RDB14600 - pMRX-IP-GFP-LC3-RFP-LC3 delta G

Fluorescent probe to evaluate autophagic flux by the rat LC3 fused with GFP and RFP.

Distribution information

Catalog number	RDB14600
Resource name	pMRX-IP-GFP-LC3-RFP-LC3 delta G
Clone info.	Fluorescent probe to evaluate autophagic flux. Expression vector of rat microtubule-associated protein 1 light chain 3 beta (LC3) fused with GFP and RFP. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3deltaG. The GFP-LC3 is degraded by autophagy, while RFP-LC3deltaG remains in the cytosol as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Please refer DISCUSSION section of Kaizuka, T., Mol. Cell 64, 835-849, 2016 for the difference between pMRX-IP-GFP-LC3-RFP and this clone. Expression was confirmed by the depositor with immunoblotting and fluorescent microscopy. Please refer Saitoh, T. et al., FEBS Lett., 532 (1-2): 45-51, (2002) for the construction of pMRX-IP vector.
Vector backbone	pMRX-IP (Plasmid)
Selectable markers	Amp^r (E. coli), Puro^r (mammalian cells).
Gene/insert name	Rat LC3 cDNA Aequorea victoria GFP cDNA Discosoma sp. RFP cDNA
Depositor\|Developer	Mizushima, Noboru \|

Other clones in our bank	Rat Map1lc3b (KEGG orthology K10435) \|

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	(1) BIOLOGICAL RESOURCE is limited to for academic research in non-profit organization. (2) The RECIPIENT of BIOLOGICAL RESOURCE shall obtain a prior written consent on use of it from the DEPOSITOR. (3) Any use of the BIOLOGICAL RESOURCE for profit purposes by a non-profit organization or any use of the BIOLOGICAL RESOURCE by a profit organization requires a separate license from the DEPOSITOR prior to distribution. (4) In publishing research results obtained by use of the BIOLOGICAL RESOURCE, a citation of the original publication designated by the DEPOSITOR (Kaizuka, T. et al. Mol. Cell, 64 (4): 835-849, 2016) and an acknowledgment to the DEPOSITOR are requested. (5) The RECIPIENT must contact the DEPOSITOR in case of application for any patents or commercial use with the results from the use of BIOLOGICAL RESOURCE.
Additional terms and conditions for distribution	Regarding pMRX vector: In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of literatures for pMX and pMRX vectors (Kitamura, T., Exp. Hematol. 31 (11): 1007-1014, 2003; Saitoh T., J. Biol. Chem. 278 (38): 36005-36012, 2003) is requested.
Ordering	Please visit Information of Request for Distribution.[link] UCSD's MTA is also required. Please visit Additional Forms section. Order form [Credit Card Payment] [Bank Transfer Payment] Material Transfer Agreement (MTA for use for not-for-profit academic purpose) [Word] Schedule A (GFP-A) [Link] Approval form by depositor [Word]
Remarks	((Additional form)) Approval Form(FormD) Remember that you will be working with samples containing infectious virus. UCSD's MTA is also required. Please visit Additional Forms section. Please use Schedule A (GFP-A) additionally. Please obtain written approval of the depositor.

Distribution information (domestic users)

