TLCV2
(Plasmid #87360)

• Purpose: LentiCRISPR v2 was modified into an all-in-one dox inducible system. The addition of doxycycline induces Cas9-2A-eGFP. The U6 promoter drives constitutive sgRNA expression.
• Depositing Lab: Adam Karpf
• Publication: Barger et al Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi: 10.3390/cancers11020251.

Backbone

• Vector backbone: lentiCRISPR v2
• Backbone manufacturer: Feng Zhang lab
• Backbone size w/o insert (bp): 16733
• Modifications to backbone: LentiCRISPR v2 was modified into an all-in-one inducible system, doxycycline induces Cas9-P2A-GFP expression. sgRNA and puromycin expression are driven by constitutive promoters. The following modifications were made to lentiCRISPR v2: the EF1 promoter was removed and replaced with a tight TRE promoter; P2A-puromcycin was removed and replaced with T2A-eGFP; and a EF1 promoter-puromycin-P2A-rtTA cassette was added.
• Vector type: Mammalian Expression, Lentiviral, CRISPR ; Doxycycline inducible; eGFP reporter
• Selectable markers: Puromycin

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: Cas9-2A-eGFP
• Promoter: Tight TRE promoter

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: LNCX [5'-AGCTCGTTTAGTGAACCGTCAGATC-3']
• 3′ sequencing primer: EF1a-R [5'CACGGCGACTACTGCACTTA-3']

Resource Information

• Addgene Notes:
  • Addgene Diagnostic Digest
  • Addgene Diagnostic Digest
• Articles Citing this Plasmid:
  • 47 References

Terms and Licenses

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

Depositor Comments

Clone sgRNAs into TLCV2 using the same protocol provided with lentiCRISPR v2 (Plasmid #52961, protocol listed under the RESOURCE INFORMATION section). After you have inserted your sgRNA, you should sequence this region with one of the following primers: hU6-F (5'-GAGGGCCTATTTCCCATGATT-3') or LKO.1 5’(5'- GACTATCATATGCTTACCGT-3').

Information for Cloning Grade DNA (Catalog # 87360-DNA.cg) ( Back to top)

Purpose

Cloning grade DNA is suitable for use in PCR, cloning reactions, or transformation into E. coli. The purity and amount is not suitable for direct transfections.

Delivery

• Amount: 2 µg
• Guaranteed Concentration: 100 ng/µl +/- 5 ng/µl
• Pricing: $105 USD
• Storage: DNA can be stored at 4℃ (short term) or -20℃ (long term).

Terms and Licenses

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

Quality Control

Addgene has verified this plasmid using Next Generation Sequencing. Results are available here