pKD‐SepRS-­EFSep-­5x tRNASep (B40 OTS)
(Plasmid #52054)

• Purpose: Used in phosphoprotein synthesis. Expresses the Sep-accepting tRNA (tRNASep), the Sep-tRNA synthetase (SepRS) and an engineered EF-Tu (EFSep)
• Depositing Lab: Jesse Rinehart
• Publication: Rinehart Lab Phosphoprotein synthesis components (unpublished)

Backbone

• Vector backbone: pKK223-3
• Backbone manufacturer: Pharmacia
• 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): Top10
• Growth instructions: Strain grows slowly.
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: SepRS
• Alt name: sepS
• Species: Methanocaldococcuc maripaludis
• Insert Size (bp): 1613
• 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: #45 M13pUC-Reverse

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
• Promoter: Ptac

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: #61 pBAD-R

Gene/Insert 3

• Gene/Insert name: 5x tRNA Sep

Cloning Information for Gene/Insert 3

• Cloning method: Unknown
• 5′ sequencing primer: #207 KanR2-F
• 3′ sequencing primer: #35 LacI-R

Resource Information

• Supplemental Documents:
  • Users Guide for Phosphoprotein reagents.pdf
• Articles Citing this Plasmid:
  • 3 References

Terms and Licenses

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

Depositor Comments

This plasmid is the same as pKD-SepRS-EFSep, Addgene plasmid #34623, with the addition of the 5xtRNA-Sep insert.

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