Resource data sheet
Prev.

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).
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 Kobayashi, Toshihide | Abe, Mitsuhiro |

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)
Ordering Please visit Information of Request for Distribution. 
Order form 
[Credit Card Payment]  [Bank Transfer or Check Payment]
Material Transfer Agreement (MTA) [Word]
Schedule A (GFP-A) [Link]
Remarks
Please use Schedule A (GFP-A) additionally.
Distribution information (domestic users) [open/close]


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

Ordering Information [in Japanese] [in English]

References and tips

Electronic file

Featured content

Featured content Sphingolipid signaling pathway (English text)
Featured content Organelle marker (English text)

References

original Abe, M., Dynamics of sphingomyelin-and cholesterol-enriched lipid domains during cytokinesis. Methods Cell Biol. 137: 15-24 (2017). PMID 28065303.
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.

User reports and related articles (go to bottom)


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 RDB13497_A5I3p1-1.pdf

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

Primer: T7_20bp RDB13497_A5I3a.seq
>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 RDB13497_A5I3b.seq
>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 RDB13497_A5I3c.seq
>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

References

original Abe, M., Dynamics of sphingomyelin-and cholesterol-enriched lipid domains during cytokinesis. Methods Cell Biol. 137: 15-24 (2017). PMID 28065303.
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.

2019.03.11

GNP_filter3_RDBDEP_html_190307.pl