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URL: http://www.addgene.org/198752

Proper Citation: RRID:Addgene_198752

Insert Name: Cre-PhoCI-ERT2-ERT2

Organism: Synthetic

Bacterial Resistance: Ampicillin

Defining Citation: PMID:36307419

Vector Backbone Description: Backbone Marker:Vlatka Zoldos; Backbone Size:8020; Vector Backbone:#71666 pdCas9-DNMT3A-EGFP; Vector Types:Mammalian Expression, Bacterial Expression, Lentiviral; Bacterial Resistance:Ampicillin

Comments: Cloning was performed using a third generation lentiviral plasmid as a backbone (pdCas9-DNMT3A-EGFP (Vlatka Zoldoš, #71666)) digested with NheI-EcoRI and ligated to the following DNA fragments: TREGV 3rd generation promoter (amplified by PCR using TRE-KRAB-dCas9-IRES-GFP (Eric Lander, #85556) as a template and digested with NheI-XmaI), PhoCl-ERT2-Cre-ERT2-PhoCl sequence (pCAG-ERT2-PhoCl-Cre-PhoCl-ERT2 (Robert Campbell, #87694)) digested with XmaI-NotI, and NotI-EcoRI adapter. From construct PhoCl-ERT2-Cre-ERT2-PhoCl, the sequence Cre-PhoCl-ERT2 was amplified. Cre sequence from the original construct lacked the canonical first 16 aminoacids (MANLLTVHQNLPALPV), so we restored them. The synthesized fragment was ligated into the pCAG-ERT2-PhoCl-Cre-PhoCl-ERT2 vector upon PacI and EcoRI digestion. Using this as a template, we amplified Cre-PhoCl-ERT2 sequence to generate a new fragment flanked by PacI and AscI restriction sites. An additional ERT2 domain was synthetized by PCR with AscI and EcoRI ends and one 5′-GSGSGGG flexible linker. Upon digestion, the two fragments were ligated into the pCAG-ERT2-PhoCl-Cre-PhoCl-ERT2 vector upon PacI and EcoRI digestion. Finally, Cre-PhoCl-ERT2(x2) fragment was jumped into the PiggyBac XLone vector system25. To this end, XLone plasmid backbone was PCR and Cre-PhoCl-ERT2(x2) cassette ligated upon NheI-NotI digestion.

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