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Please review the QC test results indicated by check icon below as well as clone information before placing your order.

pET28/EqtII(8-69)-EGFP-His6

Marker of sphingomyelin in cell membrane. Bacterial expression plasmid of nontoxic Eqinatoxin II.

Catalog number RDB13497
Resource name pET28/EqtII(8-69)-EGFP-His6
Clone info. Marker of sphingomyelin in cell membrane. Bacterial expression plasmid of nontoxic Eqinatoxin II. To use the recombinant protein, oxidization after the purification is required to prevent its cytolytic activity. Refer Abe, M., Methods Cell Biol. 137: 15-24 (2017).
Comment V8C K69C mutations. Optimized for expression in E. coli.
Please refer to the following note by the depositor:
- the expression of this protein was lesser than other protein expression vectors,
- induction was performed at 30 centigrade OVERNIGHT,
- the volume of cultivation was 500 to 1000 mL to obtain enough amount.
Please refer the Methods Cell Biol. 137 :15-24, 2017 (PMID: 28065303) for the method of expression and purification of the protein.
Vector backbone pET-28b (Plasmid)
Size of vector backbone 5.4 kb
Selectable markers Kan^r
Gene/insert name Actinia equina Equinatoxin II cDNA Aequorea victoria GFP cDNA
Depositor|Developer Kobayashi, Toshihide | Abe, Mitsuhiro |
Other clones in our bank

External Database
Actinia equina Equinatoxin II
    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 literature designated by the DEPOSITOR is requested. (Makino, A., FASEB J. 29 (2): 477-493, 2015)
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            Material Transfer Agreement (MTA for use for not-for-profit academic purpose) [Word]
            This RESOURCE contains a fluorescent protein owned or licensed by Global Life Sciences Solutions Operations UK Ltd. (Cytiva). Please refer to Schedule B and use Schedule A (download the Schedule A and Schedule B [link]).
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            提供条件 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(Makino, A., FASEB J. 29 (2): 477-493, 2015)。
            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。営利目的利用についてはお問い合わせください。
            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]
            GFPリソースを含みます。Schedule Bをご一読の上Schedule A (英語版のみ) の提出をお願いします (Schedule AとSchedule Bを入手[link])。

            Catalog # Resource name Shipping form Fee (non-profit org.)
            RDB13497 pET28/EqtII(8-69)-EGFP-His6 DNA solution

            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

            Original reference

            original Makino, A., Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis. FASEB J. 29 (2): 477-493 (2015). PMID 25389132.

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

            Featured content

            Featured content Visualization of Organelles (English text)
            Featured content Sphingolipid signaling pathway (English 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 RDB13497_A5I3p1-1.pdf check

