LOCUS       sequence-431822-        6596 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 6596)
  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 6596)
  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: LTB4R-DuET
FEATURES             Location/Qualifiers
     source          1..6596
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(705..722)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    711..935
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      981..1409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1068..1087)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1278..1299
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1418..1438)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1423..1752
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1603..1738
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1665..1684
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1819..2613
                     /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(1873..1892)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2483..2502
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2787..2908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2824..2843)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2878..2897
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2957..2973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2957..2973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2970..2992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2981..2997
                     /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(3005..3011,3012..3029,3030..3035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3050..3071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3188..3205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3359..3947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3439..3458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4118..4909,4910..4978))
                     /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     4741..4760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4979..5083)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5158..5177)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5349..5728
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5729..5932
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5883..5903
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5977..5996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5977..5995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 accatggaag actaatatgt cactgtgctt ccctaggtat ccatctgaag gccacagggc
       61 atttcacctg atcttcgagg cagtaacagg attccttttg ccatttttgg cagtggtggc
      121 ctcatatagc gatatcggac ggcggctcca ggcgcggcgc ttcaggagat cccgccggac
      181 tggaagactg gtggtactca tcatcttgac atttgcagca ttctggctgc cgtatcatgt
      241 tgtgaacctg gcagaagcag gcagggcact cgcagggcaa gccgcaggct tgggtctggt
      301 cgggaagagg ttgagcctgg cccggaacgt gctcatcgcc ctcgcttttc tgagtagctc
      361 agtgaaccct gtgctttatg cttgtgcagg aggcggcttg ctcagatcag ccggtgtggg
      421 atttgtggcg aagctgttgg agggcactgg gagcgaagcc tcttccactc ggcgcggcgg
      481 tagcttgggc cagacagcac ggagtggccc tgccgctctg gaacccggac ctagcgagag
      541 tctcacggcc tcaagccctt tgaaattgaa cgagctcaac atcgataccg gtggacgcac
      601 cggtgaattc gatgaggcca gcaccaccgt gtctaagacc gagacttctc aggtggcccc
      661 tgcctaactc gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc
      721 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
      781 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
      841 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
      901 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
      961 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1021 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1081 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1141 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1201 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1261 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1321 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1381 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     1441 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     1501 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1561 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     1621 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     1681 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     1741 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     1801 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     1861 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     1921 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     1981 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     2041 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     2101 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     2161 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2221 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2281 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2341 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     2401 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     2461 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     2521 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     2581 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     2641 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     2701 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     2761 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     2821 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2881 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     2941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3001 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3781 actacggcta cactagaaga acggtatttg gtatctgcgc tctgctgaag ccagttacct
     3841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5101 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     5161 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5221 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacatttg
     5281 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     5341 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     5401 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     5461 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     5521 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     5581 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     5641 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     5701 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     5761 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     5821 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     5881 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
     5941 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
     6001 ccaagctggc tagcgtttaa acttaagctt agcgccacca tgaagacgat catcgccctg
     6061 agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc cagcatcgat
     6121 atgaatacaa catcttctgc tgcccctcca tccctcgggg tagaattcat aagcttgctg
     6181 gcgattatac tgctgagcgt cgccttggcc gtcggcctcc ccggtaactc ctttgtggtg
     6241 tggtctatac tcaagagaat gcagaagagg tcagtgacag ctctgatggt gctgaatctg
     6301 gcactggccg atttggcagt cctgctgacc gcccctttct tccttcattt tctggcccag
     6361 ggaacctgga gttttgggct ggctggatgt cggctttgtc actacgtctg tggagtgtcc
     6421 atgtacgctt ccgtgctgtt gattacagct atgagcctcg atcgaagcct ggcggtggcc
     6481 agacctttcg tctcccagaa attgcggaca aaggccatgg cccgaagggt gctcgcaggc
     6541 atctgggtgc tgtcctttct gttggccact cctgtgctgg cctacagaac cgtagt
//
