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pTTCC
(Plasmid #164264)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 164264 Standard format: Plasmid sent in bacteria as agar stab 1 $75

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pLEI250.2
  • Backbone manufacturer
    Lei Wang, Universitat Stuttgart
  • Backbone size w/o insert (bp) 5371
  • Total vector size (bp) 7778
  • Modifications to backbone
    BbsI restriction site removed
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Kanamycin
  • Growth Temperature
    37°C
  • Growth Strain(s)
    NEB Stable
  • Growth instructions
    use 30 ug/ml kanamycin for selection in E. coli; use 23 ug/ml for selection in T. thermophilus
  • Copy number
    Unknown

Gene/Insert 1

  • Gene/Insert name
    Cas9
  • Species
    Synthetic; Synthesized, codon optimized Cas9 gene from Geobacillus sp. LC300
  • Insert Size (bp)
    3264
  • GenBank ID
    AKU27017.1
  • Promoter Pcel

Cloning Information for Gene/Insert 1

  • Cloning method Restriction Enzyme
  • 5′ cloning site NdeI (not destroyed)
  • 3′ cloning site HindIII (not destroyed)
  • 5′ sequencing primer NA
  • (Common Sequencing Primers)

Gene/Insert 2

  • Gene/Insert name
    gRNA scaffold
  • Species
    Synthetic; Geobacillus sp. LC300 derived gRNA; spacer cloned between BbsI restriction sites
  • Insert Size (bp)
    194
  • Promoter trc

Cloning Information for Gene/Insert 2

Resource Information

Depositor Comments

The plasmid carries a thermostable CRISPR-Cas9 genome editing system - i.e., a Cas9 enzyme and associated gRNA. It was designed for genome editing in Thermus thermophilus.
A spacer can be inserted by restriction ligation cloning via BbsI restriction site, as explained in supplementary document.
Homologous recombination constructs can be cloned via KpnI restriction site.

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:

    pTTCC was a gift from Gudmundur Hreggvidsson (Addgene plasmid # 164264 ; http://n2t.net/addgene:164264 ; RRID:Addgene_164264)
  • For your References section:

    Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant. Adalsteinsson, Bjorn Thor, Kristjansdottir, Thordis, Merre, William, Helleux, Alexandra, Dusaucy, Julia, Tourigny, Mathilde, Fridjonsson, Olafur & Hreggvidsson, Gudmundur Oli. Scientific Reports volume 11, Article number: 9586 (2021) 10.1038/s41598-021-89029-2