pcDNA5 FRT/TO SF-HERC2 C4762S (ShB-R)
(Plasmid #55614)

• Purpose: Dox-inducible expression of full length, ShB-resistant, 3xFLAG/STREP tagged C4762S (catalytically inactive) HERC2 in Flp-In T-Rex cells
• Depositing Lab: David Chan
• Publication: Chan et al J Biol Chem. 2014 May 22. pii: jbc.M114.569392.

Backbone

• Vector backbone: pcDNA5 FRT/TO
• Backbone manufacturer: Life Technologies
• Backbone size w/o insert (bp): 5137
• Total vector size (bp): 19689
• Modifications to backbone: The first PmeI site at the beginning of the MCS was replaced with a NheI site
• Vector type: Mammalian Expression
• Selectable markers: Hygromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Because of the size of the insert, DH5alpha cells transformed with this plasmid grows much slower, and the yield of DNA from a miniprep is usually low.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: 3xFLAG tagged HERC2 (C4762S)
• Alt name: HERC2
• Species: H. sapiens (human)
• Insert Size (bp): 14640
• Mutation: C4762 mutated to serine to render HERC2 catalytically inactive. Also, Silent mutations have been incorporated into the ORF of HERC2 to render it refractory to the shRNA described in the associated publication.
• GenBank ID: NM_004667.5
• Entrez Gene: HERC2 (a.k.a. D15F37S1, MRT38, SHEP1, jdf2, p528)
• Tags / Fusion Proteins:
  • 3xFLAG (N terminal on insert)
  • STREP (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: PmeI (not destroyed)
• 5′ sequencing primer: CGC AAA TGG GCG GTA GGC GTG
• 3′ sequencing primer: TAG AAG GCA CAG TCG AGG C

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

There is a unique XhoI between the N-terminal 3xFLAG/STREP tag and the beginning of the HERC2 ORF. When cut with XhoI, PmeI, the untagged HERC2 ORF can be excised.

There is a TEV-protease site inserted between the N-terminal 3xFLAG/STREP tag and the HERC2 ORF.

Silent mutations have been incorporated into the ORF of HERC2 to render it refractory to the shRNA described in the associated publication.