Addgene - pMAL-EFSep Plasmid Data

Plasmid 34627: pMAL-EFSep

Gene/insert name:
EF-Sep
Alt name:
tufB
Alt name:
EF-Tu
Insert size:
1184
Species:
E. coli
Entrez Gene:
tufB (B21_03809)
Fusion protein or tag:
6xHIS
Terminal:
C terminal on insert
Fusion protein or tag:
MBP
Terminal:
N terminal on backbone
Mutation:
His67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, Asn274 to Trp, and Val 351 Leu
Vector backbone:
pMAL-c2x
(Search Vector Database)
Backbone manufacturer:
NEB
Vector type:
Bacterial Expression
Backbone size w/o insert (bp):
6700
Cloning site 5':
EcoRI
Site destroyed during cloning:
Unknown
Cloning site 3':
Pst
Site destroyed during cloning:
Unknown
5' sequencing primer:
MBP_F List of Sequencing Primers
3' sequencing primer:
pBAD_rev
Bacterial resistance(s)
Ampicillin
Growth strain(s)
DH5alpha
Growth temperature (℃):
37
High or low copy:
High Copy
Sequence: View sequences (2)
Principal Investigator:
Jesse Rinehart
Principal Investigator:
Dieter Soll
Terms and Licenses:
MTA

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.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.

Article: Expanding the genetic code of Escherichia coli with phosphoserine. Park et al (Science. 2011 Aug 26;333(6046):1151-4. PubMed)

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication. Also, please include the text "Addgene plasmid 34627" in your Materials and Methods section.

Price: $65.00

Available to academic and non-profits only

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This is commonly requested with

34928: Top10∆serB

34929: BL21∆serB

16331: pCS2FLAG