LOCUS       sequence-430895-        6740 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 6740)
  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 6740)
  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: DRD3-DuET
FEATURES             Location/Qualifiers
     source          1..6740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      97..685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     586..605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     839..856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    973..994
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1009..1014,1015..1032,1033..1039)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1047..1063
                     /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     1052..1074
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1071..1087
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1071..1087
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1136..1257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1147..1166)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1201..1220
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1431..2225)
                     /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(1542..1561)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2152..2171
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2292..2621)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2306..2441
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2360..2379)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2606..2626
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2635..3063)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2745..2766)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2957..2976
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3109..3333
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3322..3339
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(4788..4807)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4789..4807)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4852..5055)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4881..4901)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5056..5435
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5607..5626
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5701..5805
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5806..5874,5875..6666)
                     /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(6024..6043)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
       61 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
      121 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
      181 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
      241 cgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
      301 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
      361 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
      421 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
      481 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
      541 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
      601 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
      661 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
      721 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
      781 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
      841 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
      901 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      961 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     1021 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     1081 ctatgaccat gattacgcca agctctagct agaggtcgac ggtatacaga catgataaga
     1141 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     1201 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttggg
     1261 gtgggcgaag aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg
     1321 gaaaacgatt ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtga
     1381 tggcaggttg ggcgtcgctt ggtcggtcat ttcgaacccc agagtcccgc tcagaagaac
     1441 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     1501 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     1561 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     1621 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     1681 tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     1741 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc
     1801 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     1861 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     1921 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     1981 tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     2041 tcctgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     2101 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     2161 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     2221 atcatgcgaa acgatcctca tcctgtctct tgatcagatc cgaaaatgga tatacaagct
     2281 cccgggagct ttttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     2341 agctcagagg cagaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     2401 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     2461 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     2521 ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
     2581 gctggggagc ctggggactt tccacaccct aactgacaca cattccacag aattaattcg
     2641 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2701 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     2761 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2821 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2881 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2941 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3001 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3061 cgtggggata ccccctagag ccccagctgg ttctttccgc ctcagaagcc atagagccca
     3121 ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg tcctgcccca
     3181 ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt tcctcatttt
     3241 attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg ggggaggggc
     3301 aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg gtttaaacgg
     3361 gccctctaga ttaggcaggg gccacctgag aagtctcggt cttagacacg gtggtgctgg
     3421 cctcatcgaa ttcaccggtg cgtccaccgg tatcgatgca agacaggatc ttgaggaagg
     3481 ctttccggaa ctcgatattg aaggtggtat agatcacagg gttgagggcg ctattcacgt
     3541 agcccagcca tgtcgtggca ctgtaaagct ctggggacac gtggcatgtc tggcagtggg
     3601 tattgagaac atgggtcaag aagaagggca gccagcagac aatgaaggcc ccaagcacaa
     3661 tggccaccat ttgggttgcc ttcttctccc gaagtggcac tccccgaggt tgcaggggcc
     3721 ccagcttcaa agatgtcgat aatctgccat tgctgagttt tcgaacttct aagctgagct
     3781 tgggcgctat ggtgggactc agggaattcc gagtcttctc ctctcttttc aactctcctc
     3841 ctctttcttg gaagcctggt ccacccaagg cagtgtcctg gcagatgctg tagtaacgct
     3901 tcagctccag atgtgccggg tcaggagaga gggtttgctg ggggaagcca ggcttgacac
     3961 tgttgcactg actgttctgt cgagtgagga tccttttccg tctcctttgt ttcagcacca
     4021 catagattct ggcatagaca aggacagtca ctccaaaggg caggtagaag gacaccactg
     4081 aagagtagat gacaaaatca gggttggaga tggagcagac agtggggtcc tctgtggtat
     4141 taaagccaaa cagaagaggg caggacacag caaaggccag tacccagacg gccgtgatca
     4201 tgagggccac gcgccgacag gagctctgtc ccgtgccatg ctggtagtga acgggcatga
     4261 ccactgcagt gtacctgtct atgctgatgg cacagagatt aaggatgctg gctgtacaca
     4321 tcatgacatc cagggtgaca aaaacatcac agcaaatgcg gctgaaattc cagactccac
     4381 ctgtcacctc caggtatacc acccagggca tcaccaaggt ggccaccagc aagtctgcca
     4441 cagccaggct cactactaag tagttggtgg tagtctgcag ggcccgctcc ttcagcacag
     4501 ccatgcacac caggccattg ccgaagacga tggccaggat gagcgcgcag taggagaggg
     4561 catagtaggc atgtgggcgg gcctggctgg cacctgtgga gttctctgcc ccacaggtgt
     4621 agttcaggtg gccactcagc tggctcagag atgccatatc gatgctggcg tcatcatcgt
     4681 ccttgtagtc ggcgaatacc aggcagaaga tgtagctcag ggcgatgatc gtcttcatgg
     4741 tggcgcggcc gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt
     4801 cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct
     4861 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt
     4921 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat
     4981 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg
     5041 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg
     5101 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
     5161 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     5221 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac
     5281 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     5341 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     5401 tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc
     5461 gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc
     5521 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     5581 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     5641 taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg
     5701 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5761 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5821 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5881 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5941 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     6001 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     6061 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6121 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6181 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6241 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6301 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6361 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6421 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6481 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6541 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6601 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6661 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6721 taatttaaaa ggatctaggt
//
