pVS2
(Plasmid #33144)

• Depositing Lab: Michael Rosbash
• Publication: Vodala et al Mol Cell. 2008 Jul 11;31(1):104-13.

Backbone

• Vector backbone: pVS1
• Backbone manufacturer: Addgene plasmid #33143
• Modifications to backbone: CEN6ARSH4 element from pRS415 (Christianson et al., 1992) was amplified using primers flanked with AatII sites, digested with AatII and cloned into the AatII site of parent vector carrying 256 copies of LacO (Straight et al., 1998).
• Vector type: Yeast Expression
• Selectable markers: LEU2

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): Stbl2
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: LacO (256 copies)
• Species: E. coli
• Insert Size (bp): 10000

Gene/Insert 2

• Gene/Insert name: GAL-GFP-kd200-GALpA reporter
• Mutation: GFP is mutated such that amino acids 64-73 (FSYGVQCFSR) are replaced with IYKIHNQQLANDYYLYVL such that the fluorophore is non-functional, but the protein is still recognized by antibody
• Promoter: bidirectional GAL1-GAL10 promoter

Cloning Information for Gene/Insert 2

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: GFP-kd200-R
• 3′ sequencing primer: GFP forward

Terms and Licenses

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

Depositor Comments

Addgene cannot verify or guarantee the presence of all 256 copies of LacO. This element is unstable.

For use in two-plasmid-based gene-nuclear periphery tethering assay with either plasmids pAK67 (Addgene #33142).

pVS2 contains 256 LacO repeats and a GAL-GFP-GALpA-reporter gene (pVS1 [Addgene #33143] is identical but does not contain the reporter gene). The reporter was previously characterized and contains a GFP open reading frame flanked by the GAL1 promoter and GAL1 3′ UTR ([Abruzzi et al., 2006] and [Dower et al., 2004]).

pKA67 expresses both LacI-GFP and Nup49-GFP to mark the first plasmid as well as the nuclear periphery with GFP.