pcDNA5 FRT/TO SF-HERC2 (ShB-R)
(Plasmid #55613)

• Purpose: Dox-inducible expression of full length, ShB-resistant, 3xFLAG/STREP tagged wild-type HERC2 in Flp-In TREx 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 of pcDNA5 FRT/TO 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 (WT)
• Alt name: HERC2
• Species: H. sapiens (human)
• Insert Size (bp): 14640
• Mutation: 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 (unknown if destroyed)
• 5′ sequencing primer: CGC AAA TGG GCG GTA GGC GTG
• 3′ sequencing primer: TAG AAG GCA CAG TCG AGG C

Resource Information

• Articles Citing this Plasmid:
  • 3 References

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.