pPB-VPR-dSpCas9[StaPL(TI)]
(Plasmid #111508)

• Purpose: Expresses a VPR transcriptional activator fused to the nuclease-deactivated S. pyogenes Cas9, which is governed by an inserted StaPL(TI) module, in mammalian cells. [TI = telaprevir inhibited].
• Depositing Lab: Michael Lin
• Publication: Jacobs et al Nat Methods. 2018 Jul;15(7):523-526. doi: 10.1038/s41592-018-0041-z. Epub 2018 Jul 2.

Backbone

• Vector backbone: pPB
• Backbone manufacturer: System Biosciences
• Total vector size (bp): 13958
• Vector type: Mammalian Expression
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: VPR-dSpCas9[StaPL(TI)]
• Species: H. sapiens (human); Hepatitis C Virus (HCV) genotype 1a, Entacmaea quadricolor, Streptococcus pyogenes, Herpes simplex virus, Epstein-Barr virus
• Insert Size (bp): 7323
• Mutation: The HCV NS3 protease carries F43L, Q80K, and S122R, and D168Y mutations. The VPR domain's internal NLS sequence was mutated to a GGSGGSGGS linker. SpCas9 contains D10A and H840A mutations.
• GenBank ID: NC_004102
• Promoter: PGK

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CCACCTTCTACTCCTCCC
• 3′ sequencing primer: ATTAACCCTCACTAAAGGG

Resource Information

• Supplemental Documents:
  • VPR-dSpCas9_StaPL(TI)_.gb
• A portion of this plasmid was derived from a plasmid made by: the Vladislav Verkhusha lab [TagBFP, Ref. 1], the George Church lab [VPR, Ref. 2], and the Stanley Qi lab [dSpCas9, Ref. 3].

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
  • piggyBac Limited Use Label License
• Industry Terms:
  • Not Available to Industry

Depositor Comments

[Ref. 1]
SUBACH O.M., GUNDOROV I.S., YOSHIMURA M., SUBACH F.V., ZHANG J., GRUENWALD D., SOUSLOVA E.A., CHUDAKOV D.M., & VERKHUSHA V.V. 2008. Conversion of Red Fluorescent Protein into a Bright Blue Probe. Chem Biol 15 (10):1116-24.

[Ref. 2]
CHAVEZ A., SCHEIMAN J., VORA S., PRUITT B.W., TUTTLE M., P R IYER E., LIN S., KIANI S., GUZMAN C.D., WIEGAND D.J., TER-OVANESYAN D., BRAFF J.L., DAVIDSOHN N., HOUSDEN B.E., PERRIMON N., WEISS R., AACH J., COLLINS J.J. & CHURCH G.M. 2015. Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12: 326-328.

[Ref. 3]
GAO Y., XIONG X., WONG S., CHARLES E.J., LIM W.A. & QI L.S. 2016. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods 13: 1043-1049.