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opCas12a-T8N (pZZ34, TAT-6MycNLS-opCas12a-2SV40NLS-sfGFP)
(Plasmid #199605)

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This material is available to academics and nonprofits only.
Item Catalog # Description Quantity Price (USD)
Plasmid 199605 Standard format: Plasmid sent in bacteria as agar stab 1 $85
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Backbone

  • Vector backbone
    Twinstrep-SUMO-huLwCas13a
  • Backbone size w/o insert (bp) 6086
  • Total vector size (bp) 11057
  • 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 Cas9-8N, 6ｘMyc NLS from pRG232 (Addgene#149723) was subcloned into Cas9-6S to replace the N-terminus 4ｘSV40.. To generate Cas9-T8N, PCR mutagenesis with overlapping DNA oligonucleotides complementary to the Cas9-8N backbone was used to incorporate the TAT equences into Cas9-8N. To generate opCas12a-T8N, opAsCas12a from pRG232 was subcloned into Cas9-T8N to replace Cas9.
  • 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
    opCas12a-T8N
  • Insert Size (bp)
    4971
  • Tag / Fusion Protein
    • 6xHis, Twin-strep, SUMO

Cloning Information

  • Cloning method Ligation Independent Cloning
  • 5′ sequencing primer SUMO: GGACTCCTTAAGATTCTTG
  • 3′ sequencing primer T7-Term: GCTAGTTATTGCTCAGCGG
    • (Common Sequencing Primers)

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

For opCas12a-T8N bacterial expression, host stain Rosetta 2(DE3)pLysS was used.

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:

    opCas12a-T8N (pZZ34, TAT-6MycNLS-opCas12a-2SV40NLS-sfGFP) was a gift from Junwei Shi (Addgene plasmid # 199605 ; http://n2t.net/addgene:199605 ; RRID:Addgene_199605)

  • For your References section:

    Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Zhang Z, Baxter AE, Ren D, Qin K, Chen Z, Collins SM, Huang H, Komar CA, Bailer PF, Parker JB, Blobel GA, Kohli RM, Wherry EJ, Berger SL, Shi J. Nat Biotechnol. 2023 Apr 24. doi: 10.1038/s41587-023-01756-1. 10.1038/s41587-023-01756-1 PubMed 37095348
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