pcDNA3.1/V5-His-TN-DROSHA
(Plasmid #106252)

• Purpose: Express TN-DROSHA in mammalian cells
• Depositing Lab: Joshua Mendell
• Publication: Chang et al Genome Res. 2015 Sep;25(9):1401-9. doi: 10.1101/gr.193607.115.

Backbone

• Vector backbone: pcDNA3/V5-His
• Backbone manufacturer: invitrogen
• Backbone size w/o insert (bp): 5514
• Total vector size (bp): 9533
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Drosha Ribonuclease III
• Alt name: Drosha
• Species: H. sapiens (human)
• Insert Size (bp): 4017
• Mutation: E1045Q and E1222Q mutations
• GenBank ID: NM_001100412
• Entrez Gene: DROSHA (a.k.a. ETOHI2, HSA242976, RANSE3L, RN3, RNASE3L, RNASEN)
• Promoter: CMV
• Tag / Fusion Protein:
  • V5-His (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CMV forward
• 3′ sequencing primer: BGH reverse

Resource Information

• A portion of this plasmid was derived from a plasmid made by: pcDNA4/TO/cmycDrosha (Thomas Tuschl)

Terms and Licenses

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

Depositor Comments

Please note that DROSHA mutation numbering (E1045Q and E1222Q) is modeled on Heo et al., Mol Cell 32: 276–284. These mutations correspond to E1008Q and E1185Q in this construct. In addition, a series of silent mutations across bp 1203 - 1221render the encoded DROSHA mRNA resistant to siRNAs that target that site.