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pET28a/Cas9-Cys
(Plasmid #53261)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 53261 Standard format: Plasmid sent in bacteria as agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pET28a
  • Backbone manufacturer
    Novagen
  • Backbone size w/o insert (bp) 5369
  • Total vector size (bp) 9542
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Kanamycin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    Unknown

Gene/Insert

  • Gene/Insert name
    Cas9-Cys
  • Species
    S. pyogenes
  • Insert Size (bp)
    4173
  • Mutation
    Additional Cysteine is added to the C-terminal region of Cas9
  • Promoter T7 Promoter
  • Tags / Fusion Proteins
    • 6X His tag (N terminal on backbone)
    • HA epitope (C terminal on insert)
    • NLS (C terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site Not I (not destroyed)
  • 3′ cloning site Not I (not destroyed)
  • 5′ sequencing primer TAA TAC GAC TCA CTA TAG GG
  • 3′ sequencing primer GCT AGT TAT TGC TCA GCG
  • (Common Sequencing Primers)

Resource Information

Depositor Comments

A sequence encoding Cas9 with a cysteine at the C-terminus was prepared by PCR
amplification using a previously described Cas9 plasmid (Cho et al, Nat Biotechnol. 2013)(Addgene: 43945) as the template and then cloned into the pET28-(a) vector (Novagen, Merk Millipore, Germany), which
includes a sequence encoding a His-tag at the N-terminus.

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:

    pET28a/Cas9-Cys was a gift from Hyongbum Kim (Addgene plasmid # 53261 ; http://n2t.net/addgene:53261 ; RRID:Addgene_53261)
  • For your References section:

    Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. Genome Res. 2014 Apr 2. 10.1101/gr.171264.113 PubMed 24696462