LOCUS       sequence-430869-        6473 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 6473)
  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 6473)
  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: MRGPRE-DuET
FEATURES             Location/Qualifiers
     source          1..6473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     437..456
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(675..779)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(854..873)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1045..1424
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1425..1628
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1579..1599
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1673..1692
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1673..1691
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2874..2891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2880..3104
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3150..3578
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3237..3256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3447..3468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3587..3607)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3592..3921
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3772..3907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3834..3853
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3988..4782
                     /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(4042..4061)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4652..4671
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4956..5077
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4993..5012)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5047..5066
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5126..5142)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5126..5142)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5139..5161)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5150..5166
                     /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(5174..5180,5181..5198,5199..5204))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5219..5240
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5357..5374)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5528..6116)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5608..5627)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6287..6473,1..605,606..674))
                     /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 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
       61 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
      121 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
      181 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
      241 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
      301 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
      361 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
      421 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
      481 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
      541 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
      601 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
      661 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
      721 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
      781 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
      841 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
      901 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      961 gcttgaccga catttgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     1021 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     1081 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1141 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1201 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1261 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     1321 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     1381 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     1441 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1501 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1561 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1621 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
     1681 ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttagcg ccaccatgaa
     1741 gacgatcatc gccctgagct acatcttctg cctggtattc gccgactaca aggacgatga
     1801 tgacgccagc atcgatatgg aaccgcgaga agccgggcag catgttggag cggccaacgg
     1861 tgcccaggag gatgtggcct tcaacctcat tatcctcagc ttgactgagg gtctggggct
     1921 gggtgggctc cttggtaacg gggctgtgct ctggctgctg tcctctaatg tgtacaggaa
     1981 cccatttgcg atataccttc tggatgtggc gtgcgccgac ttgattttcc tggggtgcca
     2041 tatggtagct attgtacccg acctgctgca ggggcggctc gacttcccag gatttgtcca
     2101 gacttctctg gctacactca ggtttttctg ttacattgtt ggcctctccc ttctcgcagc
     2161 ggtttccgtc gaacaatgtt tggcagctct gtttcctgcc tggtattcat gcagaaggcc
     2221 acggcatctt actacctgtg tgtgcgctct cacatgggct ttgtgtctgc tgctgcacct
     2281 gcttctctca ggcgcttgca ctcagttttt cggtgagccg tctaggcacc tttgcaggac
     2341 actgtggctg gttgctgctg tgctgctggc gctgctgtgt tgcacaatgt gcggcgcctc
     2401 tctgatgttg ctccttcggg tggaaagagg gccacaacgc ccccctccga ggggctttcc
     2461 tggactcatc ctgcttacgg ttctcctgtt tctcttctgt ggcctgcctt tcggcatcta
     2521 ctggctctct cggaacctcc tttggtacat tccccactat ttttaccact tctccttcct
     2581 gatggccgcc gtgcactgtg ccgctaagcc tgtagtttat ttttgccttg gaagcgctca
     2641 aggacgcaga ctccccctta ggttggtgtt gcagcgagct ctgggggatg aagctgaatt
     2701 gggagctgtg cgcgagactt ctaggcgggg ccttgtggat atcgcggcaa tcgataccgg
     2761 tggacgcacc ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca
     2821 ggtggcccct gcctaactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2881 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2941 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3001 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3061 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3121 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3181 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3241 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3301 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3361 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3421 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3481 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3541 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3601 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3661 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3721 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3781 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3841 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3901 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3961 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4021 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4081 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4141 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4201 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4261 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4321 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4381 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4441 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4501 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4561 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4621 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4681 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4741 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4801 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4861 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4921 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4981 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5041 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5101 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5161 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5221 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5281 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5341 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5401 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5461 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5521 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5581 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5641 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5701 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5761 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5821 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5881 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5941 ggtggcctaa ctacggctac actagaagaa cggtatttgg tatctgcgct ctgctgaagc
     6001 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6061 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6121 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6181 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6241 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6301 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6361 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6421 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag cca
//
