LOCUS       sequence-430759-        6824 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 6824)
  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 6824)
  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: CCKAR-DuET
FEATURES             Location/Qualifiers
     source          1..6824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(160..179)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      973..1561
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1462..1481
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1715..1732
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1849..1870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1885..1890,1891..1908,1909..1915)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1923..1939
                     /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     1928..1950
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1947..1963
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1947..1963
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2012..2133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2023..2042)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2077..2096
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2307..3101)
                     /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(2418..2437)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3028..3047
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3168..3497)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3182..3317
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3236..3255)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     3482..3502
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3511..3939)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3621..3642)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3833..3852
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    3985..4209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4198..4215
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     primer_bind     complement(5748..5767)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5749..5767)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(5812..6015)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(5841..5861)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        6016..6395
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     6567..6586
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6661..6765
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6766..6824,1..10,11..802)
                     /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"
ORIGIN
        1 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
       61 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
      121 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
      181 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
      241 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
      301 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
      361 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
      421 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
      481 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
      541 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
      601 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
      661 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
      721 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      781 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
      841 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
      901 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      961 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1021 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1081 ggtaactggc ttcagcagag cgcagatacc aaataccgtt cttctagtgt agccgtagtt
     1141 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1201 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1261 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1321 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1381 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1441 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1501 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1561 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1621 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1681 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1741 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1801 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     1861 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     1921 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     1981 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     2041 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     2101 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     2161 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     2221 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     2281 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     2341 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     2401 ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc
     2461 acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc
     2521 ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg
     2581 agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga
     2641 tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg
     2701 tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg
     2761 gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc
     2821 aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg
     2881 cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt cagggcaccg
     2941 gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg
     3001 gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa
     3061 gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct
     3121 gtctcttgat cagatccgaa aatggatata caagctcccg ggagcttttt gcaaaagcct
     3181 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggcaga ggcggcctcg
     3241 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     3301 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     3361 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     3421 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     3481 caccctaact gacacacatt ccacagaatt aattcgcgtt aaatttttgt taaatcagct
     3541 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3601 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3661 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3721 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3781 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3841 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3901 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
     3961 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
     4021 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
     4081 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
     4141 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
     4201 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactcg agttaggcag
     4261 gggccacctg agaagtctcg gtcttagaca cggtggtgct ggcctcatcg aattcaccgg
     4321 tgcgtccacc ggtatcgatc tgaggcggca cggatgcgga catgtggctg tagctgaagc
     4381 gtgaaaggga tgcgccggtt gtgcctcctt cctcttcctc tcccacctct ccgcgtgccc
     4441 ccggcgggcc aggatttggg cagcaaggaa acgtagccat gaaaccgagt ctgaatcttt
     4501 tgttcatgaa gcagtatatg atggggttaa cacaacttga cgtgtagctc agaagcagga
     4561 taaagctgat gggagtgccg gagagtcgcc gctcagcact ggcggtgtca tacgctctcc
     4621 aggcgttggc gctaaatatt ggcatccagc acaaaaagaa cagaacaact atgactatca
     4681 acatcctaat gactctcttt ttagccatca ggttggcggc agagctatta ctgcggatcc
     4741 tatttgccct ggaggaactt cccgtggaca gctgcctcag ctcaagtttc ctcggggggc
     4801 gagtcttctg cagataacag ccatcggaat cctcgtattt gccactggaa gtagtgctag
     4861 gcttcctttc tttagcactc ttcttctggg acgcctcgaa ttttataccc tgatacagct
     4921 ccagagatat gaggccgtac gcaaccatca ttacaatgcc gggaatcaga aacaggatga
     4981 gcagcaggaa cgtatgccag ctttgctgca tgacatcatt tggcaagagg aacctacaca
     5041 tgttagcggt ctggttatta ttttttgtga agggaaccag attactgtaa atgggatatg
     5101 gtgtcatgat tgtaaagctc agacaccaag ttgctgcaat caccttaaga gcatggctct
     5161 tagtctgcca caccctactt tggagaggct tgcaaatagc gccatagcgc tccagggata
     5221 tggcaacgag gttgaaagtg ctgacggaca cactcgtgcc catgaagtag gtggtcgtct
     5281 tgcacacagc gctgccaaag atgaaatctt tcaagagatt ggggatcaag ttgaatggca
     5341 tacagaacag acaaagcatc aaatcactaa cagccagaga gagcagaaag atattagtca
     5401 cagttctcat cctcttgttc cttatcaaca cggtgatgac caaagtattt cccagaacgg
     5461 aaagcaggaa gatcagagaa tagagcagta tctgaacggc aggctgccac tctttagagg
     5521 gccggggctg gtccaggcag aaaagggtct cgttttcgag gccaagctcg caaggtgggg
     5581 taatgttgga gccattcacg aggagactgt caaccacgtc catatcgatg ctggcgtcat
     5641 catcgtcctt gtagtcggcg aataccaggc agaagatgta gctcagggcg atgatcgtct
     5701 tcatggtggc gctaagctta agtttaaacg ctagccagct tgggtctccc tatagtgagt
     5761 cgtattaatt tcgataagcc agtaagcagt gggttctcta gttagccaga gagctctgct
     5821 tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt
     5881 acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat
     5941 ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg
     6001 ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg
     6061 aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac
     6121 gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta
     6181 ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac
     6241 atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt
     6301 taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat
     6361 tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg cccgtacatc
     6421 gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaaatgt cggtcaagcc
     6481 ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca acacacaagc
     6541 agggagcaga tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     6601 taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg
     6661 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     6721 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     6781 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcc
//
