pTH744-CEN-RLuc/slowmaxCFLuc
(Plasmid #38224)

• Depositing Lab: Tobias von der Haar
• Publication: Chu et al EMBO J. 2014 Jan 1;33(1):21-34. doi: 10.1002/embj.201385651. Epub 2013 Dec 19.

Backbone

• Vector backbone: pCENBEVY-U
• Backbone size w/o insert (bp): 7122
• Total vector size (bp): 9692
• Modifications to backbone: A mutated GCN4 leader sequence (containing uORF 1 as the sole remaining uORF) was introduced into the BamHI site of this vector. mRNAs expressed from the TDH3 (GPD) promoter therefore contain inefficient 5'-UTRs.
• Vector type: Yeast Expression
• Selectable markers: URA3

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: We have observed that plasmids containing the codon maximised Firefly luciferase variant or portions there of cause slow growth in some E. coli strains (eg Top10). However, this does not appear to affect plasmid yield.
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: Firefly Luciferase
• Alt name: FLuc
• Species: Photinus pyralis
• Insert Size (bp): 1644
• Mutation: All codons of the original FLuc gene were exchanged for those isoaccepting codons decoded by the most abundant tRNAs in yeast. The last three codons of the full-length Firefly luciferase gene have been deleted to yield a luciferase variant which is fully functional, but no longer localises to peroxisomes.
• GenBank ID: AF043450
• Promoter: TDH3 (=GPD)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: SalI (not destroyed)
• 5′ sequencing primer: n/a
• 3′ sequencing primer: M13f

Gene/Insert 2

• Gene/Insert name: Renilla luciferase
• Alt name: RLuc
• Species: Synthetic; Renilla reniformis
• Insert Size (bp): 942
• GenBank ID: AF043450.1
• Promoter: ADH1

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: XmaI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: n/a
• 3′ sequencing primer: M13r

Resource Information

• A portion of this plasmid was derived from a plasmid made by: The RLuc gene originated from a plasmid kindly provided by Dr David Bedwell (University of Alabama, Birmingham). Salas-Marco J, Bedwell DM (2004) Molecular and Cellular Biology 24:7769-7778.

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Luciferase Limited Use Label License
  • Renilla Luciferase Limited Use Label License
• Industry Terms:
  • Not Available to Industry