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PurposeBacterial expression plasmid for SpCas9 & sgRNA (need to clone spacer into BsaI sites): T7-humanSpCas9-NLS-3xFLAG-T7-BsaIcassette-Sp-sgRNA
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 65767 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepACYCDuet-1
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Backbone manufacturerEMD Millipore
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Vector typeBacterial Expression, CRISPR
Growth in Bacteria
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Bacterial Resistance(s)Chloramphenicol, 25 μg/mL
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Growth Temperature37°C
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Growth Strain(s)XL1 Blue
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Copy numberLow Copy
Gene/Insert
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Gene/Insert namemammalian codon-optimized Streptococcus pyogenes Cas9-NLS-3XFlag, and SpCas9 gRNA
- Promoter T7 (x2)
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Tags
/ Fusion Proteins
- NLS (C terminal on insert)
- 3x FLAG (C terminal on insert)
Cloning Information
- Cloning method Gibson Cloning
- 5′ sequencing primer ACYC-DuetUP1
- 3′ sequencing primer oBK390 (5'-GCAAATTCGACCCGGTCGTCG-3') (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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A portion of this plasmid was derived from a plasmid made byThe pACYCDuet-1 plasmid backbone was provided by The University of Western Ontario
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Article Citing this Plasmid
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
Use BsaI sites to insert sgRNA spacer sequence into the vector.
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:
BPK764 was a gift from Keith Joung (Addgene plasmid # 65767 ; http://n2t.net/addgene:65767 ; RRID:Addgene_65767) -
For your References section:
Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369