgccctga gctacatctt ctgcctggta ttcgccgact acaaggacga tgatgacgcc
     1801 agcatcgata tggagcccct gtttccggcc cccttttggg aagtcatcta tggctcacat
     1861 ctgcagggga acctgagtct gctgtccccc aaccattcac tgcttcctcc ccatctgttg
     1921 ctgaacgctt ctcatggagc attcctgcct ctcggattga aggtaactat cgtcggcttg
     1981 tatctcgccg tctgcgtggg cggcctcctg ggtaactgtc tggttatgta cgttattctc
     2041 cggcacacta aaatgaagac agcaacaaac atttacatct ttaatctggc actggctgat
     2101 acactggtat tgttgacgct gcctttccag gggaccgata ttctcctggg attctggcct
     2161 ttcgggaacg ccctgtgtaa gactgtaata gcaatagatt attacaacat gtttactagc
     2221 acattcacgc tgacagccat gagtgtggat agatacgtgg ctatctgcca ccccattcga
     2281 gcactggacg tgcgcacctc ctcaaaagcc caagcagtca atgtggccat ctgggcactg
     2341 gccagtgtgg ttggtgtgcc cgtagcgata atgggcagcg cccaagtgga ggacgaagag
     2401 atcgaatgcc tcgtggagat tccaactcca caggactatt ggggccctgt ctttgccatc
     2461 tgtatctttt tgttctcttt catcgtgcca gtactcgtca tctcagtatg ttacagcctg
     2521 atgattcgca ggctccgagg cgttagactg ctgagtggaa gcagggagaa ggaccgcaac
     2581 ctccgacgca tcacgaggct ggtcctcgtg gttgtggcgg tgttcgtggg ctgttggacc
     2641 cccgtgcagg tcttcgttct cgcccaggga cttggggtgc agccgtcaag cgagacagca
     2701 gtggctatac tgcgcttttg caccgcactg gggtacgtca acagctgcct gaacccaatt
     2761 ctttacgctt ttctcgatga gaatttcaag gcctgcttcc ggaaattctg ttgtgcttca
     2821 gccctgcgac gggatgtaca agtctcagac agagtgaggt caattgccaa ggacgtggca
     2881 ttggcttgta agaccagtga gacagtaccc cggccagcca tcgataccgg tggacgcacc
     2941 ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct
     3001 gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     3061 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3121 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3181 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3241 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3301 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3361 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3421 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3481 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     3541 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     3601 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     3661 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     3721 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
     3781 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3841 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3901 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3961 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4021 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4081 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
     4141 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4201 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4261 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4321 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4381 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4441 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4501 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4561 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4621 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4681 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4741 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4801 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4861 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4921 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4981 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5041 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5101 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     5161 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5221 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     5281 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     5341 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     5401 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5461 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5521 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5581 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5641 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5701 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5761 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5821 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5881 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5941 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6001 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6061 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6121 ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc cagttacctt
     6181 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     6241 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     6301 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6361 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6421 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6481 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6541 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6601 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
//
