LOCUS       sequence-430902-        6455 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 6455)
  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 6455)
  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: GPR141-DuET
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(9..15,16..33,34..39))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    54..75
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(192..209)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(363..951)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(443..462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1122..1913,1914..1982))
                     /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     1745..1764
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1983..2087)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2162..2181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2353..2732
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2733..2936
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2887..2907
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2981..3000
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2981..2999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4164..4181)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4170..4394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4440..4868
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4527..4546)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4737..4758
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4877..4897)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4882..5211
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5062..5197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5124..5143
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5278..6072
                     /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(5332..5351)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5942..5961
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6246..6367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6283..6302)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6337..6356
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6416..6432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6416..6432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6429..6451)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    join(6440..6455,1)
                     /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)."
ORIGIN
        1 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
       61 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
      121 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
      181 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
      241 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      301 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      361 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      481 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      541 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      601 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      661 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      721 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      781 cctaactacg gctacactag aagaacggta tttggtatct gcgctctgct gaagccagtt
      841 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      901 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      961 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1021 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1081 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1141 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1201 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1261 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1321 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1381 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1441 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1501 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1561 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1621 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1681 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1741 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1801 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1861 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1921 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1981 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2041 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     2101 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     2161 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     2221 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     2281 tttgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     2341 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     2401 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     2461 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     2521 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     2581 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     2641 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     2701 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     2761 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     2821 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     2881 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
     2941 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
     3001 agacccaagc tggctagcgt ttaaacttaa gcttagcggc cgcgccacca tgaagacgat
     3061 catcgccctg agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc
     3121 cagcatcgat atgcctgggc ataatacttc tcgcaattca tcttgtgatc ccatcgtcac
     3181 cccgcatctc atttccctgt actttatcgt gctgatcggg ggactggtgg gcgtcatctc
     3241 catcctgttt ctcctcgtga aaatgaacac gagatccgtc acaactatgg ctgtgataaa
     3301 tctggtggtg gtgcacagtg ttttccttct gaccgtaccc tttagactta cctacctgat
     3361 aaagaaaaca tggatgttcg ggctgccctt ctgtaagttt gtaagcgcga tgctgcatat
     3421 tcacatgtat ctgacattcc tcttttatgt cgttatcctg gtaaccaggt atctcatctt
     3481 tttcaagtgc aaggataagg tcgaatttta tcggaagctg cacgccgtgg cggcctcagc
     3541 tggcatgtgg acacttgtca tcgtgatcgt tgtgcccctg gtggtgtcac gatatggtat
     3601 ccacgaggag tacaacgagg agcactgctt taagttccat aaagagctgg cttacaccta
     3661 cgtcaagatt attaattaca tgattgtgat ttttgtgata gccgtggccg ttatactcct
     3721 ggtgtttcag gtgtttatta ttatgttgat ggtgcagaag ctgcggcatt ccctcctgtc
     3781 ccaccaggag ttctgggccc aactgaaaaa cctctttttt atcggagtga ttctcgtatg
     3841 tttcctgcca tatcagtttt tcaggatcta ttacctgaat gtggtcacac atagtaacgc
     3901 atgcaattcc aaggtggcct tctataatga aatcttcctt agcgtgacag ctatttcatg
     3961 ctacgacctc cttctgttcg tttttggcgg gagccactgg ttcaagcaga agattattgg
     4021 actttggaac tgtgttctct gccgcatcga taccggtgga cgcaccggtg aattcgatga
     4081 ggccagcacc accgtgtcta agaccgagac ttctcaggtg gcccctgcct aatctagagg
     4141 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4201 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4261 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4321 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4381 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     4441 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4501 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4561 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4621 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4681 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4741 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4801 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4861 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4921 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4981 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     5041 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5101 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     5161 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     5221 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     5281 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     5341 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     5401 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     5461 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     5521 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5581 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5641 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5701 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5761 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     5821 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5881 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5941 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     6001 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     6061 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     6121 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     6181 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     6241 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     6301 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     6361 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     6421 agctgtttcc tgtgtgaaat tgttatccgc tcaca
//
