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URL: http://www.addgene.org/34627
Proper Citation: RRID:Addgene_34627
Insert Name: EF-Sep
Organism: E. coli
Bacterial Resistance: Ampicillin
Defining Citation: PMID:21868676
Vector Backbone Description: Backbone Marker:NEB; Backbone Size:6700; Vector Backbone:pMAL-c2x; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Comments: To construct pMAL-EFSep the gene encoding EF-Sep, was cloned between the NdeI and BamHI sites in the pET20b plasmid (Novaven) to add a C-terminal His6 tag. This fusion construct was then PCR-amplified using primers adding MfeI and PstI restriction sites. The PCR product was cloned in-frame between EcoRI and PstI in pMALc2x (New England Biolabs) to add an N-terminal maltose binding protein (MBP) tag. To prevent possible enzymatic dephosphorylation of O-phospho-L-serine (Sep) in vivo, the gene encoding phosphoserine phosphatase (serB), which is catalyzing the last step in serine biosynthesis, was deleted from Escherichia coli strains Top10 (Top10∆serB - Addgene #34928) and BL21 (BL21∆serB - Addgene #34929). These strains are required hosts when using this plasmid.
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Source: Addgene
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