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提供条件	1. 非営利団体による学術研究に限る。2. 利用者は事前に寄託者の提供承諾書を得る。3. 当該リソースの非営利団体の営利目的での利用および営利団体の利用については、事前に寄託者との間で、別途使用許諾に係る契約の締結が必要である。4. 研究成果の公表にあたって寄託者の指定する文献(Kaizuka, T. et al. Mol. Cell, 64 (4): 835-849, 2016)を引用し、謝辞の表明を必要とする。5. 利用者が本件リソースを使用して得られた研究成果に基づき特許等の申請、及び事業活動を行う場合は、寄託者と別途協議を行う。
付加的提供条件	pMRXベクターについて：利用者は、研究成果の公表にあたって pMXならびにpMRXベクターの文献（Kitamura, T., Exp. Hematol. 31 (11): 1007-1014, 2003; Saitoh T., J. Biol. Chem. 278 (38): 36005-36012, 2003) の引用を必要とする。
提供依頼	手続きの詳細は、「提供申込みについて[link]」をご覧ください。別途、UCSDとのMTAも必要です。「申込みに必要な書式」をご覧ください。提供依頼書 [Word] 提供同意書 (MTA、非営利機関による非営利学術研究用)[Word] Schedule A（GFP-A, 英語版のみ） [Link] 寄託者による提供承諾書 [Word]
備考	【追加提供依頼書】承諾書(FormD) このリソースはウイルス粒子産生用のため、取扱いに注意が必要です。別途、UCSDとのMTAも必要です。「申込みに必要な書式」をご覧ください。 Schedule A（GFP-A, 英語版のみ）が追加で必要です。寄託者の書面による提供承諾が必要です。

References and tips

Sequence information

Catalog #	Resource name	Shipping form	Fee (non-profit org.)
RDB14600	pMRX-IP-GFP-LC3-RFP-LC3 delta G	DNA solution

original	Morishita, H., A new probe to measure autophagic flux in vitro and in vivo. Autophagy 13 (4): 757-758 (2017). PMID 28121224.
original	Kaizuka, T., An Autophagic Flux Probe that Releases an Internal Control. Mol Cell. 2016 Nov 17;64(4):835-849 (2016). PMID 27818143.

Featured content	Dnaconda's recommendation EXR0088j (Japanese text)
Featured content	Dnaconda's recommendation EXR0088e (English text)
Featured content	Autophagy (English text)

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.

