Irreversible Loading of Mcm onto Chromatin @bullet Kuipers Et Al. Jcb Supplemental Material

Abstract

Figure S1. Plasmids and cell lines constructed. (A) RMCE, recombinase-mediated cassette exchange (recombinase sites that were not used in this study). Figures using each construct are indicated. (B) Generic plasmid map of all Mcm constructs. The backbone of each construct is pBlueScriptII. FRT1 and FRT3, heterotypic FRT sites that can be used for RMCE (Wirth and Hauser, 2004). pTet, tet-regulatable promoter. pTK, thymidine kinase promoter. TKzeo, encodes a fusion protein of thymidine kinase for negative selection (not used in this study) and zeomycin resistance for positive selection. (C) All cell lines are derived from cell line " Hyg-16, " which contains a constitutively expressed " tet-off " tet transactivator (tTA) expressed from a bicistronic (IRES) vector with the hygro-mycin resistance gene (Izumi and Gilbert, 1999). These cells were further stably transformed with the E. coli Biotin ligase gene BirA, which confers blastoci-din resistance (de Boer et al., 2003). This parental cell line was then used to create all the indicated cell lines named after the two fluorescent protein constructs that were stably introduced. The RFP-PCNA, H2B-mCherry, and H2B-eGFP plasmids confer G418 resistance.

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