yHS1-M7A,H102A
(Plasmid #159152)

• Purpose: Cytosolic labile heme reporter
• Depositing Lab: Amit Reddi
• Publication: Hanna et al Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):7539-44. doi: 10.1073/pnas.1523802113. Epub 2016 May 31.

Backbone

• Vector backbone: p415GPD
• Backbone manufacturer: M. Funk
• Backbone size w/o insert (bp): 6900
• Total vector size (bp): 8600
• 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: yHS1-M7A,H102A
• Species: S. cerevisiae (budding yeast)
• Insert Size (bp): 1758
• Mutation: Mutated both Met 7 and His 102 of the cyt b562 module of yHS1 to Ala (M7A and H102A, respectively), creates weak affinity heme sensor. Mutated His 63 to Asn (H63N) to test possible role in heme binding, found no effect.
• Promoter: GPD

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: XbaI (not destroyed)
• 3′ cloning site: HindIII (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)