MIGR1-cyclin E-AA
(Plasmid #47498)

• Purpose: MSCV-based vector encodes human cyclin E mutated within N- and C-terminal Cdc4 phosphodegrons
• Depositing Lab: Alex Minella
• Publication: Xu et al Oncogene. 2013 Jul 22. doi: 10.1038/onc.2013.289.

Backbone

• Vector backbone: MIGR1
• Backbone size w/o insert (bp): 6521
• Total vector size (bp): 7805
• Modifications to backbone: none
• Vector type: Mammalian Expression, Retroviral ; MSCV-IRES-GFP
• Selectable markers: GFP

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Top10
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: cyclin E1
• Species: H. sapiens (human)
• Insert Size (bp): 1284
• Mutation: threonines 62 and 380 to alanine (T62A T380A)
• Entrez Gene: CCNE1 (a.k.a. CCNE, pCCNE1)
• Promoter: LTR

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Bgl2 (destroyed during cloning)
• 3′ cloning site: EcoR1 (not destroyed)
• 5′ sequencing primer: MSCV

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

Human cyclin E1 (CCNE1) cDNA was mutated at N- and C-terminal Cdc4 phosphodegrons (T62A and T380A, see references) to disrupt interaction with Fbw7 ubiquitin ligase. cDNA is inserted into MIGR1 (MSCV-based retroviral vector), permitting transduction of hematopoietic cells.

Cloned cDNA contains approximately 100 bp from CCNE1 3' UTR.

GFP is co-expressed with cDNA via IRES, permitting enrichment for transduced cells using flow sorting.

References:
Koepp DM, Schaefer LK, Ye X, Keyomarsi K, Chu C, Harper JW et al (2001). Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science 294: 173-177.

Strohmaier H, Spruck CH, Kaiser P, Won KA, Sangfelt O, Reed SI (2001). Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413: 316-322.