Overexpression of Sst2 and Kel1 in Yeast

The plasmids used for overexpression of Sst2 or Kel1 by the ADH1 promoter were constructed using the NEB Gibson Assembly Cloning Kit (E2611S; NEB) as advised by the manufacturer’s instructions. All plasmids were built using the pRSII416 vector backbone (Plasmid #: 35456; Addgene [Chee & Haase, 2012 (link)]). The vector backbone was linearized with SacI-HF (R3156S; NEB) and ApaI (R0114S; NEB) restriction enzymes before Gibson assembly. Primers were constructed using the online NEBuilder assembly tool (v2.6.0, https://nebuilder.neb.com/) and are listed in Table S3. The forward and reverse 3XFLAG sequences were obtained from p3xFLAG-CMV-14 and synthesized as oligos for PCR amplification with primers in Table S3. The 1 kilobase of DNA upstream of the ADH1 was amplified from genomic DNA to provide the ADH1 promoter.

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Simke W.C., Johnson C.P., Hart A.J., Mayhue S., Craig P.L., Sojka S, & Kelley J.B. (2022). Phosphorylation of RGS regulates MAP kinase localization and promotes completion of cytokinesis. Life Science Alliance, 5(10), e202101245.

Publication 2022

NEB Gibson Assembly Cloning Kit

Apai Backbone Genomic Oligos Plasmid Primers Restriction enzymes Vector

Corresponding Organization : University of Maine

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Variable analysis

independent variables

Overexpression of Sst2 or Kel1 by the ADH1 promoter

dependent variables

Not explicitly mentioned

control variables

Use of the pRSII416 vector backbone
Linearization of the vector backbone with SacI-HF and ApaI restriction enzymes before Gibson assembly
Synthesis of the forward and reverse 3XFLAG sequences from p3xFLAG-CMV-14 and use as oligos for PCR amplification
Amplification of 1 kilobase of DNA upstream of the ADH1 to provide the ADH1 promoter

controls

No positive or negative controls were explicitly mentioned in the provided information

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