pKD-SepRS-EFSep
(Plasmid #34623)

• Depositing Labs: Jesse Rinehart, Dieter Söll
• Publication: Park et al Science. 2011 Aug 26;333(6046):1151-4.

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, 50 μg/mL
• 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

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

Resource Information

• Supplemental Documents:
  • pKD_SepRS_EFSep
• Addgene Notes:
  • diagnostic digest
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

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