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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 Kanemaki, Masato |
 
Other clones in our bank Oryza sativa LOC4335696 | Arabidopsis thaliana AXR3 (KEGG orthology K14484) | Aequorea victoria |

Distribution information

Please check terms and conditions set forth by the depositor, which are specified in the RIKEN BRC Catalog and/or Web Catalog.
cDNA Request [in Japanese] [in English]
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.
(Japanese text [open/close])
Remarks ((Additional form)) GFP-A
Please use Schedule A (GFP-A) additionally [Link]
(Japanese text [open/close])

Catalog # Resource name Shipping form Fee (non-profit org.)
RDB08468 pNHK60 DNA solution

Sequence information

RIKEN BRC has sequenced portions of this material for quality test. Primers and the results are shown below.

Data are summerized on test sheet below.

Test sheet RDB08468_A5Dep1.pdf

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

Primer: CMV-Forward RDB08468_A5Dea.seq
>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 RDB08468_A5Deb.seq
>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 RDB08468_A5Dec.seq
>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 RDB08468_A5Ded.seq
>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: SV40pro_F_V2 RDB08468_A5Dee.seq
>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 plasmidSequencing and PCR primers for primer information.

References and tips

Electronic file

Electronic file Sequence RDB08468z.seq from Depositor
Electronic file PDF RDB08468.pdf from Depositor

Featured content

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

References

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 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.

2017.10.23

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