Primer: EGFP-C Region: insert mid (ratLC3, mRFP1 5) Sequence file: RDB14600_A7Baa.seq
>D04280A1_A7Ba_1_EGFP-C_A04_01_ABI24.ab1 1 ACGGGGGCAC ATCTCGGCAT GGACGAGCTG TACAGGGTGG CGGTGGCTCG GGCGGTGGTG 61 GGTCGGGTGG CGGCGGATCT ATCGATGAAT TGAATTCCAT GCCGTCCGAG AAGACCTTCA 121 AACAGCGCCG GAGCTTCGAA CAAAGAGTGG AAGATGTCCG GCTCATCCGG GAGCAGCACC 181 CCACCAAGAT CCCAGTGATT ATAGAGCGAT ACAAGGGTGA GAAGCAGCTG CCCGTCCTGG 241 ACAAGACCAA GTTCCTTGTA CCTGATCACG TGAATATGAG CGAACTCATC AAGATAATTA 301 GAAGGCGCCT GCAGCTCAAT GCTAACCAAG CCTTCTTCCT CCTGGTGAAT GGGCACAGCA 361 TGGTGAGTGT GTCCACACCC ATCTCTGAAG TGTACGAGAG CGAGAGAGAT GAAGACGGCT 421 TCCTGTACAT GGTCTATGCC TCCCAGGAGA CGTTCGGGAT GGCCTCCTCC GAGGACGTCA 481 TCAAGGAGTT CATGCGCTTC AAGGTGCGCA TGGAGGGCTC CGTGAACGGC CACGAGTTCG 541 AGATCGAGGG CGAGGGCGAG GGCCGCCCCT ACGAGGGCAC CCAGACCGCC AAGCTGAAGG 601 TGACCAAGGG CGGCCCCCTG CCCTTCGCCT GGGACATCCT GTCCCCTCAG TTCCAGTACG 661 GCTCCAAGGC CTACGTGAAG CACCCCGCCG ACATCCCCGA CTACTTGAAG CTGTCCTTCC 721 CCGAGGGCTT CAAGTGGGAG CGCGTGATGA ACTTCGAGGA CGGCGGCGTG GTGACCGTGA 781 CCCAGGACTC CTCCCTGCAG GACGGCGAGT TCATCTACAA GTGAAGCTGC GCGGCACCAA 841 CTTCCCCTCC GACGGCCCCG TAATGCAGAA GAGACCATGG GCTGGGAGGC CTCCACGAGC 901 CGGATGTTAC CCCGAGGACG GCGCCCTGAG GCGAGATCAG ATGAGCTGAA GCTGAAGGAC 961 GGCGGCACTA CGACGCGAGG TCAGACCACT ACATGGCCAG AGCCGGTGCA GCTGCCCGGC 1021 GCTACAAGAA CGGACTCAAG CTGGAACATC ACCTCCCACA ACGAAGACTA CACCAATCGT 1081 GAACAGTACG AGGCGGCGAG GGCGCCACTC ACGGCATCAT GAATTGAATC ATGGCTCGGA 1141 AGACTCAACG GCCGACTGAA CAAGTGATAA GTTCCGTCAT CTGGCAC //
Primer: mRFP1-inner1 Region: insert mid (mRFP1 3', ratLC3deltaG, IRES) Sequence file: RDB14600_A7Bab.seq
>D04280A1_A7Ba_1_mRFP1-inner1_E10_13_ABI24.ab1 1 GCGCGAGGGG GGGGCTGACG TGACCCAGGA CTCCTCCCTG CAGGACGGCG AGTTCATCTA 61 CAAGGTGAAG CTGCGCGGCA CCAACTTCCC CTCCGACGGC CCCGTAATGC AGAAGAAGAC 121 CATGGGCTGG GAGGCCTCCA CCGAGCGGAT GTACCCCGAG GACGGCGCCC TGAAGGGCGA 181 GATCAAGATG AGGCTGAAGC TGAAGGACGG CGGCCACTAC GACGCCGAGG TCAAGACCAC 241 CTACATGGCC AAGAAGCCCG TGCAGCTGCC CGGCGCCTAC AAGACCGACA TCAAGCTGGA 301 CATCACCTCC CACAACGAGG ACTACACCAT CGTGGAACAG TACGAGCGCG CCGAGGGCCG 361 CCACTCCACC GGCGCCATCG ATGAATTGAA TTCCATGCCG TCCGAGAAGA CCTTCAAACA 421 GCGCCGGAGC TTCGAACAAA