pKT0210
(Plasmid #8724)

• Depositing Lab: Kurt Thorn
• Publication: Sheff et al Yeast 2004 Jun;21(8):661-70.

Backbone

• Vector backbone: pFA6a-link
• Backbone size w/o insert (bp): 4738
• Vector type: Yeast Expression
• Selectable markers: S. pombe HIS5

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: yECFP A206R
• Alt name: Codon-optimized GFP variant
• Alt name: pKT210 (pFA6a–link–yEmCFP–SpHIS5)
• Mutation: yEGFP with F64L, S65T, Y66W, N146I, M153T, V163A, A206R
• Tag / Fusion Protein:
  • Codon optimized linker (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: PacI (not destroyed)
• 3′ cloning site: AscI (not destroyed)
• 5′ sequencing primer: SP6

Terms and Licenses

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

Depositor Comments

Full name of this plasmid is pFA6a-link-yECFPA206R-SpHIS5. yECFP A206R is created by mutagenesis from a codon-optimized green fluorescent protein - yEGFP1 (Cormack et al. 1997). This yEGFP variant is cloned into pDH5, replacing YFP. An codon-optimized linker was added into the yEGFP variant to improve expression level. The selectable marker, Sz. pombe HIS5 (SpHIS5), complements S.
cerevisiae HIS3. The yEmCFP and yEmCitrine proteins contain the A206R mutation to block the weak dimerization of the parent GFP (see Zacharias et al., Science 2002 296: 913-916).

The yeast GFP used to create this plasmid contained a point mutation M233I that is present in all GFP variants derived from this original yEGFP. The mutation had no effect on fluorescence (see associated publication for more details).