PurposeMSCV-based vector encodes human cyclin E mutated within N- and C-terminal Cdc4 phosphodegrons
Full plasmid sequence is not available for this item.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||47498||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 6521
- Total vector size (bp) 7805
Modifications to backbonenone
Vector typeMammalian Expression, Retroviral ; MSCV-IRES-GFP
Growth in Bacteria
Copy numberHigh Copy
Gene/Insert namecyclin E1
SpeciesH. sapiens (human)
Insert Size (bp)1284
Mutationthreonines 62 and 380 to alanine (T62A T380A)
Entrez GeneCCNE1 (a.k.a. CCNE, pCCNE1)
- Promoter LTR
- Cloning method Restriction Enzyme
- 5′ cloning site Bgl2 (destroyed during cloning)
- 3′ cloning site EcoR1 (not destroyed)
- 5′ sequencing primer MSCV (Common Sequencing Primers)
Terms and Licenses
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.
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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:MIGR1-cyclin E-AA was a gift from Alex Minella (Addgene plasmid # 47498 ; http://n2t.net/addgene:47498 ; RRID:Addgene_47498)
For your References section:Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism. Xu Y, Swartz KL, Siu KT, Bhattacharyya M, Minella AC. Oncogene. 2013 Jul 22. doi: 10.1038/onc.2013.289. 10.1038/onc.2013.289 PubMed 23873023