GAGTGGAAGA TGTCCGGCTC ATCCGGGAGC AGCACCCCAC 481 CAAGATCCCA GTGATTATAG AGCGATACAA GGGTGAGAAG CAGCTGCCCG TCCTGGACAA 541 GACCAAGTTC CTTGTACCTG ATCACGTGAA TATGAGCGAA CTCATCAAGA TAATTAGAAG 601 GCGCCTGCAG CTCAATGCTA ACCAAGCCTT CTTCCTCCTG GTGAATGGGC ACAGCATGGT 661 GAGTGTGTCC ACACCCATCT CTGAAGTGTA CGAGAGCGAG AGAGATGAAG ACGGCTTCCT 721 GTACATGGTC TATGCCTCCC AGGAGACGTT CTAAGGATCC CAGTGTGGTG GTACGGGAAT 781 TCCTGCAGGC CTCGAGGGCC GGCGCGCCGC GGCCGCTACG TAAATTCCGC CCCCCCCCCC 841 CCTCTCCCTC //
Primer: MMLV-pol_f2 Region: pol region, insert 5' (EGFP, ratLC3 5') Sequence file: RDB14600_A7Bac.seq
>D04280A1_A7Ba_1_MMLV-pol_F2_C04_07_ABI24.ab1 1 TACGGGGGGG GTGGACATCC TCTAGACTGC CGGATCTCCC GCCGCCACCA TGGTGAGCAA 61 GGGCGAGGAG CTGTTCACCG GGGTGGTGCC CATCCTGGTC GAGCTGGACG GCGACGTAAA 121 CGGCCACAAG TTCAGCGTGT CCGGCGAGGG CGAGGGCGAT GCCACCTACG GCAAGCTGAC 181 CCTGAAGTTC ATCTGCACCA CCGGCAAGCT GCCCGTGCCC TGGCCCACCC TCGTGACCAC 241 CCTGACCTAC GGCGTGCAGT GCTTCAGCCG CTACCCCGAC CACATGAAGC AGCACGACTT 301 CTTCAAGTCC GCCATGCCCG AAGGCTACGT CCAGGAGCGC ACCATCTTCT TCAAGGACGA 361 CGGCAACTAC AAGACCCGCG CCGAGGTGAA GTTCGAGGGC GACACCCTGG TGAACCGCAT 421 CGAGCTGAAG GGCATCGACT TCAAGGAGGA CGGCAACATC CTGGGGCACA AGCTGGAGTA 481 CAACTACAAC AGCCACAACG TCTATATCAT GGCCGACAAG CAGAAGAACG GCATCAAGGT 541 GAACTTCAAG ATCCGCCACA ACATCGAGGA CGGCAGCGTG CAGCTCGCCG ACCACTACCA 601 GCAGAACACC CCCATCGGCG ACGGCCCCGT GCTGCTGCCC GACAACCACT ACCTGAGCAC 661 CCAGTCCGCC CTGAGCAAAG ACCCCAACGA GAAGCGCGAT CACATGGTCC TGCTGGAGTT 721 CGTGACCGCC GCCGGGATCA CTCTCGGCAT GGACGAGCTG TACAAGGGTG GCGGTGGCTC 781 GGGCGGTGGT GGGTCGGGTG GCGGCGGATC TATCGATGAA TTGAATTCCA TGCCGTCCGA 841 GAAGACCTTC AAACAGCGCC GGAGCTTCGA ACAAAGAGTG GAAGATGTCC GGCTCATCCG 901 GGAGCAGCAC CCCACCAAGA TCCCAGTGAT TATAGAGCGA TACAAGGGTG AGAAGCAGCT 961 GCCCGTCCTG ACAGACCAAG TTCCTTGTAC CTGATCACGT GAATATGAGC GAACTCATCA 1021 AGATAATTTA GAAGCGCTGC AGCTCAATGC TAACCAAGCC TTCTTCTCTG CTGATGGGCA 1081 CAGCATGTGA GTGTGTCCAC ACCCATCTCT GAGTGTACGA AGCGAGAGAT GAGACGCTCC 1141 TGTACATGTC TATGCTCCAG GAGAAACGTT CCGG //
Primer: EGFP-N Region: insert 5' (EGFP 5', pol region, gag, MMLV psi) Sequence file: RDB14600_A7Bad.seq
