pRW006-SM-destination-pro35S-Cas9
(Plasmid #104439)

• Purpose: (Empty Backbone) Destination vector expressing plant-codon-optimized Cas9 under 35S promoter, with sgRNAs be shuffled in; seed coat specific red fluorescence for screening trangene free
• Depositing Lab: Detlef Weigel
• Publication: Wu et al Plant Methods. 2018 Aug 2;14:65. doi: 10.1186/s13007-018-0330-7. eCollection 2018.

Backbone

• Vector backbone: pZ001
• Backbone manufacturer: Lampropoulos et al
• Backbone size (bp): 4114
• Modifications to backbone: Cassettes of pro::pcoCas9 and At2S3::mcherry are engineered into the backbone; and the cassette of ccdB-Chloramphenicol is replaced with proLacZ::LacZ expressing unit
• Vector type: Bacterial Expression, Plant Expression, CRISPR
• Promoter: 35S
• Selectable markers: red fluorescence

Growth in Bacteria

• Bacterial Resistance(s): Spectinomycin, 50 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Cloning Information

• Cloning method: Restriction Enzyme

Terms and Licenses

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

Depositor Comments

There are two additional BsaI cloning sites in the Cas9 sequences, but they should not affect the cloning process, since GoldenGate cloning can assemble four or more fragments into a backbone. The additional BsaI sites create four overhangs that are very different from the overhangs for integrating the sgRNAs, and there should be no major issues with fragment assembly or cloning efficiency.