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pNHK60

Expression clone for an auxin-based degron (AID) system, GFP-aid-NLS.

Catalog number RDB08468
Resource name pNHK60
Alternative name OsTIR1-9Myc::IRES::GFP-aid-NLS (kan/neoR)
Clone info. Expression clone for an auxin-based degron (AID) system. GFP-aid-NLS. Refer Supplementary figure 10 (pIRES OsTIR1-EGFP-IAA17) of Nat. Methods, 6, 917-922, 2009; PMID: 19915560.
Please note that (1) the degron system may not work well in T-antigen expressing cells such as HEK293T because pNHK60 has the SV40 replication origin and the expression level of GFP-degron is quite high, (2) the depositor found that degradation is efficient in stable cells rather than in transiently transiently transfected cells. Accession numbers, OsTIR1 (GeneID 4335696); AtIAA17 (GeneID 839568). To make 0.5 M stock solution, dissolve indole-3-acetic acid (IAA) (Sigma-Aldrich, 45533) or 1-naphthaleneacetic acid (NAA) (Sigma-Aldrich, 35745) in 100 % DMSO. IAA solution can be made in 100 % ethanol. Aliquots can be kept for 6 months in the dark at -20oC (Devrekanli, A., Kanemaki, M.T. Methods in Molecular Biology, 1369, 257-278, 2016).
Vector backbone pIRES-EGFP (Plasmid)
Selectable markers Kan^r
Gene/insert name Oryza sativa OsTIR1 cDNA Arabidopsis thaliana AtIAA17 cDNA Aequorea victoria GFP cDNA
Depositor|Developer Kanemaki, Masato |
Other clones in our bank

External Database
Oryza sativa OsTIR1 Arabidopsis thaliana AtIAA17 Aequorea victoria GFP

        Reference sequence
          

        Distribution information

        Please check terms and conditions set forth by the depositor, which are specified in the RIKEN BRC Catalog and/or Web Catalog.
        Terms and conditions set forth by the DEPOSITOR In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of the following literature(s) designated by the DEPOSITOR is requested (Nishimura, K., Nat. Methods., 6, 917-922 (2009)). This DNA clone is used for academic research purpose only. This is not to be used on a commercial basis. For use of this DNA clone except for internal academic research, the RECIPIENT must contact a NIG license representative at chizai@nig.ac.jp.
        Ordering Please visit Information of Request for Distribution.[link] 
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        Material Transfer Agreement (MTA for use for not-for-profit academic purpose) [Word]
        Schedule A (GFP-A) [Link]
        Remarks
        Please use Schedule A (GFP-A) additionally.
        Distribution information (domestic users) [open/close]


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        RDB08468 pNHK60 DNA solution

        Ordering Information [in Japanese] [in English]
        Please review the QC test results indicated by check icon below as well as clone information before placing your order.

        References and tips

        Electronic file

        Sequence RDB08468z.seq
        Remarks, protocol and/or map (pdf) RDB08468.pdf

        Original reference

        original Nishimura, K., An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods. 6(12), 917-922, (2009). PMID 19915560.
        original Kanemaki, M., Method for inducing degradation of protein in mammalian cell. Patent Abstracts WO2010125620A1.

        Further references such as user reports and related articles (go to bottom)

        Featured content

        Featured content Auxin Inducible Degron (AID) System (English text)
        Featured content Auxin Inducible Degron (AID) System (Japanese text)

        Sequence information

        RIKEN BRC has sequenced portions of this material for quality test.
        Please review the QC test results indicated by check icon as well as clone information before placing your order.

        Test sheet RDB08468_A5Dep1-2.pdf check

        Nucleotide sequence of a portion of this resource (if available).

