Plasmid 33144: pVS2

• Gene/Insert 1
• Gene/insert name: LacO (256 copies)
• Insert size: 10000
• Species: E. coli
• 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
• Vector backbone: pVS1
  (Search Vector Database)
• Backbone manufacturer: Addgene plasmid #33143
• Vector type: Yeast Expression
• 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).
• Cloning information for Gene/Insert 2
• Promoter: bidirectional GAL1-GAL10 promoter
• Cloning method: Ligation Independent Cloning
• 5' sequencing primer: GFP-kd200-R
• 3' sequencing primer: GFP forward
• Bacterial resistance(s) Ampicillin
• Growth strain(s) Stbl2
• Growth temperature (℃): 30
• High or low copy: Unknown
• Selectable markers: LEU2
• Sequence: View sequences (4)
• Map: View map
• Principal Investigator: Michael Rosbash
• Terms and Licenses: MTA

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.

Article: The nuclear exosome and adenylation regulate posttranscriptional tethering of yeast GAL genes to the nuclear periphery. Vodala et al (Mol Cell. 2008 Jul 11;31(1):104-13. PubMed)