>D04280A1_A7Ba_1_EGFP-N_E04_13_ABI24.ab1 1 GAATCAACCC GGGTTTAAAG CTCCTCGCCC TTGCTCACCA TGGTGGCGGC GGGAGATCCG 61 GCAGTCTAGA GGATGGTCCA CCCCCGGGGT CGGCAGCCTT CACGTGGGCG GCGTGTATCC 121 AAGCTGCGAT GCCGTCTACT TTGAGGGCGG TGGGGGTGGT CAGCAGGACT GTGTAAGGTC 181 CTTTCCAGCG AGGTTCTAGG TTCTTAGTCT GGTGTCGGCG GACCCACACT GTGTCGCCGA 241 CTCGGTAAGG GTGAGGTACC ACCGGTCGGT CCAGTTGTTC TTGGTAGGCT GCCGCCAGAG 301 GTCTCCAGAC TTCGTGCTGG ACTAAGTAGA GAGCCTGTAA GTGAGCTTGG AGAGAGGGGC 361 TGTTAGTAAC TCTTGTCATG TCAGGGTCAG GGAAGTTTAC AAGGGGCGGG GGTGCCCCAT 421 ATAAGATCTC ATATGGCCAT ATGGGGGCGC CTAGAGAAGG AGTGAGGGCT GGATAAAGGG 481 AGGATCGAGG CGGGGTCGAA CGAGGAGGTT CAAGGGGGAG AGACGGGGCG GATGGAGGAA 541 GAGGAGGCGG AGGCTTAGGG TGTACAAAGG GCTTGACCCA GGGAGGGGGG TCAAAAGCCA 601 AGGCTTCCCA GGTCACGATG TAGGGGACCT GGTCTGGGTG TCCATGCGGG CCAGGTGAAA 661 AGACCTTGAT CTTAACCTGG GTGATGAGGT CTCGGTTAAA GGTGCCGTCT CGCGGCCATC 721 CGACGTTAAA GGTTGGCCAT TCTGCAGAGC AGAAGGTAAC CCAACGTCTC TTCTTGACAT 781 CTACCGACTG GTTGTGAGCG ATCCGCTCGA CATCTTTCCA GTGACCTAAG GTCAAACTTA 841 AGGGAGTGGT AACAGTCTGG CCCGGGCCCA TATTTTCAGA CAAATACAGA AACACAGTCA 901 GACAGAGACA ACACAGAACG ATGCTGCAGC AGACAAGACG CGCGGCGCGG CTTCGGTCCC 961 AAACCGAAAG CAAAAAATTC AGACGGAGGC GGGAACTGTT TTAAGTTCTC GTCTCCTACC 1021 AGGAACCAAC AATTATCCCT CCTCTAAGGG GGGGTGCACC CAAAGAAAGT CCAAAACCGA 1081 TTCGGGAATT TTTGGAACCT CAAGTCGGGG CCACAAAAAC GGCCCTCGAA GTCCCTGGGA 1141 ACGTCTCAGA TGCCGCGGGG TTTCGAACTC GTTCAGGTTC CCACCAACCG GGGGTCGCGC 1201 CCCCGGATAT //
Primer: pMXs-3LTRup_R2 Region: PuriR, IRES Sequence file: RDB14600_A7Bae.seq
>D04280A1_A7Ba_1_pMXs-3LTRup_R2_G04_19_ABI24.ab1 1 GGCATATAAA ATCTTTTATT TTATCGTCGA GCCATGGGGT CGTGCGCTCC TTTCGGTCGG 61 GCGCTGCGGG TCGTGGGGCG GGCGTCAGGC ACCGGGCTTG CGGGTCATGC ACCAGGTGCG 121 CGGTCCTTCG GGCACCTCGA CGTCGGCGGT GACGGTGAAG CCGAGCCGCT CGTAGAAGGG 181 GAGGTTGCGG GGCGCGGAGG TCTCCAGGAA GGCGGGCACC CCGGCGCGCT CGGCCGCCTC 241 CACTCCGGGG AGCACGACGG CGCTGCCCAG ACCCTTGCCC TGGTGGTCGG GCGAGACGCC 301 GACGGTGGCC AGGAACCACG CGGGCTCCTT GGGCCGGTGC GGCGCCAGGA GGCCTTCCAT 361 CTGTTGCTGC GCGGCCAGCC GGGAACCGCT CAACTCGGCC ATGCGCGGGC CGATCTCGGC 421 GAACACCGCC CCCGCTTCGA CGCTCTCCGG CGTGGTCCAG ACCGCCACCG CGGCGCCGTC 481 GTCCGCGACC CACACCTTGC CGATGTCGAG CCCGACGCGC GTGAGGAAGA GTTCTTGCAG 541 CTCGGTGACC CGCTCGATGT GGCGGTCCGG