        Primer: CMV_Forward
        Sequence file: RDB08468_A5Dea.seq check
        >08468_08468_A5De_CMV-Forward_G08_20.ab1
            1 CATCAACTCC GCCCCATTGA CGCAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA
           61 AGCAGAGCTG GTTTAGTGAA CCGTCAGATC CGCTAGCGCT ACCGGACTCA GATCTCGAGA
          121 TGACGTACTT CCCGGAGGAG GTGGTGGAGC ACATCTTCAG CTTCCTGCCG GCGCAGCGCG
          181 ACCGCAACAC GGTCTCGCTC GTCTGCAAGG TGTGGTACGA GATCGAGAGG CTGAGCCGCC
          241 GCGGCGTCTT CGTGGGCAAC TGCTACGCCG TGCGCGCCGG CCGCGTCGCC GCGCGGTTCC
          301 CCAACGTGCG GGCGCTCACG GTGAAGGGGA AGCCCCACTT CGCCGACTTC AACCTCGTGC
          361 CCCCCGACTG GGGCGGCTAC GCGGGGCCGT GGATCGAGGC GGCCGCGAGG GGATGCCACG
          421 GCCTGGAGGA GCTCAGGATG AAGCGGATGG TGGTGTCCGA CGAGAGCCTC GAGCTGCTGG
          481 CTCGCTCGTT CCCGCGGTTC AGGGCTCTTG TTCTTATCAG CTGCGAGGGG TTCAGCACTG
          541 ACGGGCTAGC CGCCGTCGCG AGCCATTGCA AGCTTCTGAG GGAGTTGGAT TTGCAGGAAA
          601 ATGAAGTGGA GGATCGAGGG CCTAGGTGGC TTTCCTGCTT CCCTGATTCC TGCACATCAC
          661 TTGTCTCATT GAATTTTGCC TGCATCAAAG GGGAGGTTAA TGCTGGTTCA CTGGAGAGAC
          721 TTGTTAGCAG GTCCCCAAAC CTGCGGAGTT TGAGGCTGAA TCGATCTGTA TCGGTAGATA
          781 CACTTGCAAA GATACTACTG CGTACCCCTA ACTTGGAGGA TTTGGGGACA GGGAATTTGA
          841 CAGATGACTT CCAAACTGAG TCCTACTTTA AGCTTACCAG TGCTCTGGAG AAATGCAAGA
          901 TGTTGAGGAG TTTGTCTGGA TTCTGGGATG CTTCTCCTGT TTGCCTGTCA TTTATCTACC
          961 CCCCTGTGTG CTCAACTGAC AGGATTGAAC TTGAGCTATG CACCCACACT TGATGCTTCT
         1021 GACCCTTACA AAAATGATTA GCCGCTGTGT GAAGCTCCAA CGCCTTTGGG TACTGGATGT
         1081 ATCTCGGGAC AAGGGCTGCA GTTGATTGCC TCAAGCTTGC AAGACTTTGC AAGAACTCTC
        1141 AGGGATATTT TTGCAT
        //
        Primer: ME-1290Rv
        Sequence file: RDB08468_A5Deb.seq check
        >08468_08468_A5De_ME-1290Rv_E04_13.ab1
            1 ATGCAATTCA ATTTTATGTT TCAGGTTCAG GGGGAGGTGT GGGAGGTTTT TTAAAGCAAG
           61 TAAAACCTCT ACAAATGTGG TATGGCTGAT TATGATCTAG AGTCGCGGCC GCTGGGTACC
          121 TTAAACCTTA CGTTTCTTTT TAGGGACCTT TCTTTTCTTC TTGGGGGCGC CAGCACCAGC
          181 TCTGCTCTTG CACTTCTCCA TCGCCCTCGG AGCGAGACCA ATGGCATCCG ATCCTTTCAT
          241 GAGACGTAAA CGCTTGCATG TATCGACGAA CATTGGCCAA GGAACGTCGC CGACGAGCAT
          301 CCAATCACCG TCTTTGTCTT CATAAGAGGG AACATAGTCC CAGCTATTCA CCAAATCCAT
          361 CAATTTCCTC TCATTCATGA AGTCTATCAT TCCTTCTTCT CCTCCATGTT TGCCCATGGT
          421 AAAAGAGCTG AACATGTTGG ACAAAGCATT AGAAAGCTCA TCGTAGCTTT TATACATCCT
          481 CAAATCGATT TTCCTCAAGT ACGGTGCTCC GTCCATTGAT ACCTTCACGA ACGCCGCCGC
          541 CTCCGGGCCA CCGCTTGATT TTTGGCAGGA AACCATCACG TTCTTCCGGT ATGATCTCAC
          601 CGGTGGCCAT CCCACAACTT GTGCCTTGGC CGGAGGTTTG GCTGGATCTT TAGGACAAGC
          661 ACTCTTCTCC TTGGAATCAA AAGTCACGAC GTCATGAGTC GTAGATCCTT CCTTGTTTGC
          721 AGGCTCATTA TTCAGATTTA GCTTCAGATC AACCGTCTCT GAGAACCCTC TCTTGTTTCC
          781 GGTTACCGGA GCCACTGTAT CTCCACCGGG AAGACCAAGA CACAGCTCAG TCTCCCTCAG
          841 ATTCAGCTCG ACACTGCCCA TCATGGGCCC GGCACCCGCT CCAGCGCCTG CACCAGCTCC
          901 CTTGTACAGC TCGTCCATGC CGAGAGTGAT CCCGGCGGCG GTCACGAACT CCAGCAGGAC
          961 CATGTGATCG CGCTTCTCGT TGGGGTCTTT TGCTCAGGCG GACTGGTGCT CAGGTAGTGG
         1021 TTTGTCGGGC AGCAGCACGG GGTCGTCGCG ATGGGGGGTG TTTCTGCTGT AGTGATCGGC
         1081 GAGCTGCACG CTGCCGTCTT CGAATGGTGT GGCCGGAATC CTTGAGGTTC ACCCTTTGAT
        1141 GACCGGAT
        //
        Primer: IRES-F
        Sequence file: RDB08468_A5Dec.seq check
        >08468_08468_A5De_IRES-F_A09_03.ab1
            1 CGTTCCCTTT AATGTGTTTA GTCGAGGTTA AAAACGTCTA GGCCCCCCGA ACCACGGGGA
           61 CGTGGTTTTC CTTTGAAAAA CACGATGATA ATATGGCCAC AACCATGGTG AGCAAGGGCG
          121 AGGAGCTGTT CACCGGGGTG GTGCCCATCC TGGTCGAGCT GGACGGCGAC GTAAACGGCC
          181 ACAAGTTCAG CGTGTCCGGC GAGGGCGAGG GCGATGCCAC CTACGGCAAG CTGACCCTGA
          241 AGTTCATCTG CACCACCGGC AAGCTGCCCG TGCCCTGGCC CACCCTCGTG ACCACCCTGA
          301 CCTACGGCGT GCAGTGCTTC AGCCGCTACC CCGACCACAT GAAGCAGCAC GACTTCTTCA
          361 AGTCCGCCAT GCCCGAAGGC TACGTCCAGG AGCGCACCAT CTTCTTCAAG GACGACGGCA
          421 ACTACAAGAC CCGCGCCGAG GTGAAGTTCG AGGGCGACAC CCTGGTGAAC CGCATCGAGC
          481 TGAAGGGCAT CGACTTCAAG GAGGACGGCA ACATCCTGGG GCACAAGCTG GAGTACAACT
