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:
  • 40 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