LOCUS       sequence-431823-        6614 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 6614)
  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 6614)
  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: LTB4R2B-DuET
FEATURES             Location/Qualifiers
     source          1..6614
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     237..257
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(266..694)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(376..397)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     588..607
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    740..964
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     953..970
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(2293..2312)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2294..2312)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2357..2560)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2386..2406)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2561..2940
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3112..3131
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3206..3310
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3311..3379,3380..4171)
                     /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(3529..3548)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4342..4930
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4831..4850
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5084..5101
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5218..5239
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5254..5259,5260..5277,5278..5284)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5292..5308
                     /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     5297..5319
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5316..5332
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5316..5332
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5381..5502
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5392..5411)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5446..5465
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5676..6470)
                     /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(5787..5806)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6397..6416
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6537..6614,1..252))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6551..6614,1..72)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(join(6605..6614,1..10))
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac tgggcggagt
       61 taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat tgagatgcat
      121 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg ttgctgacta
      181 attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact ttccacaccc
      241 taactgacac acattccaca gaattaattc gcgttaaatt tttgttaaat cagctcattt
      301 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      361 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
      421 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
      481 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
      541 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
      601 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
      661 cccgccgcgc ttaatgcgcc gctacagggc gcgtggggat accccctaga gccccagctg
      721 gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct
      781 tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta
      841 ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct
      901 tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc
      961 acagtcgagg ctgatcagcg ggtttaaacg ggccctctag attaggcagg ggccacctga
     1021 gaagtctcgg tcttagacac ggtggtgctg gcctcatcga attcaccggt gcgtccaccg
     1081 gtatcgatca ggtcccattc ggggccatcc ttttccatcc cacctcctgg atctccatta
     1141 cccctgccct gtccaacgac cttcagctgg ggggtggtcc gaagttccat agtcccctct
     1201 ctgcttcttc cacccccccg ggcttcacca gacccctcaa acagccgagt gaggaaccgc
     1261 gggcctgccc tgggcagaag gtcgccggct gtaaagacat acagaactgg gttaactgaa
     1321 gagctgaaga aggcaagagc tgtcgtgccg gcgcgtgcgg cctggcctgc ccccccgagt
     1381 ttggcaagcg ctccttcagg gggagccaag gcagccactg cctgcaacag attaactgcg
     1441 tggtatgggg cccacagcag tccgaaagcc agcacaatag cagagaccag tcgtcccact
     1501 ctggctccat ggcgcccact gccccaccgt gcgcccctga gtctggccag ggtaacgcta
     1561 taacacccga gcatgagccc gaaggggagc acaaaggcgg tcagggtttc gagagacagg
     1621 tgagcagcag cgtgcaccgg gcttggatgg cacagctgac agacccgatc cctccacagg
     1681 tgtctataaa ctgcagcagg cacggccaag agaagggctg ccagccatac ggcaaggaga
     1741 agccgtcgtg ccaacgctgg agatctcagg cggggggcca ggaagggtcg tgtcacagcc
     1801 aaacaccgct gcagagaaag cagcccagtc aggagcacag aggcatacat actcagggcg
     1861 caaacatagt acacagcctt acaccctgcc tgtcccagcg gccaagcctg ccttgtcaga
     1921 aatgcaacga acaagggagt cagcagaagg acggctccgt cagcgagggc caggtgcagc
     1981 accagagtgg cggccagtgg tcgtcctcgc gctggccgcc atccagccaa gctccaaaca
     2041 acgaagccat tgccggggag acccaagagt gcagcgagca aaaggaaagc ggttccggtg
     2101 gccctagatg ttttccagga cagcagagtt tcattgccag gaggcctata gcagacactc
     2161 atatcgatgc tggcgtcatc atcgtccttg tagtcggcga ataccaggca gaagatgtag
     2221 ctcagggcga tgatcgtctt catggtggcg cggccgctaa gcttaagttt aaacgctagc
     2281 cagcttgggt ctccctatag tgagtcgtat taatttcgat aagccagtaa gcagtgggtt
     2341 ctctagttag ccagagagct ctgcttatat agacctccca ccgtacacgc ctaccgccca
     2401 tttgcgtcaa tggggcggag ttgttacgac attttggaaa gtcccgttga ttttggtgcc
     2461 aaaacaaact cccattgacg tcaatggggt ggagacttgg aaatccccgt gagtcaaacc
     2521 gctatccacg cccattgatg tactgccaaa accgcatcac catggtaata gcgatgacta
     2581 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     2641 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     2701 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     2761 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggg cgggggtcgt
     2821 tgggcggtca gccaggcggg ccatttaccg taagttatgt aacgcggaac tccatatatg
     2881 ggctatgaac taatgacccc gtaattgatt actattaata actagtcaat aatcaatgtc
     2941 aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa acgcctaacc ctaagcagat
     3001 tcttcatgca aatgtcggtc aagccttgcc ttgttgtagc ttaaattttg ctcgcgcact
     3061 actcagcgac ctccaacaca caagcaggga gcagatactg gcttaactat gcggcatcag
     3121 agcagattgt actgagagtg caccataggg gatcgggaga tctcccgatc cgtcgacgtc
     3181 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3241 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3301 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3361 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3421 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3481 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3541 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3601 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3661 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3721 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3781 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3841 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3901 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3961 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     4021 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     4081 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     4141 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     4201 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4261 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4321 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4381 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4441 ttttccgaag gtaactggct tcagcagagc gcagatacca aataccgttc ttctagtgta
     4501 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4561 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4621 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4681 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4741 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4801 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4861 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4921 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4981 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5041 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5101 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     5161 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     5221 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     5281 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     5341 cgccaagctc tagctagagg tcgacggtat acagacatga taagatacat tgatgagttt
     5401 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     5461 attgctttat ttgtaaccat tataagctgc aataaacaag ttggggtggg cgaagaactc
     5521 cagcatgaga tccccgcgct ggaggatcat ccagccggcg tcccggaaaa cgattccgaa
     5581 gcccaacctt tcatagaagg cggcggtgga atcgaaatct cgtgatggca ggttgggcgt
     5641 cgcttggtcg gtcatttcga accccagagt cccgctcaga agaactcgtc aagaaggcga
     5701 tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca
     5761 gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag
     5821 cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
     5881 atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg
     5941 cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga
     6001 tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc
     6061 gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca
     6121 gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
     6181 acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg
     6241 caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc
     6301 agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg
     6361 aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc
     6421 tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
     6481 cctcatcctg tctcttgatc agatccgaaa atggatatac aagctcccgg gagctttttg
     6541 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggcagag
     6601 gcggcctcgg cctc
//