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

            Primer: T7_20bp
            Sequence file: RDB13497_A5I3a.seq check
            >13497_13497_A5I3_5_T7_20bp_G04_19.ab1
                1 TCGGTCGCTC CATCTAGAAT AATTTTGTTT AACTTTAAGA AGGAGATATA CCGGTACCAT
               61 GAGCGCGGAT GTGGCTGGTG CATGCATAGA TGGCGCCTCT CTGAGTTTCG ACATCCTCAA
              121 GACCGTGTTG GAAGCTCTGG GGAATGTCAA ACGGAAGATT GCCGTAGGAG TGGACAACGA
              181 AAGCGGTAAG ACATGGACTG CCCTGAATAC GTACTTCAGG TCTGGAACCA GCGACATTGT
              241 TCTGCCCCAC AAAGTCCCAC ATGGGTGTGC ACTCCTGTAC AATGGGCAGA AAGATCGAGG
              301 ACCTGTTGCG ACTGGTGCAG TTGGGGTATT GGCCTATCTC ATGTCAGATG GCAACACCCT
              361 TGCTGTGCTG TTTTCCGTCC CCTATGACTA CAACTGGTAT TCCAACTGGT GGAATGTGCG
              421 CATCTACAAG GGCAAACGTA GAGCCGATCA ACGCATGTAT GAGGAGCTTT ACTACAACCT
              481 GTCCCCGTTT AGGGGCGACA ATGGATGGCA CACACGGAAT CTCGGGTATG GCCTGAAGAG
              541 CAGAGGCTTC ATGAACAGCA GTGGCCATGC CATCCTGGAG ATTCACGTGT CCAAGGCTGG
              601 TGGAGGGTCA GGCGGCGGAT CTGAATTCGG AGGAGGAGGA GGAGGATCCA TGGTGAGCAA
              661 GGGCGAGGAG CTGTTCACCG GGGTGGTGCC CATCCTGGTC GAGCTGGACG GCGACGTAAA
              721 CGGCCACAAG TTCAGCGTGT CCGGCGAGGG CGAGGGCGAT GCCACCTATG GCAAGCTGAC
              781 CCTGAAGTTC ATCTGCACCA CCGGCAAGCT GCCCGTGCCC TGGCCCACCC TCGTGACCAC
              841 CCTGACCTAC GGCGTGCAGT GCTTCAGCCG CTACCCCGAC CACATGAAGC AGCACGACTT
              901 CTTCAAGTCC GCCATGCCCG AAAGGCTACG TCCAGGAGCG CACCATCTTC TTCAAAGGAC
              961 GACGGCAACT ACAAGACCCG CGCCCGAGGT GAAGTTCGAG GCGACACCCT GGTGAAACCG
             1021 CATCGAGCTG ATGGGCATCG ACTTTCAATG AAGACCGCCA CCATCCTGGG GCACAAGCTG
            1081 CAGTACACTT ACACAGCCAA CGACCGATCT TATTATTCAA TGGTCA
            //
            Primer: T7_terminator
            Sequence file: RDB13497_A5I3b.seq check
            >13497_13497_A5I3_5_T7_terminator_H04_22.ab1
                1 ACCTACGTCT TTCGGGCTTT GTTAGCAGCC GGATCTCAGT GGTGGTGGTG GTGGTGAAGC
               61 TTCTTGTACA GCTCGTCCAT GCCGAGAGTG ATCCCGGCGG CGGTCACGAA CTCCAGCAGG
              121 ACCATGTGAT CGCGCTTCTC GTTGGGGTCT TTGCTCAGGG CGGACTGGGT GCTCAGGTAG
              181 TGGTTGTCGG GCAGCAGCAC GGGGCCGTCG CCGATGGGGG TGTTCTGCTG GTAGTGGTCG
              241 GCGAGCTGCA CGCTGCCGTC CTCGATGTTG TGGCGGATCT TGAAGTTCAC CTTGATGCCG
              301 TTCTTCTGCT TGTCGGCCAT GATATAGACG TTGTGGCTGT TGTAGTTGTA CTCCAGCTTG
              361 TGCCCCAGGA TGTTGCCGTC CTCCTTGAAG TCGATGCCCT TCAGCTCGAT GCGGTTCACC
              421 AGGGTGTCGC CCTCGAACTT CACCTCGGCG CGGGTCTTGT AGTTGCCGTC GTCCTTGAAG
              481 AAGATGGTGC GCTCCTGGAC GTAGCCTTCG GGCATGGCGG ACTTGAAGAA GTCGTGCTGC
              541 TTCATGTGGT CGGGGTAGCG GCTGAAGCAC TGCACGCCGT AGGTCAGGGT GGTCACGAGG
              601 GTGGGCCAGG GCACGGGCAG CTTGCCGGTG GTGCAGATGA ACTTCAGGGT CAGCTTGCCA
