pEDA5_GFPmut3_Y66H
(Plasmid #80085)

• Purpose: Positive control plasmid for Nicking Mutagenesis. Constitutively expresses a non-fluorescent variant of GFPmut3 (Y66H) that is mutated to fluorescent (H66Y) in the protocol.
• Depositing Lab: Timothy Whitehead
• Publication: Wrenbeck et al Nat Methods. 2016 Nov;13(11):928-930. doi: 10.1038/nmeth.4029. Epub 2016 Oct 10.

Backbone

• Vector backbone: pET29b(+)
• Backbone manufacturer: Novagen
• Backbone size w/o insert (bp): 3583
• Total vector size (bp): 4303
• Modifications to backbone: - Swapped antibiotic resistance from KanR to AmpR - Promoter is proB (see doi: 10.1371/journal.pone.0109105)
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Plasmid must be prepared fresh and avoid multiple freeze/thaw cycles for use as a positive control in Nicking Mutagenesis.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: GFPmut3_Y66H
• Species: Synthetic; Aequorea victoria
• Insert Size (bp): 720
• Mutation: changed Tyrosine 66 to Histidine
• Promoter: proB
• Tag / Fusion Protein:
  • 6x-His tag (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NdeI (not destroyed)
• 3′ cloning site: XhoI (not destroyed)
• 5′ sequencing primer: pED_2ND
• 3′ sequencing primer: T7 term

Resource Information

• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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