LOCUS       sequence-430880-        6656 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 6656)
  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 6656)
  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: OPRD1-DuET
FEATURES             Location/Qualifiers
     source          1..6656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     537..556
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(775..879)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(954..973)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1145..1524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1525..1728
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1679..1699
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1773..1792
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1773..1791
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3157..3174)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3163..3387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3433..3861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3730..3751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3870..3890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3875..4204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4055..4190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4117..4136
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4271..5065
                     /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(4325..4344)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4935..4954
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5239..5360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5276..5295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5330..5349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5409..5425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5409..5425)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5422..5444)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5433..5449
                     /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(5457..5463,5464..5481,5482..5487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5502..5523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5640..5657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5811..6399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5891..5910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6570..6656,1..705,706..774))
                     /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 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
       61 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
      121 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
      181 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
      241 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
      301 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
      361 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
      421 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
      481 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
      541 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
      601 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
      661 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
      721 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
      781 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
      841 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
      901 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
      961 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
     1021 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga catttgcatg
     1081 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
     1141 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
     1201 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     1261 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     1321 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     1381 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     1441 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     1501 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
     1561 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     1621 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
     1681 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
     1741 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
     1801 gctggctagc gtttaaactt aagcttagcg ccaccatgaa gacgatcatc gccctgagct
     1861 acatcttctg cctggtattc gccgactaca aggacgatga tgacgccagc atcgatatgg
     1921 aaccagcccc tagtgcaggc gcagaattgc agccccctct gtttgccaac gcaagtgacg
     1981 cctacccttc cgcgtgcccc agcgcagggg ctaacgccag cggccctccg ggggcaagaa
     2041 gcgctagcag tctggccctg gctattgcca ttaccgccct gtattccgcc gtgtgtgcag
     2101 tgggacttct gggtaacgtg ctggtgatgt tcgggattgt gcgctacacc aagatgaaaa
     2161 ccgcaaccaa catttacatt tttaacctgg ctttggccga cgcactggcc accagtaccc
     2221 tgccattcca aagcgccaaa tacctgatgg aaacgtggcc gtttggggag ttgctgtgta
     2281 aggccgtgct gagcattgat tactacaaca tgtttacctc catctttact ctgacaatga
     2341 tgagtgtgga tcggtatatt gcagtctgcc acccagtcaa ggccctggat ttccggactc
     2401 ccgcaaaggc caaactgata aatatatgta tctgggtcct ggcctcaggc gtgggagtac
     2461 ccattatggt gatggctgtg acacggcccc gggatggggc cgttgtctgc atgcttcaat
     2521 ttcccagccc ctcctggtac tgggataccg tcactaagat ctgtgtcttc ctgtttgcgt
     2581 ttgtggtacc aatcctgatc ataacggtct gctacgggct gatgttgctg aggctgagga
     2641 gtgtgaggct cctgagtggg agtaaagaaa aagatcgaag tctgcgccgg attacacgga
     2701 tggttctggt agtggtgggc gcgttcgtgg tgtgttgggc ccccatccac atattcgtga
     2761 ttgtgtggac attggtcgac attgaccgcc gagatcctct ggtggtagct gctctccatc
     2821 tgtgcatcgc cttgggctat gccaatagtt ctctgaatcc ggtgctgtat gccttcctgg
     2881 acgagaactt caagagatgt tttcggcagc tgtgtaggaa gccttgtggg cgacccgacc
     2941 cttcaagctt cagccgagct cgcgaagcaa cagcaagaga gagggtaacc gcctgtactc
     3001 ccagcgatgg gcccgggggt ggagccgccg cgatcgatac cggtggacgc accggtgaat
     3061 tcgatgaggc cagcaccacc gtgtctaaga ccgagacttc tcaggtggcc cctgcctaac
     3121 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3181 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3241 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3301 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3361 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3421 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3481 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3721 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3781 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3841 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3901 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3961 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4021 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4081 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4141 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4201 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     4261 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4321 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4381 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4441 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4501 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4561 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4621 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4681 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4741 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4801 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4861 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4921 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4981 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5041 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5101 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5161 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5221 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5281 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5341 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5401 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5461 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5521 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5581 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5641 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5701 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5761 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5821 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5881 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5941 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6001 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6061 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6241 tacactagaa gaacggtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagat
//
