pET28 His6-mEGFP-D4H
(Plasmid #226373)

• Purpose: Production of recombinant mEGFP-D4H, a fluorescent probe binding cholesterol
• Depositing Lab: Catherine Tomasetto
• Publication: Eichler et al Methods Mol Biol. 2025;2888:101-118. doi: 10.1007/978-1-0716-4318-1_8.

Backbone

• Vector backbone: pET28b
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 5368
• Total vector size (bp): 6389
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: D4H
• Alt name: Mutant D4 (D434S) domain of the cholesterol-dependent cytolysin perfringolysin O
• Species: Clostridium perfringens
• Insert Size (bp): 336
• GenBank ID: WP_003479450.1
• Entrez Gene: pfoA (a.k.a. I6G60_RS00735, I6G60_00735)
• Tags / Fusion Proteins:
  • His6 (N terminal on insert)
  • monomeric EGFP (N terminal on insert)

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: T7

Resource Information

• Supplemental Documents:
  • pET28 His6-mEGFP-D4H.gb
• A portion of this plasmid was derived from a plasmid made by: Mitsuhiro Abe and Toshihide Kobayashi (D4 cDNA). Abe, M., Makino, A., Hullin-Matsuda, F., Kamijo, K., Ohno-Iwashita, Y., Hanada, K., Mizuno, H., Miyawaki, A., Kobayashi, T., A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis. Mol. Cell. Biol. 32 (8): 1396-1407 (2012). PMID 22331463. https://doi.org/10.1128/MCB.06113-11

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Based on the D4H probe described in Maekawa M, Fairn GD (2015) Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol. J Cell Sci 128:1422–1433. https://doi.org/10.1242/jcs.164715