              661 TAGGTGGCAT CGCCCTCGCC CTCGCCGGAC ACGCTGAACT TGTGGCCGTT TACGTCGCCG
              721 TCCAGCTCGA CCAGGATGGG CACCACCCCG GTGAACAGCT CCTCGCCCTT GCTCACCATG
              781 GATCCTCCTC CTCCTCCTCC GAATTCAGAT CCGCCGCCTG ACCCTCCACC AGCCTTGGAC
              841 ACGTGAATCT CCAGGATGGC ATGGCCACTG CTGTTCATGA AGCCTCTGCT CTTCAGGCCA
              901 TACCCGAGAT TCCGTGTGTG CCATCCATTG TCGCCCCTAA ACGGGGACAG GTTTGTAGTA
              961 AAGCTCCTCA TACATGCGTT GATCGGGCTC TACGTTTGCC CTTGTAGATG CGCACATTTC
             1021 CACCAGTTGG GAATACCAAG TGGTAGGTCA TAGGGGGACC GAAACAGCAC AGCCAAGGTG
            1081 ATTGCCATCT GACATGAGAT AGGTCAATTA CCCCCAACCT GCCACCAGGT TCG
            //
            Primer: pGL3_RV4
            Sequence file: RDB13497_A5I3c.seq check
            >13497_13497_A5I3_5_pGL3_Rv4_A05_02.ab1
                1 GGAACTCTGG GTTGAGGCTC TCAGGGCATC GGTCGAGATC CCGGTGCCTA ATGAGTGAGC
               61 TAACTTACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC
              121 CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCCAG
              181 GGTGGTTTTT CTTTTCACCA GTGAGACGGG CAACAGCTGA TTGCCCTTCA CCGCCTGGCC
              241 CTGAGAGAGT TGCAGCAAGC GGTCCACGCT GGTTTGCCCC AGCAGGCGAA AATCCTGTTT
              301 GATGGTGGTT AACGGCGGGA TATAACATGA GCTGTCTTCG GTATCGTCGT ATCCCACTAC
              361 CGAGATATCC GCACCAACGC GCAGCCCGGA CTCGGTAATG GCGCGCATTG CGCCCAGCGC
              421 CATCTGATCG TTGGCAACCA GCATCGCAGT GGGAACGATG CCCTCATTCA GCATTTGCAT
              481 GGTTTGTTGA AAACCGGACA TGGCACTCCA GTCGCCTTCC CGTTCCGCTA TCGGCTGAAT
              541 TTGATTGCGA GTGAGATATT TATGCCAGCC AGCCAGACGC AGACGCGCCG AGACAGAACT
              601 TAATGGGCCC GCTAACAGCG CGATTTGCTG GTGACCCAAT GCGACCAGAT GCTCCACGCC
              661 CAGTCGCGTA CCGTCTTCAT GGGAGAAAAT AATACTGTTG ATGGGTGTCT GGTCAGAGAC
              721 ATCAAGAAAT AACGCCGGAA CATTAGTGCA GGCAGCTTCC ACAGCAATGG CATCCTGGTC
              781 ATCCAGCGGA TAGTTAATGA TCAGCCCACT GACGCGTTGC GCGAGAAGAT TGTGCACCGC
              841 CGCTTTACAG GCTTCGACGC CGCTTCGTTC TACCATCGAC ACCACCACGC TGGCACCCAG
              901 TTGATCGGCG CGAGATTTAA TCGCCGCGAC AATTTGCGAC GGCGCGTGCA GGGCCAGACT
              961 GGGATGTGGG CAACGCCAAA TCAGCAACGA CTGTTTGCCC CGCCCAGTTG TTTGTGCCAC
             1021 GCGGCTTGGG AATGTAAATT CAAGCTCCGT CATCGGCGCT TCACTTTTTT CCGCGTTTTC
            1081 GCAGAATCGG TGGACTGGAC CTTGAATTTC ATCAACGTCA GGA
            //

            Please visit Sequencing and PCR primers for primer information.


            References

            Original, user report and related articles

            original Makino, A., Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis. FASEB J. 29 (2): 477-493 (2015). PMID 25389132.
            protocol Abe, M., Dynamics of sphingomyelin-and cholesterol-enriched lipid domains during cytokinesis. Methods Cell Biol. 137: 15-24 (2017). PMID 28065303.

            2022.05.17

            GNP_filter3_RDBDEP_html_220516.pl