          541 ACAACAGCCA CAACGTCTAT ATCATGGCCG ACAAGCAGAA GAACGGCATC AAGGTGAACT
          601 TCAAGATCCG CCACAACATC GAGGACGGCA GCGTGCAGCT CGCCGACCAC TACCAGCAGA
          661 ACACCCCCAT CGGCGACGGC CCCGTGCTGC TGCCCGACAA CCACTACCTG AGCACCCAGT
          721 CCGCCCTGAG CAAAGACCCC AACGAGAAGC GCGATCACAT GGTCCTGCTG GAGTTCGTGA
          781 CCGCCGCCGG GATCACTCTC GGCATGGACG AGCTGTACAA GGGAGCTGGT GCAGGCGCTG
          841 GAGCGGGTGC CGGGCCCATG ATGGGCAGTG TCGAGCTGAA TCTGAGGGAG ACTGAGCTGT
          901 GTCTTGGTCT TCCCGGTGGA GATACAGTGG CTCCGGTAAC CGGAAACAAG AGAGGGTTCT
          961 CAGAGACGGT TGATCTGAAG CTAAATCTGA ATAATGAGCC TGCAAACAAG GAAAGGATCT
         1021 ACGACTCATG ACGTCGTGAC TTTTGATTCC AAGGAGAGGA GTGCTTTGTC CTAAAGATCC
         1081 AGCAATCTTC GGGCCAAGGC ACAAGTTGTG GGATGCACGA TGAGATCATA CCGGAAGAAC
        1141 GTGATGATTT CCTGGCCCAG A
        //
        Primer: IRES-R
        Sequence file: RDB08468_A5Ded.seq check
        >08468_08468_A5De_IRES-R_B09_06.ab1
            1 GCAAAAACCG GGCCTTCGGC CAGTAACGTT AGGGGGGGGG GAGGGAGAGG GGCGGATCCC
           61 GGGGATCTCT TGAATGATCG TTCCACTTTT TAGCTAGTGG ATCCGTTCAA GTCTTCTTCT
          121 GAGATTAATT TTTGTTCACC GTTCAAGTCT TCCTCGGAGA TTAGCTTTTG TTCACCGTTC
          181 AAATCTTCTT CAGAAATCAA CTTTTGTTCA CCTCTAGAGG ATCCGTTCAA GTCTTCTTCT
          241 GAGATTAATT TTTGTTCACC GTTCAAGTCT TCCTCGGAGA TTAGCTTTTG TTCACCGTTC
          301 AAATCTTCTT CAGAAATCAA CTTTTGTTCA CCTCTAGAGG ATCCGTTCAA GTCTTCTTCT
          361 GAGATTAATT TTTGTTCACC GTTCAAGTCT TCCTCGGAGA TTAGCTTTTG TTCACCGTTC
          421 AAATCTTCTT CAGAAATCAA CTTTTGTTCA CCACTAGCAG CAGAACCGGA GTCGACTAGG
          481 ATTTTAACAA AATTTGGTGC ATCATCCCTC GCCCCAGCAG TTGTGCGGTA CACATATAAT
          541 TTCTCCACTT TAGGCAGATC TCCATGGTTT TCCTCCATTT CATTGCTACC ATCCCGCTCA
          601 TTTATGACCT CAACATTGAG CATCGGCATC TTTGACGCAA GGACTTGGCA CCCCTTTAAC
          661 GTGACATTGC AAGATGACAT CCAAAGGGAT CGCATTGTCT CGTACCTAGC AAAATTCCCC
          721 AAGAGTGCAG CATCACCAAA CGGGCTATCT CTTATCTCCA GTTTCCTTAA ATTCTTGCAT
          781 CCATTCATAA CATGCATCAT ACCCTTATCA CTGTCACCAG CAAATGCTAT AGAAAGCATC
          841 TCAAGTTGTT TTGCATATTT CCCAATATAC ATGAAAACTT TGTCTGTGAG AAGACCAGAT
          901 ATTGACAAAC GTTGTAATCC CTTGCACTCA CGAACAATAG CTCCAAAGCC TTCATCTAAT
          961 GGTTGGCTTG TCACAACATC AGGCTTCCCT GGCTCAAGAA TACAAAGTCT GAATCGTGTG
         1021 AAATTTGGAC AGTTCTTGGC GACAGTAACT AGTGCAGCAT TAGTCATTTG GTGACAGAAG
         1081 TACAGTAGTG AGTCAGTTTT GGACAGCCAG GATACTGCAC AGGTTCCTTC GTCTGGTCAC
        1141 TGCCAGATAC CCAGCTACGT TAAGAAAATC TCTGAATGTG
        //
        Primer: SV40proF_V2
        Sequence file: RDB08468_A5Dee.seq check
        >08468_08468_A5De_SV40proF_V2_F04_16.ab1
            1 GACGGGCTTT TTTGGAGGCC TAGGCTTTTG CAAGATCGAT CAAGAGACAG GATGAGGATC
           61 GTTTCGCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG
          121 GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG
          181 GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA
          241 TGAACTGCAA GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC
          301 AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC
          361 GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA
          421 TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA
          481 ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT
          541 GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT
          601 GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT
          661 GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA
          721 TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA
          781 CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG
          841 CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGGTTC GAAATGACCG ACCAAGCGAC
          901 GCCCAACCTG CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAG GTTGGGGCTT
          961 CGGAATCGTT TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA
         1021 GTTCTTTCGC CCACCCTAGG GGGAGGCTAA CTGAAACACG GAAGGAGACA TACCGGAGAC
         1081 CCGCGCTATG ACGGCATTAA AAGACCGATT AAACGCACGG TGATGGGATC GGATTGGTTC
        1141 ATACGCGGAG TCGGATTCCA GGATCTGTGG CA
        //

