RDB15088 - pcDNA5-GAPDH-HaloTag

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pcDNA5-GAPDH-HaloTag (#RDB15088)

Expression vector for the visualization of chaperone-mediated autophagy (CMA).

Alternative name	GAPDH-HT/pcDNA5/FRT
Clone info.	Expression vector of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), tagged with C-terminal HaloTag, for genomic integration. Translocation of the GAPDH-HT labeled with an appropriate fluorescent dye as a substrate of chaperone-mediated autophagy (CMA) from cytosol to lysosome can be detected. Refer Fig. 1 of Seki, T., et al., PLoS ONE 7 (2), e31232, 2012.
Comment	Expression was confirmed by the depositor with immunostaining.
Vector backbone	pcDNA5/FRT (Plasmid)
Size of vector backbone	5.1 kb
Selectable markers	Amp^r (E. coli), Hyg^r (mammalian cells).
Growth conditions	LB+Amp, 37oC
Gene/insert name	Human GAPDH cDNA
Depositor\|Developer	Seki, Takahiro \|

Distribution information

Please check terms and conditions set forth by the depositor, which are specified in the RIKEN BRC Catalog and/or Web Catalog.

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Terms and conditions for distribution In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of literature designated by the DEPOSITOR is requested (Sato, M. et al. Genes Cells 21 (8): 861-873, 2016).
Additional terms and conditions:
The use of the BIOLOGICAL RESOURCE is restricted to the academic researches conducted by non-profit organization. By using this material, the RECIPIENT agrees to be bound by the conditions of the limited use statement of the Promega Corporation.

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Ordering forms	Order form [Credit Card Payment] [Bank Transfer Payment] [Example of order form ] MTA, for use for not-for-profit academic purpose [Word] [Example of MTA ] Please visit Information of Request for Distribution.[link]
Terms and conditions for distribution	In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of literature designated by the DEPOSITOR is requested (Sato, M. et al. Genes Cells 21 (8): 861-873, 2016). Additional terms and conditions: The use of the BIOLOGICAL RESOURCE is restricted to the academic researches conducted by non-profit organization. By using this material, the RECIPIENT agrees to be bound by the conditions of the limited use statement of the Promega Corporation.

必要書類	提供依頼書 [依頼書の記入例 ] 提供同意書 (MTA, 非営利学術目的用)[Word] [MTAの記入例 ] 手続きの概要は、「提供申込みについて[link]」をご覧ください。
MTAに書く使用条件	利用者は、研究成果の公表にあたって寄託者の指定する文献を引用する (Sato, M. et al. Genes Cells 21 (8): 861-873, 2016)。 MTAに書く付加的使用条件：本件リソースの使用は学術機関での学術研究に限る。本件リソースの使用にあたって、利用者はプロメガ社の限定使用条件に従う必要がある。

Catalog #	Resource name	Shipping form	Fee (non-profit org.)
RDB15088	pcDNA5-GAPDH-HaloTag	DNA solution	JPY 9,460 (not-for-profit academic purpose) plus cost of shipping containers, dry ice (if required) and shipping charge

Please review the QC test results indicated by check

icon below as well as clone information before placing your order.

How to cite this biological resource

Materials & Methods section:

The pcDNA5-GAPDH-HaloTag was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan (cat. RDB15088).

Reference section:

Sato, M., Seki, T., Konno, A., Hirai, H., Kurauchi, Y., Hisatsune, A., Katsuki, H., Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells. Genes Cells 21 (8): 861-873 (2016). PMID 27377049. [link to RRC of NBRP]

Seki,T., Yoshino, K.I., Tanaka, S., Dohi, E., Onji, T., Yamamoto, K., Hide, I., Paulson, H.L., Saito, N., Sakai, N., Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron. PLoS One 7 (2): e31232 (2012). PMID 22363588. [link to RRC of NBRP]