GTCGACGGTG TGGCGCGTGG CGGGGTAGTC 601 GGCGAACGCG GCGGCGAGGG TGCGTACGGC CCGGGGGACG TCGTCGCGGG TGGCGAGGCG 661 CACCGTGGGC TTGTACTCGG TCATGGTTGT GGCCATATTA TCATCGTGTT TTTCAAAGGA 721 AAACCACGTC CCCGTGGTTC GGGGGGCCTA GACGTTTTTT TAACCTCGAC TAAACACATG 781 TAAAGCATGT GCACCGAGGC CCCAGATCAG ATCCCATACA ATGGGGTACC TTCTGGGCAT 841 CCTTCAGCCC CTTGTTGAAT ACGCTTGAGG AGAGCCATTT GACTCTTTCC ACAACTATCC 901 AACTCACAAC GTGGCACTGG GGTTGTGCCG CCTTTGCAGG TGTATCTTAT ACACGTGGCT 961 TTTGGGCCGC AGAGGCACCT GTCGCCAGGG TGGGGGGTTC CGCTGCCTGC AAAGGGTCGC 1021 TACAGACGTT GTTTGTCTTC AGAGCTTCCA GAGGAACTGC TTCTTCACGA CATTCAACAG 1081 ACCTTGCATT CCTTGCGAGA GGGAAGACCC CTAGGATGGC TCGTCAGAGA CCGTCAGTAT 1141 TCGGCCTCAC ATTGCCTAAA GAAACGGCCA AATATG //
Primer: p15Aori_F Region: Ori, LTR Sequence file: RDB14600_A7Baf.seq
>D04280A1_A7Ba_1_p15Aori_F_F04_16_ABI24.ab1 1 GCAGGGACGT GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC 61 AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT 121 GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT 181 CGCCGCAGCC GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCCA 241 ATACGCAAAC CGCCTCTCCC CGCGCGTTGG CCGATTCATT AATGCAACTA TGGCCATTTA 301 ATGTAAATAC TTAAGAAAAA AAACCAAATT AATTTTGATA CATGCTGCAT GTGAAAGACC 361 CCCGCTGACG GGTAGTCAAT CACTCAGAGG AGACCCTCCC AAGGAACAGC GAGACCACAA 421 GTCGGATGCA ACTGCAAGAG GGTTTATTGG ATACACGGGT ACCCGGGCGA CTCAGTCAAT 481 CGGAGGACTG GCGCCCCGAG TGAGGGGTTG TGGGCTCTTT TATTGAGCTC GGGGAGCAGA 541 AGCGCGCGAA CAGAAGCGAG AAGCGAACTG ATTGGTTAGT TCAAATAAGG CACAGGGTCA 601 TTTCAGGTCC TTGGGGCACC CTGGAAACAT CTGATGGTTC TCTAGAAACT GCTGAGGGCT 661 GGACCGCATC TGGGGACCAT CTGTTCTTGG CCCTGAGCCG GGGCAGGAAC TGCTTACCAC 721 AGATATCCTG TTTGGCCCAT ATTCAGCTGT TCCATCTGTT CTTGGCCCTG AGCCGGGGCA 781 GGAACTGCTT ACCACAGATA TCCTGTTTGG CCCATATTCA GCTGTTCCAT CTGTTCCTGA 841 CCTTGATCTG AACTTCTCTA TTCTCAGTTA TGTATTTTTC CATGCCTTGC AAAATGGCGT 901 TACTTAAGCT AGCTTGCCAA ACCTACAGGT GGGGTCTTTC ATTCCCCCCT TTTTCTGGAG 961 ACTAAATAAA ATCTTTTATT TTATCGTCGA GCCATGGGGT CGTGCGCTCC TTTCGGTCGG 1021 GCGCTGCGGG TCGTGGGGCG GGGCGTCAGG CACCGGGCTT GCGGGTCATG CACCAGTGCG 1081 CGTCCTCCGG CACCTCGACG TCGCGTGACG TGAAGCCGAG TCGCTCGTTA GAGGAGTTGC 1141 GGACGCGGAG TCTCTCCAGG AATGC //