LOCUS       sequence-431826-        7019 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 7019)
  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 7019)
  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: OPRM1-DuET
FEATURES             Location/Qualifiers
     source          1..7019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      288..876
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     777..796
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1030..1047
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1164..1185
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1200..1205,1206..1223,1224..1230)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1238..1254
                     /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     1243..1265
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1262..1278
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1262..1278
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1327..1448
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1338..1357)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1392..1411
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1622..2416)
                     /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(1733..1752)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2343..2362
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2483..2812)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2497..2632
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2551..2570)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2797..2817
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2826..3254)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2936..2957)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3148..3167
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3300..3524
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3513..3530
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5258..5277)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5259..5277)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5322..5525)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5351..5371)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5526..5905
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6077..6096
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6171..6275
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6276..6344,6345..7019,1..117)
                     /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(6494..6513)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
       61 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
      121 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
      181 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
      241 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
      301 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
      361 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
      421 ataccaaata ccgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
      481 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
      541 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
      601 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
      661 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      721 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      781 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      841 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      901 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      961 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     1021 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     1081 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     1141 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     1201 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     1261 acaggaaaca gctatgacca tgattacgcc aagctctagc tagaggtcga cggtatacag
     1321 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     1381 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     1441 aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     1501 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     1561 aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg
     1621 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     1681 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     1741 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     1801 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     1861 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
     1921 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     1981 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     2041 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     2101 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     2161 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     2221 gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     2281 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     2341 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     2401 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg
     2461 atatacaagc tcccgggagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
     2521 acttctggaa tagctcagag gcagaggcgg cctcggcctc tgcataaata aaaaaaatta
     2581 gtcagccatg gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag
     2641 ttaggggcgg gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg
     2701 ctggggagcc tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca
     2761 tacttctgcc tgctggggag cctggggact ttccacaccc taactgacac acattccaca
     2821 gaattaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     2881 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     2941 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3001 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3061 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3121 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3181 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3241 gctacagggc gcgtggggat accccctaga gccccagctg gttctttccg cctcagaagc
     3301 catagagccc accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct
     3361 gtcctgcccc accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat
     3421 ttcctcattt tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac
     3481 gggggagggg caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg
     3541 ggtttaaacg ggccctctag attaggcagg ggccacctga gaagtctcgg tcttagacac
     3601 ggtggtgctg gcctcatcga attcaccggt gcgtccaccg gtatcgattg ggagaggggc
     3661 ggtttctgcc tcaaggttct caagctggtg attggtccgg tcgacagtat tggcagtgct
     3721 aggatgatct ctggtattct gtcgtattcg tgtagaattc tgctgttcaa tgttagagct
     3781 tgtgggaatg cagaactccc tgaagcatcg cttgaaattc tcgtccagaa acgcgtacag
     3841 aacgggatta aggcaactgt tggtatatcc caatgctata caaaagtgcc aggaaacggt
     3901 ctggaatgtg gtttccggga tggtcaccag ggccttaatt ataacataaa tatgaatagg
     3961 ggtccagcaa acaatgaaca cagccacaac caccaggacc atccgtgtga ttcgccgaag
     4021 gttccggtcc ttttctttgg agccgctgag catgcgcacg ctcttcaggc gcaggatcat
     4081 aagaccataa caaacggtga ttatcagtac tggcatgatg aaggcgaaga tgaacacaca
     4141 gatcttcagc aagttctccc aataccacgt gggatgtgaa aatgtgaggg tacagtcgat
     4201 tgaaccttgt ctgtacttgg tagtggccat gaacatcacc ggcaagccga tagcgctaga
     4261 cagaatccaa ttgcacacat tgatgatctt ggcattccta ggggtcctga aatcaagagc
     4321 tttgacaggg tggcatacgg cgatatagcg gtctactgac attgtacaca aggtaaaaat
     4381 ggaggtgaac atgttataat aatcaattga gattacgatt ttgcacagaa tggttccgaa
     4441 gggccatgtg cccatcagat aattcacgga ttgaaacggc agtgtactgg ttgcaagggc
     4501 atccgccagg gccagattaa agatgtaaat attggttgcg gttttcatct ttgtgtatct
     4561 gacgataacg tacatgacga gaaaattgcc aaagagtccc acgacgcaga cgatactata
     4621 gagagccatt attgtgatag ctgtgatcat gctggggctt ccagtagggg ggcacaggct
     4681 atctcgacca ccgaggtcgg tcctattagg gccacaaggg tcggacaggt tcccgtcgag
     4741 atgactgagg ttaacccagc ttcccggaga tggtgcggga gagcaagagc tataggccaa
     4801 ggcgtctgta caatttgagg cattggtggg agcagcggaa gagtccatgg tgctcacagc
     4861 gggccgtgca ccgctgtggc tagttgccag cagcacggac acctttcttg ttcccagggc
     4921 ctcagcggcc tcctttctcc tctgccaaag ctcctgctgt ttcttgcaga atccggttcg
     4981 tgctgctccg catcgctgaa gcagcggtgc atgcgccatt ctggattctg aggctggcag
     5041 gcttggggga gggaacaaag gcgggttttg agcgaagcgg tcgagactgt atgtctcctc
     5101 ggagggcacc ctccgatgaa ggcacatatc gatgctggcg tcatcatcgt ccttgtagtc
     5161 ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg tggcgcggcc
     5221 gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt cgtattaatt
     5281 tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct tatatagacc
     5341 tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt
     5401 ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga
     5461 cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc
     5521 atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     5581 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     5641 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     5701 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     5761 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     5821 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     5881 taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc
     5941 gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc ttgccttgtt
     6001 gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga
     6061 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg
     6121 ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     6181 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     6241 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     6301 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     6361 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     6421 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     6481 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     6541 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     6601 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     6661 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     6721 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     6781 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     6841 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6901 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6961 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactgggg
//
