mito-yHS1-M7A,H102A
(Plasmid #159165)

• Purpose: Mitochondrial labile heme reporter
• Depositing Lab: Amit Reddi
• Publication: Martinez-Guzman et al J Cell Sci. 2020 May 20;133(10). pii: jcs.237917. doi: 10.1242/jcs.237917.

Backbone

• Vector backbone: p415GPD
• Backbone manufacturer: M. Funk
• Backbone size w/o insert (bp): 6900
• Total vector size (bp): 8700
• Vector type: Yeast Expression
• Selectable markers: LEU2

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: mito-yHS1-M7A,H102A
• Species: S. cerevisiae (budding yeast)
• Insert Size (bp): 1800
• Mutation: Mutated both Met 7 and His 102 of the cyt b562 module of mito-yHS1 to Ala (M7A and H102A, respectively), creates weak affinity heme sensor.
• Promoter: GPD
• Tag / Fusion Protein:
  • COX4 pre-sequence (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SpeI (not destroyed)
• 3′ cloning site: SalI (not destroyed)
• 5′ sequencing primer: T3
• 3′ sequencing primer: T7 Pro

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry

Depositor Comments

The first-generation heme sensor, HS1, consists of a heme-binding domain, the His/Met coordinating 4-alpha-helical bundle hemoprotein cytochrome b562 (Cyt b562) (13), fused to a pair of fluorescent proteins, EGFP and Katushka 2 (mKATE2)