LOCUS       sequence-430789-        6869 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 6869)
  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 6869)
  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: DRD2-DuET
FEATURES             Location/Qualifiers
     source          1..6869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      225..813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     714..733
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     967..984
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1101..1122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1137..1142,1143..1160,1161..1167)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1175..1191
                     /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     1180..1202
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1199..1215
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1199..1215
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1264..1385
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1275..1294)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1329..1348
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1559..2353)
                     /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(1670..1689)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2280..2299
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2420..2749)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2434..2569
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2488..2507)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2734..2754
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2763..3191)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2873..2894)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3085..3104
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3237..3461
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3450..3467
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5045..5064)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5046..5064)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5109..5312)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5138..5158)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5313..5692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5864..5883
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5958..6062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6063..6131,6132..6869,1..54)
                     /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(6281..6300)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
       61 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
      121 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
      181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
      241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      361 ccaaataccg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
      601 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
      661 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
      721 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
      781 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
      841 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
      901 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
      961 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     1021 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     1081 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     1141 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     1201 ggaaacagct atgaccatga ttacgccaag ctctagctag aggtcgacgg tatacagaca
     1261 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     1321 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     1381 aagttggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg
     1441 gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa
     1501 tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc
     1561 agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac
     1621 cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
     1681 tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc
     1741 cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga
     1801 cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg gctggcgcga
     1861 gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac
     1921 gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
     1981 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag
     2041 atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag
     2101 tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg
     2161 ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg
     2221 ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
     2281 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat
     2341 cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatccg aaaatggata
     2401 tacaagctcc cgggagcttt ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     2461 tctggaatag ctcagaggca gaggcggcct cggcctctgc ataaataaaa aaaattagtc
     2521 agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta
     2581 ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     2641 gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac
     2701 ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagaa
     2761 ttaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2821 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2881 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2941 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3001 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3061 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3121 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3181 acagggcgcg tggggatacc ccctagagcc ccagctggtt ctttccgcct cagaagccat
     3241 agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc ccttgctgtc
     3301 ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg atgcaatttc
     3361 ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg aaggcacggg
     3421 ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg atcagcgggt
     3481 ttaaacgggc cctctagact cgagttaggc aggggccacc tgagaagtct cggtcttaga
     3541 cacggtggtg ctggcctcat cgaattcacc ggtgcgtcca ccggtatcga tacagtggag
     3601 aatcttcaga aatgcttttc gaaattcgat attgaatgtg gtgtagatta tggggtttac
     3661 cgcactattc acataaccca gccaagtaaa ggcgctatag agcactggtg gaatgttgca
     3721 atcacaatga atgttcagga tgtgggtgat aaaaaagggc agccaacaaa tgatgaacac
     3781 cccgagtaca atggccagca tttgggtggc cttcttctcc ttttgctgtg aaagttttct
     3841 ccgactcatt gttttcagag aggtgcgagt tttgccgtta ggcatagttt ggatctcgaa
     3901 aatcttagcg attttcgggt gatctttcgc gtgcccgttc ttttcaggct tggccggtga
     3961 gtccggggta gagtggagtc catgatgtga ggggtcgggc agtgtgagtt ggtgatgtga
     4021 gggcggaatg gggctgtacc gggtgcgctc aggggggctg gtgctgctga gcatctccat
     4081 ctccagctcc tgtgcgcgcc ttgcggcttc cactctccgc ctattaacgg ggaagctccc
     4141 attggatttc atgatgacgg tacacagttt catgtcttcc gggtgggtac agttgccctt
     4201 cagtggagct ctcagatgag cgcggaaagc tctagatgac cgcttggtgt tcacccgttt
     4261 tctccgccgc ctgaggacaa tgtatatttt aatgtacacc aacaatgtaa caataaatgg
     4321 gacgtaaaag ctcacaattg atgaatacac cacaaaggcg ggattggcaa taatacactc
     4381 attttggtct gcgttgttca gcccgaacag cagagggcaa ctaatcgtaa aggacagcac
     4441 ccaaacgatg gaaatcataa ccgtcaccct ccgcttgctt gaatatcggg tattgtaaag
     4501 cataggcatt gctacggcag tatagcggtc gatggagatg gcacagaggt tcaatattga
     4561 agccgtacac atcataacat ccagggttac aaagatatcg caatgtatcc ttgagaactt
     4621 ccactcgcca acaacctcca agtaaaccac ccaaggcatg accagggtag ccaccagcaa
     4681 gtctgctacc gcaagtgaga cgatgaggta gttggtagtt gtctggagag ccttttcccg
     4741 gcttactgcc atgcaaacca gcacgttccc gaacacaata actgcaatga ggagggtgag
     4801 cagtgtggcg tagtaattgt agtgaggccg atctgcttta ccatctgatc cattaaacgg
     4861 ccgggaccag ttttgccttt ccagatcatc gtcataccat gacagattca gaggatccat
     4921 atcgatgctg gcgtcatcat cgtccttgta gtcggcgaat accaggcaga agatgtagct
     4981 cagggcgatg atcgtcttca tggtggcgct aagcttaagt ttaaacgcta gccagcttgg
     5041 gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg ttctctagtt
     5101 agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc catttgcgtc
     5161 aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg ccaaaacaaa
     5221 ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa ccgctatcca
     5281 cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac taatacgtag
     5341 atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg ccaggcgggc
     5401 catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac ttgatgtact
     5461 gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa agtccctatt
     5521 ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc gttgggcggt
     5581 cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata tgggctatga
     5641 actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg tcaacgcgta
     5701 tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag attcttcatg
     5761 caaatgtcgg tcaagccttg ccttgttgta gcttaaattt tgctcgcgca ctactcagcg
     5821 acctccaaca cacaagcagg gagcagatac tggcttaact atgcggcatc agagcagatt
     5881 gtactgagag tgcaccatag gggatcggga gatctcccga tccgtcgacg tcaggtggca
     5941 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6001 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     6061 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6121 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6181 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6241 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6301 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6361 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6421 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6481 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6541 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6601 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6661 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6721 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6781 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6841 acacgacggg gagtcaggca actatggat
//
