Insertion of gene sequence was conducted either by using restriction enzymes from New England Biolabs, Gibson Assembly (E5510S; New England Biolabs) or In-Fusion HD Cloning Plus (638909; Takara). Site-directed mutagenesis was performed either by whole-plasmid PCR followed by circularization or by QuikChange Multi Site-Directed Mutagenesis Kit (200515; Agilent). Successful cloning was confirmed by sequencing for all constructs. A bipartite NLS from Xenopus laevis nucleoplasmin (KRPAATKKAGQAKKKK; Dingwall et al., 1988 (link); Robbins et al., 1991 (link); Sołtysik et al., 2019 (link)) was used.
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Genetic Construct Generation and Nuclear Localization
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Corresponding Organization : Yale University
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Variable analysis
independent variables
- Method of gene sequence insertion (restriction enzymes, Gibson Assembly, or In-Fusion HD Cloning Plus)
- Method of site-directed mutagenesis (whole-plasmid PCR followed by circularization or QuikChange Multi Site-Directed Mutagenesis Kit)
- Successful cloning of constructs
- Confirmation of successful cloning by sequencing for all constructs
- Positive control: Successful cloning of constructs
- Negative control: Not explicitly mentioned
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