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

QC test results

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	RDB15088_A7F8p1-3.pdf

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

Primer: CMV_Forward (Pr0016) Region: CMV pro,T7 pro,insert 5'(hGAPDH 5') Sequence file: RDB15088_A7F8a.seq
>D04515A1_A7F8-1_CMV-Forward_A09_01_ABI08.ab1 1 ATTTTCACTT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT 61 ATAAGCAGAG CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT 121 ACGACTCACT ATAGGGAGAC CCAAGCTGGC TAGCGTTTAA ACTTAAGCTT GGTACCGAGC 181 TCGGATCCAC TAGTCCAGTG TGGTGGAATT CTGCAGATAT CACCATGGGG AAGGTGAAGG 241 TCGGAGTCAA CGGATTTGGT CGTATTGGGC GCCTGGTCAC CAGGGCTGCT TTTAACTCTG 301 GTAAAGTGGA TATTGTTGCC ATCAATGACC CCTTCATTGA CCTCAACTAC ATGGTTTACA 361 TGTTCCAATA TGATTCCACC CATGGCAAAT TCCATGGCAC CGTCAAGGCT GAGAACGGGA 421 AGCTTGTCAT CAATGGAAAT CCCATCACCA TCTTCCAGGA GCGAGATCCC TCCAAAATCA 481 AGTGGGGCGA TGCTGGCGCT GAGTACGTCG TGGAGTCCAC TGGCGTCTTC ACCACCATGG 541 AGAAGGCTGG GGCTCATTTG CAGGGGGGAG CCAAAAGGGT CATCATCTCT GCCCCCTCTG 601 CTGATGCCCC CATGTTCGTC ATGGGTGTGA ACCATGAGAA GTATGACAAC AGCCTCAAGA 661 TCATCAGCAA TGCCTCCTGC ACCACCAACT GCTTAGCACC CCTGGCCAAG GTCATCCATG 721 ACAACTTTGG TATCGTGGAA GGACTCATGA CCACAGTCCA TGCCATCACT GCCACCCAGA 781 AGACTGTGGA TGGCCCCTCC GGGAAACTGT GGCGTGATGG CCGCGGGGCT CTCCAGAACA 841 TCATCCCTGC CTCTACTGGC GCTGCCAAGG CTGTGGGCAA AGGTCATCCC TGAGCTGAAC 901 GGGAAGCTCA CTGGCATGGC CTTCCGTGTC CCCACTGCCA ACGTGTCAGT GGGTGGACCT 961 GACCTGCCGT CTAGAAAACC CTGGCCCAAT ATGATGACAT CAAGAAAGTT GTGGAGCAGC 1021 GTCGGAGGAC CCCTTCAAGG CATCCTTGGC TACACTGAGC ACAGATGATC TCCTCTGACC 1081 TTC //
Primer: HaloTag-N_R (Pr0492) Region: insert mid(HaloTag,TEV site,hGPDH 3') Sequence file: RDB15088_A7F8b.seq
>D04515A1_A7F8-1_HaloTag-N_R_B09_02_ABI08.ab1 1 ATAGCATCAT ATGGGGGTCG ATGGAAGCCA GTACCGATTT CGGATCCATC GTTATCGCTC 61 TGAAAGTACA GATCCTCAGT GGTTGGCTCG AGAGCTCGAG ATCCCTCCTT GGAGGCCATG 121 TGGGCCATGA GGTCCACCAC CCTGTTGCTG TAGCCAAATT CGTTGTCATA CCAGGAAATG 181 AGCTTGACAA AGTGGTCGTT GAGGGCAATG CCAGCCCCAG CGTCAAAGGT GGAGGAGTGG 241 GTGTCGCTGT TGAAGTCAGA GGAGACCACC TGGTGCTCAG TGTAGCCCAG GATGCCCTTG 301 AGGGGGCCCT CCGACGCCTG CTTCACCACC TTCTTGATGT CATCATATTT GGCAGGTTTT 361 TCTAGACGGC AGGTCAGGTC CACCACTGAC ACGTTGGCAG TGGGGACACG GAAGGCCATG 421 CCAGTGAGCT TCCCGTTCAG CTCAGGGATG ACCTTGCCCA CAGCCTTGGC AGCGCCAGTA 481 GAGGCAGGGA TGATGTTCTG GAGAGCCCCG CGGCCATCAC GCCACAGTTT CCCGGAGGGG 541 CCATCCACAG TCTTCTGGGT GGCAGTGATG GCATGGACTG TGGTCATGAG TCCTTCCACG 601 ATACCAAAGT TGTCATGGAT GACCTTGGCC AGGGGTGCTA