PB-Puro-U6-gRNA-pA plasmid
(Plasmid #247671)

• Purpose: spCas9 gRNA is driven by human U6 promoter, with the gRNA cassette between PuroR and pA, making it detectable in polyA-based RNA-seq.
• Depositing Lab: Tae Hyun Kim
• Publication: pb-Puro-gRNA-pA plasmid - gRNA is readable in RNAseq (unpublished)

Backbone

• Vector backbone: pUC-GW-Amp
• Backbone manufacturer: Genewiz
• Backbone size w/o insert (bp): 2671
• Total vector size (bp): 4067
• Modifications to backbone: Piggybac 5'TR - TK promoter - PuroR - U6 - BBSI - gRNA scaffold - SV40 pA signal - Piggybac 3'TR cassette was cloned into the backbone.
• 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: High Copy

Gene/Insert

• Gene/Insert name: Puromycin resistant gene
• gRNA/shRNA sequence: NA - scaffold, BBSI cloning
• Promoter: TK promoter

Cloning Information

• Cloning method: Gene Synthesis
• 5′ sequencing primer: ACTGGCCGTCGTTTTAC

Resource Information

• Supplemental Documents:
  • PB-Puro-U6-gRNA-pA.gb

Terms and Licenses

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

Depositor Comments

This piggyBac-based spCas9 gRNA expression vector allows both transient gRNA expression and stable integration of the gRNA cassette in the presence of piggyBac transposase. The puromycin resistance marker enables rapid selection. gRNA cloning follows the Zheng lab’s BbsI-based PX330 plasmid protocol. Because the gRNA expression cassette is located between the PuroR gene and the polyadenylation signal, CRISPR gRNAs can be detected by polyA-based RNA-seq, including bulk and single-cell RNA-seq.

5’ – CACCGNNNNNNNNNNNNNNNNNNN – 3’
3’ – CNNNNNNNNNNNNNNNNNNNCAAA – 5’