Skip to main content
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

pKD-SepRS-EFSep
(Plasmid #34623)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 34623 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pKK223-3
  • Backbone manufacturer
    Pharmacia
  • Backbone size w/o insert (bp) 4300
  • Modifications to backbone
    pKD is derived from pKK223-3 (Pharmacia). The ampicillin (Amp) resistance gene was replaced with a kanamycin (Kan) resistance gene. The original multiple cloning site (MCS; NcoI-EcoRI-SacI) was modified by adding an additional ribosome binding site (RBS) and a second MCS (NdeI-BamHI-SalI-HindIII), thus enabling simultaneous protein expression from two genes, both under the control of the same tac promoter.
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Kanamycin
  • Growth Temperature
    30°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    High Copy

Gene/Insert 1

  • Gene/Insert name
    SepRS
  • Alt name
    sepS
  • Species
    Methanocaldococcuc maripaludis
  • Insert Size (bp)
    1613
  • Entrez Gene
    MMP0688 (a.k.a. MMP0688)
  • Promoter Ptac

Cloning Information for Gene/Insert 1

  • Cloning method Restriction Enzyme
  • 5′ cloning site NcoI (not destroyed)
  • 3′ cloning site SacI (not destroyed)
  • 5′ sequencing primer M13_pUC-Rev
  • (Common Sequencing Primers)

Gene/Insert 2

  • 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, and Asn274 to Trp
  • Entrez Gene
    tufB (a.k.a. B21_03809)
  • Promoter Ptac (from SepRS- operon like)

Cloning Information for Gene/Insert 2

  • Cloning method Restriction Enzyme
  • 5′ cloning site BamHI (not destroyed)
  • 3′ cloning site SalI (not destroyed)
  • 5′ sequencing primer pJR_F2
  • 3′ sequencing primer pBAD_rev
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

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.

Certain elements of this plasmid are covered under US patent 9,090,928 to Yale University

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:

    pKD-SepRS-EFSep was a gift from Jesse Rinehart & Dieter Söll (Addgene plasmid # 34623 ; http://n2t.net/addgene:34623 ; RRID:Addgene_34623)
  • 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