pVP64
(Plasmid #22287)

• Depositing Lab: Louise Prakash
• Publication: Pages et al Genes Dev. 2009 Jun 15. 23(12):1438-49.

Backbone

• Vector backbone: YCplac111
• Backbone size w/o insert (bp): 6112
• 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: Unknown

Gene/Insert

• Gene/Insert name: REV1
• Species: S. cerevisiae (budding yeast)
• Insert Size (bp): 4640
• Entrez Gene: REV1 (a.k.a. YOR346W)
• Tag / Fusion Protein:
  • 4xHA (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 3′ cloning site: BglII (between insert and tag) (not destroyed)
• 5′ sequencing primer: M13_pUC-rev
• 3′ sequencing primer: M13_puc-for

Terms and Licenses

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

Depositor Comments

The REV1 ORF plus 1050 nucleotides (nt) of its upstream region that include the REV1 promoter and 500nt of the downstream region that include the REV1 transcription terminator was cloned in the vector YCplac111, which carries the LEU2 gene, the autonomous replicating sequence ARS1, and the centromeric CEN4 region.

A BglII restriction site was added by site-directed mutagenesis right before the stop codon of REV1. A 33-mer oligonucleotide duplex containing the HA-coding sequence and a BamHI and a BglII restriction site was inserted into the BglII restriction site, adding an HA tag in fusion with REV1, and regenerating a unique BglII restriction site. This step was repeated three times in order to get four HA tags in fusion with REV1.