Cas9-T6N (pZZ12, TAT-4MycNLS-Cas9-2SV40NLS-sfGFP)
(Plasmid #199604)

• Purpose: Expression of cell permeable spCas9 in E. Coli.
• Depositing Lab: Junwei Shi
• Publication: Zhang et al Nat Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1.

Backbone

• Vector backbone: pC013 - Twinstrep-SUMO-huLwCas13a
• Backbone size w/o insert (bp): 6086
• Total vector size (bp): 11171
• Modifications to backbone: To generate the Twinstrep-SUMO-Cas9-6S (Cas9-6S) expression vector for recombinant Cas9 protein expression in bacteria, the protein coding sequence “4 x SV40 NLS-Cas9-2 x SV40 NLS-sfGFP” from (Addgene#88921) was subcloned into Twinstrep-SUMO-huLwCas13a (Addgene#90097). To generate Cas9-6N, 4ｘMyc NLS from pRG232 (Addgene#149723) was subcloned into Cas9-6S to replace the N-terminus 4ｘSV40. To generate Cas9-T6N, PCR mutagenesis with overlapping DNA oligonucleotides complementary to the Cas9-6N backbone was used to incorporate the TAT equences into Cas9-6N.
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Cas9-T6N
• Insert Size (bp): 5085

Cloning Information

• Cloning method: Ligation Independent Cloning
• 5′ sequencing primer: SUMO: GGACTCCTTAAGATTCTTG
• 3′ sequencing primer: T7-Term: GCTAGTTATTGCTCAGCGG

Resource Information

• Supplemental Documents:
  • ZHANG_a7d_1_pZZ12_pLann.gbk
  • pZZ12 (TAT-4MycNLS-Cas9-2SV40NLS-sfGFP).gb

Terms and Licenses

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

Depositor Comments

For Cas9-T6N bacterial expression, host stain Rosetta 2(DE3)pLysS was used.