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PurposeExpresses a thermostable Cas9 enzyme and gRNA in Thermus thermophilus
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 164264 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepLEI250.2
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Backbone manufacturerLei Wang, Universitat Stuttgart
- Backbone size w/o insert (bp) 5371
- Total vector size (bp) 7778
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Modifications to backboneBbsI restriction site removed
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Vector typeBacterial Expression
Growth in Bacteria
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Bacterial Resistance(s)Kanamycin, 50 μg/mL
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Growth Temperature37°C
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Growth Strain(s)NEB Stable
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Growth instructionsuse 30 ug/ml kanamycin for selection in E. coli; use 23 ug/ml for selection in T. thermophilus
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Copy numberUnknown
Gene/Insert 1
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Gene/Insert nameCas9
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SpeciesSynthetic; Synthesized, codon optimized Cas9 gene from Geobacillus sp. LC300
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Insert Size (bp)3264
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GenBank IDAKU27017.1
- 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
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Gene/Insert namegRNA scaffold
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SpeciesSynthetic; Geobacillus sp. LC300 derived gRNA; spacer cloned between BbsI restriction sites
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Insert Size (bp)194
- Promoter trc
Cloning Information for Gene/Insert 2
- Cloning method Gibson Cloning
- 5′ sequencing primer NA (Common Sequencing Primers)
Resource Information
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Supplemental Documents
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
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.
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.
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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 BT, Kristjansdottir T, Merre W, Helleux A, Dusaucy J, Tourigny M, Fridjonsson O, Hreggvidsson GO. Sci Rep. 2021 May 5;11(1):9586. doi: 10.1038/s41598-021-89029-2. 10.1038/s41598-021-89029-2 PubMed 33953310