LOCUS       sequence-431803-        7246 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 7246)
  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 7246)
  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: TH1532-DuET
FEATURES             Location/Qualifiers
     source          1..7246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      337..925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     826..845
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1079..1096
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1213..1234
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1249..1254,1255..1272,1273..1279)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1287..1303
                     /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     1292..1314
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1311..1327
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1311..1327
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1376..1497
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1387..1406)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1441..1460
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1671..2465)
                     /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(1782..1801)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2392..2411
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2532..2861)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2546..2681
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2600..2619)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2846..2866
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2875..3303)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2985..3006)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3197..3216
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3349..3573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3562..3579
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5395..5421)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      5487..5496
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(5534..5553)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5535..5553)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5598..5801)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5627..5647)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5802..6181
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6353..6372
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6447..6551
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6552..6620,6621..7246,1..166)
                     /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(6770..6789)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
       61 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
      121 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
      181 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
      241 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
      301 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
      361 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
      421 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
      481 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
      541 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
      601 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
      661 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
      721 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
      781 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
      841 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
      901 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      961 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     1021 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     1081 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     1141 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     1201 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     1261 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     1321 ctatgaccat gattacgcca agctctagct agaggtcgac ggtatacaga catgataaga
     1381 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     1441 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttggg
     1501 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg
     1561 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga
     1621 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac
     1681 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     1741 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     1801 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     1861 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     1921 tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     1981 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     2041 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     2101 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     2161 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     2221 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     2281 tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     2341 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     2401 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     2461 atcatgcgaa acgatcctca tcctgtctct tgatcagatc cgaaaatgga tatacaagct
     2521 cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     2581 agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     2641 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     2701 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     2761 ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
     2821 gctggggagc ctggggactt tccacaccct aactgacaca cattccacag aattaattcg
     2881 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2941 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3001 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3061 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3121 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3181 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3241 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3301 cgtggggata ccccctagag ccccagctgg ttctttccgc ctcagaagcc atagagccca
     3361 ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca
     3421 ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt
     3481 attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc
     3541 aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg gtttaaacgg
     3601 gccctctaga gcggccgctt aggcaggggc cacctgagat gtctcggtct tagacacggt
     3661 tgtgctggcc tcatccgagg aggcggtacc gacgtggctc agagacactt gcttgtgata
     3721 ggtggcggca ggtccaggag gtcctgttct gccggtcagt gcggcagaag cctcaggata
     3781 atcgcctgcg gccatagcgc cgccagattt gtggcctctt ctaggtctgc aacagcaccg
     3841 gctggtgaac ctccgccagc tttccacggt cttgccgctc caaatccaaa cgccgctggt
     3901 gatgcccacg accaggcaca taaagtactt cagcatcagg acccagtact ctggcttggc
     3961 tctaggctgg cctgtatcgt ggccaggaca ggcacatgtc agagcggctt cccagctctc
     4021 tctgtagtgc tgttcgtaca ggtagcaagc caccacgata gaggcgggca ctgtgtacag
     4081 cagggtgaag atgccgatgc ggatcatcag tttttccagc ttgtcggtct tggtgccgcc
     4141 ttgcttgatc acgcttctga tccggaacag ggacacaaag ccggccagca aaaacagggt
     4201 gcccacgagc agatacagca ccagaggtcc cagcacgaag cctctcaggc tgttcaggtt
     4261 ctggttgccc acgtaacaga ttccggccac aggatcgccg tccacgctgg acagagccag
     4321 ggctgtaata gacttcacgc tggggatcag ccaggcggcc agatgaaaat actgggcgta
     4381 gccggcaatg gcctcgttgc cccacttcat tccagcggcc agaaaccagg tcaggctcag
     4441 gatgacccac caaatggagc tggccatgcc gaagaagtac accagcagaa acacgatggt
     4501 gcacagagcg gggcctgttg tctcgtagtg gatgtggttg tgctctctgc tacaggcaac
     4561 gctggcatgt cccacaacca gccgcacgag aaatcccagg gacacgcaca ggtaacaggc
     4621 gctcaggaag atgataggcc gctcggggta tctgaaccgt tccatgtcga tcaggaaggt
     4681 ggccacggtt gtagaggtgc tgatgaagca cagcacgctc caaaggccga tccagaaggt
     4741 ggcgaaggtt ctctcgtcgg cgctgaagct aggctggtaa caaggaacgg cgcaatttgg
     4801 cacctggccg gtccgcactt tgttgtacag aggatggctc tctttcagga tgggcacgaa
     4861 gggttctctg cacttgcaca cgaaaggtcc gccagcagga cattctccgc cagaagcagg
     4921 agcacctgga ggtccaggca gtgtaggctt ggctggaaaa ggtctaggtg gagcggtggt
     4981 ggcctcgctt ctgttgtagt ccatgcacag cacctcggca tctctgccca gcacaggcag
     5041 tctatcacag ctcatccgct caggccaggc gaagccgtac tgtctcatca gagggctaca
     5101 tccggctttg gccctctcgc acacggatct gcatggaggc agaggcttgt ggtagtcagg
     5161 caggcagata ggggtgtaca tggagcacag gaagaaccgc aggtcggggc tacactggat
     5221 ttccaccagg ggccaaaact ggtgcacttc cagtccggcc tcatcctggg tgtcgtggtt
     5281 gaactggttg ggcatgtgtg tcaggttgta gccgatgcct ctgcacatag gcacggtgat
     5341 ctcttggcac acaggggcct tagaggccaa ttgactccct gcaggagcga attcggcgta
     5401 gtcaggcaca tcgtaagggt agccctcgcc aggtcctgtc agcatgctca ggaacagggc
     5461 cagcagaact tgagggatac acagcgccat ggtggcggca agcttaagtt taaacgctag
     5521 ccagcttggg tctccctata gtgagtcgta ttaatttcga taagccagta agcagtgggt
     5581 tctctagtta gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc
     5641 atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc
     5701 caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac
     5761 cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact
     5821 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     5881 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     5941 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     6001 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     6061 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     6121 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt
     6181 caacgcgtat atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga
     6241 ttcttcatgc aaatgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac
     6301 tactcagcga cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca
     6361 gagcagattg tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt
     6421 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     6481 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     6541 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     6601 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6661 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6721 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6781 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6841 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6901 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6961 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     7021 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     7081 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     7141 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     7201 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataa
//
