LOCUS       sequence-430773-        7136 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 7136)
  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 7136)
  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: CHRM5-DuET
FEATURES             Location/Qualifiers
     source          1..7136
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(485..504)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(486..504)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(549..752)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(578..598)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        753..1132
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1304..1323
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1398..1502
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1503..1571,1572..2363)
                     /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(1721..1740)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2534..3122
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3023..3042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3276..3293
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3410..3431
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3446..3451,3452..3469,3470..3476)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3484..3500
                     /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     3489..3511
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3508..3524
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3508..3524
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3573..3694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3584..3603)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3638..3657
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3868..4662)
                     /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(3979..3998)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4589..4608
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4729..5058)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4743..4878
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4797..4816)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5043..5063
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(5072..5500)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5182..5203)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5394..5413
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5546..5770
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5759..5776
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
ORIGIN
        1 attcattacg ctggcgttag atgcaacata atccagcgca agccacaagt cacatgccaa
       61 tgaacccagt gcccagcgcc ccatcaggat gtacgtagta tacaggttca tgctaaagat
      121 cccgatgatc aagtcagcgc atgccaggga aaggagataa taattattca cggtcttcag
      181 ctggctgtta accttaaaag aaatcatgac caatacattg ccaacaatgg taatcagact
      241 aactactgca gtcactgcgg ctatggttat tacctcccaa agcctgtgcc tttcaagtgg
      301 ctggtggttg acgggtgtgc cgttcacagt cgtggcattg tgatatgagt cgccctccat
      361 atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct
      421 cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg
      481 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
      541 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
      601 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
      661 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
      721 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
      781 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
      841 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
      901 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
      961 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     1021 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     1081 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     1141 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     1201 caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     1261 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     1321 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     1381 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1441 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     1501 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     1561 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     1621 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1681 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1741 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1801 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1861 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1921 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     1981 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2041 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2101 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2161 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2221 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2281 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2341 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     2401 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2461 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2521 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2581 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2641 aggtaactgg cttcagcaga gcgcagatac caaataccgt tcttctagtg tagccgtagt
     2701 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2761 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2821 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2881 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2941 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3001 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3061 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3121 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3181 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3241 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3301 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3361 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3421 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     3481 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     3541 tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt ttggacaaac
     3601 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3661 atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac tccagcatga
     3721 gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc
     3781 tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt
     3841 cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc
     3901 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     3961 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     4021 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     4081 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt
     4141 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     4201 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     4261 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     4321 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     4381 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     4441 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc
     4501 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     4561 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     4621 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     4681 tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt tgcaaaagcc
     4741 taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag aggcggcctc
     4801 ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg cggaactggg
     4861 cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg actaattgag
     4921 atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac acctggttgc
     4981 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     5041 acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg ttaaatcagc
     5101 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5161 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5221 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5281 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5341 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5401 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5461 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc cctagagccc
     5521 cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc atgcctgcta
     5581 ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga
     5641 cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg
     5701 gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta
     5761 gaaggcacag tcgaggctga tcagcgggtt taaacgggcc ctctagactc gagttaggca
     5821 ggggccacct gagaagtctc ggtcttagac acggtggtgc tggcctcatc gaattcaccg
     5881 gtgcgtccac cggtatcgat tggcaatttg ctattgccct gccagtaaag cttttcttcg
     5941 acttttttct tcttccacct acacaaaagc agcatcttaa aggttttgcg aaaggtccgg
     6001 ttacaaaggg cataacagat gggattcact gttgaattaa cgtagcacag ccagtagccc
     6061 aggtgccaca gggtcacggg gacgcacttg tcacagaaag tagatacgag caccatgatg
     6121 ttataggggg tccaagtaat gataaaggcc agcagaatcg cgctcagagt ctgtgcagct
     6181 ttcctttctt taaccagcac tacccgcttg cgtttggtca tctgatggct gggattcggg
     6241 ttgagtcctt tagtagatgg ctccttggcg acggggaaag gacagggcat gatcttgacc
     6301 ttgtgacagc cattgttcgt ttcttggttg ccatcggctt tcacaaccag cctgaatttg
     6361 taggctacac atttctgaga ctttggccga tgggctgcag caggactcag cagatagttg
     6421 ggtgtgtcat aatcgctctt ttctgtctcg gctttcacaa aggtttcctc ggtttcttcc
     6481 gcgctaaact cctcgcctgg actttctttt ccctgacttt tatatacaac ctgcaggact
     6541 ggatccgtgg cgggtttgtc ctcgtcttca ctactaggat agctagagca tgtagtcagt
     6601 tgttcagcct tggcccaatt cgcgctaggc cctgtagcct gggagggttt tcctgtagtg
     6661 ctagtactcc tccgactgct ggaccatgag gcttgatttc tctctcgctg agccagagtt
     6721 ggcctggggc agcgcagaca gcttcggaag agcgcccggt gtgctggttt ccttttctcg
     6781 gctttagtca cagaatcaga cccctgaaga tcagccaaat ctttggtcct tttctctgtc
     6841 tctctgtaga tcctgcagta aaggatggtc ataacggata ccggaatata gaaagcagct
     6901 atggcggtcc caaaagttat tgtgggctca ctcaggaact ggatctggca ttcgtccagg
     6961 ggcacagtcc ttttccccac gagatactgc caacagagga tcgcaggggc ccagagtatg
     7021 aaggatatga gccaagccag tccaatcatg atgcctgctc gtttgggggt ccttttggct
     7081 ctgtacgtca ggggccgtgt aatagagaag tatctgtcaa aggatatcac gagaag
//
