LOCUS       sequence-430913-        6776 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 6776)
  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 6776)
  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: MLNR-DuET
FEATURES             Location/Qualifiers
     source          1..6776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1073..1090)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1079..1303
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1349..1777
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1436..1455)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1646..1667
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1786..1806)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1791..2120
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1971..2106
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2033..2052
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2187..2981
                     /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(2241..2260)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2851..2870
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3155..3276
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3192..3211)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3246..3265
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3325..3341)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3325..3341)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3338..3360)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3349..3365
                     /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(3373..3379,3380..3397,3398..3403))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3418..3439
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3556..3573)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3727..4315)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3807..3826)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4486..5277,5278..5346))
                     /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     5109..5128
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5347..5451)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5526..5545)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5717..6096
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6097..6300
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6251..6271
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6345..6364
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6345..6363
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ctgtatagac tctggagaag tcggccatgg gtctttgggc ccctcttgtg tagacttagc
       61 ctgtacgtag gcgaagggtg cacttatgcc acacttttgc atatgaccgc cctgtccgtc
      121 gagcggtatc tggccatctg cagacctttg cgggcacggg tcctggtgac tagaagaaga
      181 gtgcgagctc tgatcgccgt tctgtgggcc gtggccctgc ttagcgccgg acccttcctc
      241 ttcttggtgg gcgtagagca agaccccggg atcagcgttg tcccaggact caatggaacc
      301 gctcgcatcg cctcctctcc acttgccagc agcccacctc tttggctgag tcgggcccca
      361 cccccaagtc ccccaagcgg gccagagaca gctgaagctg cggcgctctt ttctcgcgag
      421 tgccggccct ctccagctca acttggagct cttcgcgtta tgctgtgggt aaccactgca
      481 tacttcttcc tgcctttcct gtgcttgagc atcctgtacg gactcatcgg tcgggaactt
      541 tggtcctcaa ggcgaccctt gaggggacct gcagctagcg gcagagagag aggtcatcgc
      601 cagaccgtcc gggtactcct ggtggttgtc ctggcgttca tcatttgttg gctgccattt
      661 cacgtcggta ggatcattta catcaacacc gaagactctc gcatgatgta tttttcacaa
      721 tattttaata tagtagccct gcaactgttt tatctgtctg ccagcataaa ccctatcctg
      781 tacaacctga tatccaagaa ataccgggca gctgcgttca aactgctcct tgctagaaag
      841 tctaggcccc ggggatttca caggtcccgg gacaccgctg gggaggtcgc aggcgacact
      901 ggtggcgata ccgtggggta cacagagaca tccgctaatg ttaagaccat ggggatcgat
      961 accggtggac gcaccggtga attcgatgag gccagcacca ccgtgtctaa gaccgagact
     1021 tctcaggtgg cccctgccta atctagaggg cccgtttaaa cccgctgatc agcctcgact
     1081 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1141 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1201 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1261 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     1321 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     1381 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1441 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1501 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     1561 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     1621 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     1681 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     1741 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     1801 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1861 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     1921 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     1981 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2041 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2101 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     2161 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2221 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2281 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2341 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     2401 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2461 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2521 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2581 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2641 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2701 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2761 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2821 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2881 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     2941 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3001 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3061 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3121 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3181 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3241 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3301 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3361 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3421 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3481 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3541 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3601 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3661 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3721 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3781 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3841 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3901 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3961 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4021 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4081 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4141 gtggcctaac tacggctaca ctagaagaac ggtatttggt atctgcgctc tgctgaagcc
     4201 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4261 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4321 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4381 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4441 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4501 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4561 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4621 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4681 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4741 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4801 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4861 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4921 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4981 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5041 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5101 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5161 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5221 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5281 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5341 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5401 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5461 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     5521 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     5581 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     5641 gacatttgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     5701 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     5761 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     5821 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     5881 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     5941 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     6001 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     6061 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     6121 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     6181 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     6241 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     6301 ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat
     6361 agggagaccc aagctggcta gcgtttaaac ttaagcttag cggccgcgcc accatgaaga
     6421 cgatcatcgc cctgagctac atcttctgcc tggtattcgc cgactacaag gacgatgatg
     6481 acgccagcat cgatatgggc tcaccttgga acggctcaga cggtccagaa ggggcacggg
     6541 aacctccttg gccggcactg ccaccctgcg acgagaggag atgtagtcct ttccctctgg
     6601 gcgcactggt gcccgtgacc gccgtgtgtc tctgtctgtt cgtcgttgga gtgtcaggca
     6661 acgttgtaac agttatgctg atcgggaggt atcgggatat gagaactacc accaacctct
     6721 atctcgggag tatggccgtc agcgaccttc tgatcctcct gggactgcct ttcgat
//
