This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at [email protected] Learn more

pMAL-EFSep
(Plasmid #34627)

Loading...

Full plasmid sequence is not available for this item.

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 34627 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pMAL-c2x
  • Backbone manufacturer
    NEB
  • Backbone size w/o insert (bp) 6700
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert

  • Gene/Insert name
    EF-Sep
  • Alt name
    tufB
  • Alt name
    EF-Tu
  • Species
    E. coli
  • Insert Size (bp)
    1184
  • Mutation
    His67 to Arg , Glu216 to Asn, Asp217 to Gly, Phe219 to Tyr, Thr229 to Ser, Asn274 to Trp, and Val 351 Leu
  • Entrez Gene
    tufB (a.k.a. B21_03809)
  • Tags / Fusion Proteins
    • 6xHIS (C terminal on insert)
    • MBP (N terminal on backbone)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site EcoRI (unknown if destroyed)
  • 3′ cloning site Pst (unknown if destroyed)
  • 5′ sequencing primer MBP_F
  • 3′ sequencing primer pBAD_rev
  • (Common Sequencing Primers)

Resource Information

  • Terms and Licenses

Depositor 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.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pMAL-EFSep was a gift from Jesse Rinehart & Dieter Söll (Addgene plasmid # 34627 ; http://n2t.net/addgene:34627 ; RRID:Addgene_34627)
  • For your References section:

    Expanding the genetic code of Escherichia coli with phosphoserine. Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ, Rinehart J, Soll D. Science. 2011 Aug 26;333(6046):1151-4. 10.1126/science.1207203 PubMed 21868676