LP102: pMAGIC (R4-R3) NLS-Sp Cas9-NLS
(Plasmid #132927)

• Purpose: pMAGIC R4-R3 entry plasmid, contains 2xNLS SpCas9 (nuclease active) for 3 or 4-component MultiSite Gateway Pro assembly.
• Depositing Lab: Christopher Newgard
• Publication: Unpublished pMVP and pMAGIC plasmids (unpublished)

Backbone

• Vector backbone: pDONR221 P4r-P3r
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 2664
• Total vector size (bp): 6876
• Vector type: CRISPR, Synthetic Biology ; pMAGIC Gateway Entry plasmid

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: humanized SpCas9
• Alt name: S. pyogenes CRISPR-Cas9
• Alt name: Cas9
• Species: Synthetic; Streptococcus pyogenes
• Insert Size (bp): 4212

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: M13F
• 3′ sequencing primer: M13R

Resource Information

• Supplemental Documents:
  • LP102_2xNLS_SpCas9.gbk

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

SpCas9 was PCR'd from lentiCRISPRv2 (Addgene #52961) and inserted into PmeI/SrFI digested KJ901 (Addgene #121835).

This entry plasmid is compatible with pMAGIC cloning platform (PMID: 30590691). The 2x-NLS SpCas9 gene encoded in this plasmid does not contain a stop codon. SpCas9 gRNAs can be expressed from plasmids KK701 (Addgene #121842), LF701 (Addgene #121817), or LG002 (Addgene # 121816).