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

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 52054 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
  • 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)
    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
  • (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
  • 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
  • (Common Sequencing Primers)

Gene/Insert 3

  • Gene/Insert name
    5x tRNA Sep

Cloning Information for Gene/Insert 3

Resource Information

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

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-­5x tRNASep (B40 OTS) was a gift from Jesse Rinehart (Addgene plasmid # 52054 ; http://n2t.net/addgene:52054 ; RRID:Addgene_52054)