LOCUS       sequence-431805-        7633 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 7633)
  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 7633)
  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: TH1533-DuET
FEATURES             Location/Qualifiers
     source          1..7633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(228..332)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(407..426)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        598..977
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        978..1181
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1132..1152
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1226..1245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1226..1244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1283..1292
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1358..1384
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(3587..3604)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3593..3817
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3863..4291
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3950..3969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4160..4181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4300..4320)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4305..4634
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4485..4620
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4547..4566
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4701..5495
                     /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(4755..4774)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5365..5384
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5669..5790
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5706..5725)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5760..5779
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5839..5855)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5839..5855)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5852..5874)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5863..5879
                     /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(5887..5893,5894..5911,5912..5917))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5932..5953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6070..6087)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6241..6829)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6321..6340)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7000..7633,1..158,159..227))
                     /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     join(7623..7633,1..9)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
       61 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
      121 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
      181 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
      241 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
      301 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
      361 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
      421 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      481 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacatttgc atgaagaatc
      541 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      601 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      661 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      721 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      781 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      841 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      901 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      961 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     1021 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     1081 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     1141 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
     1201 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
     1261 agcgtttaaa cttaagcttg ccgccaccat ggcgctgtgt atccctcaag ttctgctggc
     1321 cctgttcctg agcatgctga caggacctgg cgagggctac ccttacgatg tgcctgacta
     1381 cgccgaattc gctcctgcag ggagtcaatt gcacagcctg tttacctgcg agcccatcac
     1441 cgtgcctcgg tgcatgaaga tggcctacaa catgacattc ttccccaacc tgatgggcca
     1501 ctacgaccag tctattgccg ccgtggaaat ggaacacttc ctgcctctgg ccaacctgga
     1561 atgtagcccc aatatcgaga cattcctgtg caaggccttc gtgcctacct gcatcgagca
     1621 gatccatgtg gtgcctccat gccggaagct gtgcgagaag gtgtacagcg actgcaagaa
     1681 gctgatcgac accttcggca tcagatggcc cgaggaactg gaatgcgacc gcctgcagta
     1741 ctgtgacgag acagtgcccg tgaccttcga tccccacacc gagtttctgg gccctcagaa
     1801 aaagaccgaa caggtgcaga gagatatcgg cttctggtgc cccagacacc tgaaaacatc
     1861 tggcggccag ggctacaagt tcctgggcat cgatcagtgc gcccctccat gtcctaacat
     1921 gtacttcaag agcgacgagc ttgagttcgc caagagcttc atcggaaccg tgtccatctt
     1981 ctgcctgtgc gctaccctgt ttactttcct gaccttcctg atcgacgtgc ggcggttcag
     2041 ataccccgag aggcccatca tctactacag cgtgtgctac agcatcgtgt ccctgatgta
     2101 ctttatcggc tttctgctgg gcgacagcac cgcctgtaac aaggccgatg agaagctgga
     2161 actgggcgat accgtggtgc tgggcagtca gaacaaggcc tgcaccgtgc tgtttatgtt
     2221 gctgtacttc ttcacaatgg ccggcaccgt ttggtgggtc atcctgacca tcacctggtt
     2281 tctggccgct ggcagaaagt ggtcttgcga ggccatcgaa cagaaggccg tctggtttca
     2341 cgctgtggcc tggggcacac caggctttct gacagtgatg ctcctggcca tgaacaaggt
     2401 ggaaggcgac aacatcagcg gcgtgtgctt tgtgggcctg tacgatctgg acgccagccg
     2461 gtactttgtg ctcctgccac tgtgcctgtg tgtgttcgtg ggactgtctc tgcttctggc
     2521 cggcatcatc agcctgaacc atgtgcggca agtgatccag cacgacggcc ggaatcaaga
     2581 gaagctgaag aaattcatga tccgcatcgg cgtgttcagc ggcctgtatc tggtgcctct
     2641 ggtcacactg ctgggctgtt acgtgtacga gcaagtgaac cggatcacct gggagatcac
     2701 atgggtgtcc gaccactgca gacagtatca catcccatgt ccataccagg ccaaggccaa
     2761 agccagacct gagctggccc tgttcatgat caagtacctg atgaccctga tcgtgggcat
     2821 ctccgccgtg ttttgggtcg gaagcaagaa aacctgtacc gagtgggccg gcttcttcaa
     2881 gcggaacaga aagagggacc ccatcagcga gagcagacgg gtcctgcaag aatcctgcga
     2941 gttcttcctg aagcacaaca gcaaagtcaa gcacaagaag aagcactaca agccctccag
     3001 ccacaagctg aaagtgatca gcaagagcat gggcaccagc acaggcgcca cagccaatca
     3061 tggaacatct gccgtggcca tcaccagcca cgattacctg ggacaagaga cactgaccga
     3121 gatccagaca agccccgaaa ccagcatgcg ggaagtgaag gctgacggcg cctctacacc
     3181 tagactgaga gagcaggatt gcggcgagcc tgcttctcct gccgcctcta ttagtagact
     3241 gagcggagaa caggtggacg gcaaaggaca ggccggatct gtgtctgaga gcgccagatc
     3301 tgagggcaga atcagcccca agtccgacat caccgatacc ggactggccc agagcaacaa
     3361 tctgcaggtc ccaagctcca gcgagccttc cagcctgaag ggctctacat ctctgctggt
     3421 gcaccctgtg tctggcgtgc ggaaagaaca aggcggcgga tgccacagcg atactggtac
     3481 cgcctcctcg gatgaggcca gcacaaccgt gtctaagacc gagacatctc aggtggcccc
     3541 tgcctaagcg gccgctctag agggcccgtt taaacccgct gatcagcctc gactgtgcct
     3601 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3661 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3721 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     3781 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc
     3841 tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3901 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3961 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4021 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     4081 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4141 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     4201 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     4261 gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt
     4321 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4381 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4441 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4501 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4561 ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4621 taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga
     4681 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     4741 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4801 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4861 ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg
     4921 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4981 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     5041 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     5101 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     5161 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     5221 tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     5281 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     5341 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     5401 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     5461 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     5521 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     5581 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     5641 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa
     5701 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     5761 tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta
     5821 gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     5881 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5941 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6001 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6061 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6121 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6181 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6241 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6301 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6361 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6421 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6481 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6541 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6601 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6661 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6721 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6781 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6841 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6901 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6961 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7021 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7081 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7141 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7201 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7261 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7321 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7381 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7441 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7501 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7561 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7621 ggataatacc gcg
//
