LOCUS       sequence-431800-        6888 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 6888)
  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 6888)
  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: DRD1-DuET
FEATURES             Location/Qualifiers
     source          1..6888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      329..917
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     818..837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1071..1088
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1205..1226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1241..1246,1247..1264,1265..1271)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1279..1295
                     /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     1284..1306
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1303..1319
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1303..1319
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1368..1489
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1379..1398)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1433..1452
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1663..2457)
                     /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(1774..1793)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2384..2403
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2524..2853)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2538..2673
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2592..2611)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2838..2858
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2867..3295)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2977..2998)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3189..3208
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3341..3565
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3554..3571
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5158..5177)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5159..5177)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5222..5425)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5251..5271)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5426..5805
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5977..5996
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6071..6175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6360..6738
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     complement(6394..6413)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
       61 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
      121 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
      181 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
      241 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
      301 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
      361 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
      421 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat accgttcttc
      481 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      541 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      601 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      661 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      721 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
      781 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      841 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      901 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      961 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     1021 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     1081 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     1141 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     1201 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     1261 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     1321 atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa gatacattga
     1381 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1441 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     1501 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     1561 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     1621 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     1681 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     1741 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     1801 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     1861 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     1921 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     1981 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     2041 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     2101 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     2161 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     2221 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag
     2281 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     2341 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     2401 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     2461 aaacgatcct catcctgtct cttgatcaga tccgaaaatg gatatacaag ctcccgggag
     2521 ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     2581 ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga
     2641 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt
     2701 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
     2761 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     2821 gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt cgcgttaaat
     2881 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2941 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3001 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3061 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3121 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3181 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3241 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga
     3301 taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc caccgcatcc
     3361 ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc
     3421 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
     3481 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
     3541 gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac gggccctcta
     3601 gactcgagtt aggcaggggc cacctgagaa gtctcggtct tagacacggt ggtgctggcc
     3661 tcatcgaatt caccggtgcg tccaccggta tcgatagttg gatgttgacc gttctgtgtt
     3721 attggttgaa ttttctccag gctcacatca gtgtcataat ccaggatcac agaaagtgca
     3781 gggctcagtt tttcgagggg cctagcgatg ccggcggctt cttctttctt aaggtcttct
     3841 gaactcccaa ctgcgtgagg tatcaagtaa acaagattac actctttgct aattgaacct
     3901 cttggctcat ggtgagagga gaacatagct gcgccgttat tgttaataga tacggtctca
     3961 attgcgttat tggtggcggg acaaagccgg taacagccca gcagggtgga gaatgctttg
     4021 cgaaaatcag cattaaatgc gtatataata ggattaaggg aggaattggc ccaaccgaac
     4081 caaacgaaga cgtcaaaagt gtttgagtct atacagaaag gctgagtctc gccactgccg
     4141 cagaaaggca gtatacaatt caaaatgaag aaggggagcc agcaacacac aaacacgccc
     4201 ataatcacag agagggtctt cagaacctta gtttcgcgct taaatgacat tttgaagctg
     4261 gactctggct gggaacactc gacgggttta ccgttgccgg tcgtcgtctg acaattcttt
     4321 gcgtgcacag cggctctctc caaagccgcg atcctccgga tttgcttctg tgcgattcta
     4381 tagatccttg tgtaggtcac gatcataatc gcgacaggaa tgtaaaagga gatcactgaa
     4441 ctgctgatag cgtaggtcct ggacaggcta ctgtcacaat tgtcgatagt ctcggccaga
     4501 ctagtggcgt ttccgtcgct gggactcgtt ggtttagcct tgtgccagga caattgcaca
     4561 gggatgaaag aaatcagcac gctgagggtc caggcgactg atatcagtat aaaggcggcc
     4621 tttggggtca tcttccgctc gtatcggaag ggagagctga tggcccaata cctatcgaca
     4681 ctaatgacac agagattgag aattgaggcc gtgctacaca tgatgtcgaa tgccacccag
     4741 atattgcaaa atgacccgaa gggccagaac ccggcgatct cggccactgc tttccatggc
     4801 atcacgagga cagccaccag cagatcagag acggccagtg agatgacaaa aaagttagtc
     4861 actttggacc gcaagtgccg gaatctgatc acggcggcgc acacaagtgt attacccagg
     4921 agagtagaca ggataagcag tgacaggaag caagcggtaa gaattctaac gctaaagtcc
     4981 cgttcgacca cgaggcccgt tccgtccatc gctgaagtat tcaatgtgcg catatcgatg
     5041 ctggcgtcat catcgtcctt gtagtcggcg aataccaggc agaagatgta gctcagggcg
     5101 atgatcgtct tcatggtggc gctaagctta agtttaaacg ctagccagct tgggtctccc
     5161 tatagtgagt cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga
     5221 gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg
     5281 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat
     5341 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat
     5401 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact
     5461 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
     5521 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
     5581 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
     5641 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
     5701 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
     5761 accccgtaat tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg
     5821 cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt
     5881 cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca
     5941 acacacaagc agggagcaga tactggctta actatgcggc atcagagcag attgtactga
     6001 gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg
     6061 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6121 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6181 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6241 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6301 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     6361 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     6421 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6481 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6541 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6601 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6661 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6721 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6781 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6841 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcg
//
