LOCUS       sequence-430886-        6719 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 6719)
  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 6719)
  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: PK1-DuET
FEATURES             Location/Qualifiers
     source          1..6719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(113..217)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(292..311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        483..862
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        863..1066
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1017..1037
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1111..1130
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1111..1129
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2558..2575)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2564..2788
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2834..3262
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2921..2940)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3131..3152
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3271..3291)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3276..3605
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3456..3591
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3518..3537
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3672..4466
                     /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(3726..3745)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4336..4355
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4640..4761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4677..4696)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4731..4750
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4810..4826)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4810..4826)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4823..4845)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4834..4850
                     /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)."
     promoter        complement(join(4858..4864,4865..4882,4883..4888))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4903..4924
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5041..5058)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5212..5800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5292..5311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5971..6719,1..43,44..112))
                     /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     6594..6613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
       61 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      121 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      181 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      241 ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca gtacaatctg
      301 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
      361 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca tttgcatgaa
      421 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      481 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      541 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      601 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      661 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      721 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      781 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      841 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      901 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      961 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     1021 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
     1081 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
     1141 tggctagcgt ttaaacttaa gcttagcggc cgcgccacca tgaagacgat catcgccctg
     1201 agctacatct tctgcctggt attcgccgac tacaaggacg atgatgacgc cagcatcgat
     1261 atggagacaa caatgggctt tatggatgat aacgcgacta atacctccac gagtttcctg
     1321 tccgtgctta acccgcacgg tgcccatgcc acgtccttcc cattcaactt ctcatacagt
     1381 gactacgata tgcccctgga tgaggatgaa gacgtgacta actctcggac tttttttgcc
     1441 gccaagattg ttatcggtat ggctctcgtc ggaatcatgc ttgtctgcgg tattggaaac
     1501 tttattttta tagcggcact ggtacggtac aaaaaactgc ggaatctcac caatctgctg
     1561 attgctaacc tggcgatctc agacttcttg gtcgccattg tttgctgtcc tttcgagatg
     1621 gattactatg tggtgagaca gctgtcttgg gaacacggac atgtgctctg taccagtgtt
     1681 aactatctca ggaccgtaag tctgtatgtt agtactaacg cgctccttgc cattgctatt
     1741 gaccgatatt tggcaattgt acatccactt agaccacgga tgaagtgcca gactgccacc
     1801 gggctgatag ccctcgtgtg gacagttagt atcctgatcg ccatcccctc agcctacttt
     1861 accacagaga ccgtcctcgt gattgtaaaa tcacaggaga agatcttctg cggacaaata
     1921 tggcccgttg atcagcaact ctactacaag agctacttcc tgtttatttt cgggatcgag
     1981 ttcgtgggcc cagttgtcac catgaccctc tgttacgcta gaatcagcag agagctttgg
     2041 ttcaaagctg tacctggttt ccagacagaa cagatccgga agcgcctgag atgcaggagg
     2101 aaaactgtcc ttgtcctcat gtgtatcctt accgcctatg ttctgtgctg ggcgcctttc
     2161 tacgggttca cgatcgttcg ggatttcttc ccaacagtgt tcgtgaagga gaagcattat
     2221 ttgacagcct tttacattgt ggagtgtatt gctatgtcca actctatgat caacacgctt
     2281 tgtttcgtta ccgtcaagaa cgataccgtg aagtacttca agaaaattat gctgctgcac
     2341 tggaaggcct cttacaatgg gggaaaatct tcagccgatc tcgatctgaa aaccatcggc
     2401 atgccagcga ccgaggaggt ggattgcatt cggctcaaga tcgataccgg tggacgcacc
     2461 ggtgaattcg atgaggccag caccaccgtg tctaagaccg agacttctca ggtggcccct
     2521 gcctaatcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2581 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2641 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2701 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2761 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2821 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2881 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2941 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3001 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3061 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3121 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3181 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3241 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3301 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3361 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3421 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3481 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3541 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3601 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3661 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3721 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3781 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3841 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3901 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     3961 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4021 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4081 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4141 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4201 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4261 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4321 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4381 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4441 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4501 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4561 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4621 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4681 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4741 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4801 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4861 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4921 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4981 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5041 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5101 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5161 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5221 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5281 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5341 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5401 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5461 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5521 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5581 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5641 ctacactaga agaacggtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5701 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5761 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5821 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5881 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5941 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6001 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6061 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6121 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6181 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6241 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6301 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6361 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6421 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6481 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6541 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6601 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6661 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcaccca
//
