LOCUS       sequence-430791-        6971 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 6971)
  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 6971)
  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: DRD5-DuET
FEATURES             Location/Qualifiers
     source          1..6971
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      264..852
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     753..772
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1006..1023
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1140..1161
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1176..1181,1182..1199,1200..1206)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1214..1230
                     /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     1219..1241
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1238..1254
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1238..1254
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1303..1424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1314..1333)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1368..1387
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1598..2392)
                     /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(1709..1728)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2319..2338
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2459..2788)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2473..2608
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2527..2546)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2773..2793
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2802..3230)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2912..2933)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3124..3143
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3276..3500
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3489..3506
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5186..5205)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5187..5205)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5250..5453)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5279..5299)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        5454..5833
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6005..6024
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6099..6203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6204..6272,6273..6971,1..93)
                     /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(6422..6441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
       61 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
      121 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
      181 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
      241 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
      301 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
      361 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaataccgt tcttctagtg
      421 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
      481 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
      541 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
      601 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
      661 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
      721 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      781 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      841 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      901 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      961 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     1021 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     1081 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     1141 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     1201 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     1261 tacgccaagc tctagctaga ggtcgacggt atacagacat gataagatac attgatgagt
     1321 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     1381 ctattgcttt atttgtaacc attataagct gcaataaaca agttggggtg ggcgaagaac
     1441 tccagcatga gatccccgcg ctggaggatc atccagccgg cgtcccggaa aacgattccg
     1501 aagcccaacc tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc
     1561 gtcgcttggt cggtcatttc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
     1621 gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt
     1681 cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat
     1741 agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca
     1801 ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca
     1861 tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
     1921 gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt
     1981 tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat
     2041 cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg
     2101 gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg
     2161 cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
     2221 tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc
     2281 ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc
     2341 tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg
     2401 atcctcatcc tgtctcttga tcagatccga aaatggatat acaagctccc gggagctttt
     2461 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggcag
     2521 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     2581 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     2641 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     2701 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     2761 gggactttcc acaccctaac tgacacacat tccacagaat taattcgcgt taaatttttg
     2821 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2881 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2941 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3001 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3061 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3121 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3181 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ggggataccc
     3241 cctagagccc cagctggttc tttccgcctc agaagccata gagcccaccg catccccagc
     3301 atgcctgcta ttgtcttccc aatcctcccc cttgctgtcc tgccccaccc caccccccag
     3361 aatagaatga cacctactca gacaatgcga tgcaatttcc tcattttatt aggaaaggac
     3421 agtgggagtg gcaccttcca gggtcaagga aggcacgggg gaggggcaaa caacagatgg
     3481 ctggcaacta gaaggcacag tcgaggctga tcagcgggtt taaacgggcc ctctagactc
     3541 gagttaggca ggggccacct gagaagtctc ggtcttagac acggtggtgc tggcctcatc
     3601 gaattcaccg gtgcgtccac cggtatcgat gtgaaatccg ttgggcgtaa agggagtgat
     3661 tttatccagt gatatttcac cttcacagtc cagctcccac acagattcag cgactgggtc
     3721 gccgtcaggg ctcgtctgat aaatctggaa catccgatcg aaggggccct cctcctcgtc
     3781 gttatccacc tccctgtttc caggtgtaac agcattgggc atcatgtgga tgtatgcagc
     3841 tgcaatctct ttgtggaata cgatgtcctg gttgtaggag atgagttcat tagagatgtt
     3901 gactgtttcc acaggcgttc gtgagcaaaa atgactacaa cccagcagct gcgcgaagac
     3961 tttctgaaaa tcagcgttaa atgcgtagat tacaggattc aaagagctat ttgcccaccc
     4021 aaaccacaca aagacgtcga aagtcgtctc acttacacaa ggaaaccctg cggggggccc
     4081 ttctgggtgc ccagagcaaa atgggaccat gcaattcaga atgaagaagg ggagccaaca
     4141 gcacacgaag acgcccataa tcacgctcag agtcttgagg accttggttt ctttcttgat
     4201 agaagctctg agagaggtgt cgggagcaca agcagcggat gagcggcagg attgagcgtg
     4261 ctcagcggcg cgctccaggc tagagatcct tcgtatttga acttgagcga ttctatagat
     4321 ccttgtgtaa gtcactatca ttatagcgac cggaatgtag aaggagatca gactggaact
     4381 aatagcatag gtccgattaa ggctactatc acaattctct gcgttgacgt ctggttccca
     4441 gaaatcttcc tcccatgggg tccaattggc caggttattt ggcagatcca gcccgcccca
     4501 acttgcagcc tggtccctgt gccaattcaa ctgaacagga atgaaagaaa tcaggatgga
     4561 gagggtccag gccagtccga ccatcacaag ggccatcctc tgggtcatct ttcttttata
     4621 gcggaacggc ctgctaattg cccagtagcg atcgacggaa atcacgcaca gattgagaat
     4681 actggccgtg gaacacataa tatcaaaggc cacccaaaca tcgcaaaatg cgccaaaagg
     4741 ccaatatccg gccacttctg ccaccgcctt ccagggcatc acgaggagcg caacgaacag
     4801 gtcgctcact gccaggctca ctatgaacac gtttgtcatg ttagctcgca ggtggcgaga
     4861 gcgcacaatt gctgcacaaa ccaggacgtt gcccagcaga gtccatatta tcagaagggt
     4921 cagaagacag gcagtcacga cctgacttgg ccccaaaggt ggagcgcctg cgctgccccc
     4981 gacggcatta ccctgggcaa gttgttggta gagggcgaac tgcccgggat acgccgtacc
     5041 attgctccct ggtggcaaca tatcgatgct ggcgtcatca tcgtccttgt agtcggcgaa
     5101 taccaggcag aagatgtagc tcagggcgat gatcgtcttc atggtggcgc taagcttaag
     5161 tttaaacgct agccagcttg ggtctcccta tagtgagtcg tattaatttc gataagccag
     5221 taagcagtgg gttctctagt tagccagaga gctctgctta tatagacctc ccaccgtaca
     5281 cgcctaccgc ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt
     5341 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
     5401 cgtgagtcaa accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta
     5461 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac
     5521 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc
     5581 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     5641 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     5701 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     5761 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtc
     5821 aataatcaat gtcaacgcgt atatctggcc cgtacatcgc gaagcagcgc aaaacgccta
     5881 accctaagca gattcttcat gcaaatgtcg gtcaagcctt gccttgttgt agcttaaatt
     5941 ttgctcgcgc actactcagc gacctccaac acacaagcag ggagcagata ctggcttaac
     6001 tatgcggcat cagagcagat tgtactgaga gtgcaccata ggggatcggg agatctcccg
     6061 atccgtcgac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6181 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6421 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     6481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6721 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6961 cctcccgtat c
//