        Please visit Sequencing and PCR primers for primer information.


        References

        Original, user report and related articles

        original Nishimura, K., An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods. 6(12), 917-922, (2009). PMID 19915560.
        original Kanemaki, M., Method for inducing degradation of protein in mammalian cell. Patent Abstracts WO2010125620A1.
        reference Devrekanli, A., Conditional Budding Yeast Mutants with Temperature-Sensitive and Auxin-Inducible Degrons for Screening of Suppressor Genes. Methods in Molecular Biology 1369: 257-278 (2016). PMID 26519318.
        reference Delacour, Q., Light-Activated Proteolysis for the Spatiotemporal Control of Proteins. ACS Chem. Biol.10 (7): 1643-1647 (2015). PMID 25938742.
        reference Wend, S., A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics. Sci. Rep. 3: 2052 (2014). PMID 23787479.
        reference Farr, C.J., The alpha isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells. Nucleic Acids Res. 42 (7): 4414-4426 (2014). PMID 24476913.
        reference Nishimura, K., Rapid Depletion of Budding Yeast Proteins via the Fusion of an Auxin-Inducible Degron (AID). Curr. Protoc. Cell Biol. \64: 20.9.1-16 (2014). PMID 25181302.
        reference Kampf, M.M., Rewiring and dosing of systems modules as a design approach for synthetic mammalian signaling networks. Mol. Biosyst. 8 (6): 1824-1832 (2012). PMID 22532387.
        reference Holland, A.J., Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 109 (49): E3350-7 PMID 23150568.

        2021.11.02

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