AGCAGTTGGT GGTGCAGGAG 661 GCATTGCTGA TGATCTTGAG GCTGTTGTCA TACTTCTCAT GGTTCACACC CATGACGAAC 721 ATGGGGGCAT CAGCAGAGGG GGCAGAGATG ATGACCCTTT TGGCTCCCCC CTGCAAATGA 781 GCCCCAGCCT TCTCCATGGT GGTGAAGACG CCAGTGGACT CCACGACGTA CTCAGCGCCA 841 GCATCGCCCC ACTTGATTTT GGAGGGGATC TCGCTCCTGG AAAGATGGTG ATGGGGATTT 901 CCATTGATGA CAAGCTTTCC CGTTTCTCAG CCCTTGACGG TGCCATGGGA AATTTGCCCA 961 TGGGTGGGAA TCATATTTGG GAACATGTAA ACCCATGTAG TTTGAGGTTC CATTGAGGGG 1021 TCATTGATGG CCACCATATC AACTTACCAG AGTAAAAGGC CAGCCCCTTG GGTTGACCAG 1081 GGACGC //
Primer: BGH_rev2 (Pr0606) Region: bGHpA,insert 3'(HaloTag 3') Sequence file: RDB15088_A7F8c.seq
>D04515A1_A7F8-1_BGH_rev2_C09_03_ABI08.ab1 1 CATTAGGGTT CGGGAGGCAC GGGGGAGGGG CAAACAACAG ATGGCTGGCA ACTAGAAGGC 61 ACAGTCGAGG CTGATCAGCG GGTTTAAACG GGCCCTCTAG ACTCGAGCGG CCGCCCCAAG 121 GGGTTATGCT AGTTATTGCT CAGCGGTGGC AGCAGCCAAC TCAGCTTCCT TTCGGGCTTT 181 GTTAGCAGCC GGATCAGCTT GCATGCCTGC AGGTCGACTC TAGAATTCTA TTAACCGGAA 241 ATCTCCAGAG TAGACAGCCA GCGCGCGATC TCGCTGCCGA TCAGGTCCGG GTTGTCTTCT 301 TGCAGCAGAT TCAGACCCGG GCCGATGTCC ACAGCCTTGC AGTTAGGCAG GCTTTTGGCC 361 AGGCGAGCGG CTTCGGCCGG TGGGATCAGA ACGCCTGGGG TGCCCCAGAA CAGCAGCTTC 421 GGGACAGGGG ACTGGTGCAG CCAGTCCATG TATTCTTCGA CCAGCGCGAC GATGTTCGCT 481 GGCTCACCGG CGATTGGCAG CTCGTTTGGG AAGCGCCACA GTGGCTCGCG GTCAACAGGA 541 TTCAGGAACG GCTCGCGGTA ATGGTCCATC TCGACTTCAG TCAGCGGGCG GACGACACCC 601 ATCGGCAGCG TACCCTCGAT AAAAACGTTC TGATCGATGA TCAGCTTGCG GCCGACGTCG 661 GTGGTGCGGA AGGCCTGGAA GGTCTCGCGG GCAAATTCTG GCCATTCGTC CCAGGTCGGG 721 ATAGGGCGGA TGAACTCCAT AAATGCAATA CCTTTGACGC GCTCTGGATT GCGCTTGGCC 781 CAGTGGAAAC CCAGAGCGGA GCCCCAGTCG TGAATGACCA GGACGACCTC TTCCAGACCC 841 AGGGCTTCGA TGAAGGCATC CATGAAGCGG ACGTGGTCGT CGAAGAAATA ACCCAGGGTC 901 TGGTTTGTCG GATTTGCCCA TACCGATCAG GTCTGGGAGC AATGCAGCGA TGGGTCGGTG 961 CACATGCGGG ATGATGTTGC GCCACACGTA GAGGAGGTCG GGTTACCCGT GCCAGACAGC 1021 ACAGGGTGCA ATCGCGCGGA ACAACATCGA CGTAGTGCAT GCGCTCGCCA GGAACTTCCG 1081 ACATAA //

Please visit Sequencing and PCR primers for primer information.

References

Original, user report and related articles

original	Sato, M., Fluorescent-based evaluation of chaperone-mediated autophagy and microautophagy activities in cultured cells. Genes Cells 21 (8): 861-873 (2016). PMID 27377049. [link to RRC of NBRP]
original	Seki,T., Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron. PLoS One 7 (2): e31232 (2012). PMID 22363588. [link to RRC of NBRP]

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human GAPDH

External Database
Human GAPDH

Reference sequence