pKT0221
(Plasmid #8726)

• 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): 5014
• Vector type: Yeast Expression
• Selectable markers: KanR

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
• Alt name: Codon-optimized GFP variant
• Alt name: pKT221 (pFA6a–link–yECFP–3HA–KANr)
• Mutation: yEGFP with F64L, S65T, Y66W, N146I, M153T, V163A
• Tags / Fusion Proteins:
  • Codon optimized linker (N terminal on backbone)
  • 3HA (C 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-yECFP-3HA-KanR. yECFP 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 G418 resistance marker (KanR) was introduced into the plasmid by subcloning the BglII/EcoRI fragment of pDH3 (Hailey et al., 2002) into the plasmid, in place of SpHIS5.

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).