Full plasmid sequence is not available for this item.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||20975||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Vector backboneMSCV (murine stem cell virus)
- Backbone size w/o insert (bp) 6500
Vector typeMammalian Expression, Retroviral
Growth in Bacteria
Copy numberHigh Copy
SpeciesM. musculus (mouse)
Insert Size (bp)2100
MutationFull length cDNA with ~1.1kb 3'UTR with 2 putative polyA signals mutated to allow expression in MSCV+CD24 selection epitope (FACS surface marker). No polyA signal.
Entrez GeneHoxa9 (a.k.a. D6a9, Hox-1.7)
/ Fusion Protein
- Flag (N terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ cloning site EcoRI (unknown if destroyed)
- 3′ cloning site XhoI (unknown if destroyed)
- 5′ sequencing primer MSCV (Common Sequencing Primers)
A portion of this plasmid was derived from a plasmid made byDebbie Wolgemuth
Terms and Licenses
We discovered that, although a 1,975-nt full-length Hoxa9 clone (numbered from the protein start site; I.M.A.G.E. clone BC055059) would produce protein when transiently expressed from the murine stem cell virus (MSCV) long terminal repeat, following retroviral transduction, the HOXA9 protein could not be detected. Since this cDNA contained two putative polyadenylation signals in the 3' UTR, we removed these sites to determine if they were interfering with retrovirally mediated protein expression. The site at nt 1798 was replaced using a primer containing a KpnI sequence in place of the AATAAA sequence together with a primer containing the BglII site at nt 749 to amplify a 1,052-nt BglII-KpnI fragment by PCR. A second primer containing the KpnI site in place of the polyadenylation signal at nt 1798 was used in conjunction with a primer containing an AAT-to-CGG change in the polyadenylation signal at nt 1975 and an adjacent XhoI site for cloning to generate a 150-nt KpnI-XhoI fragment. Standard methods were used to combine these fragments to create a 1,975-nt Hoxa9 cDNA encoding an N-terminal FLAG-tagged version of the mature protein but lacking the two putative polyadenylation signals. The resulting MSCV clone produced high levels of FLAG-HOXA9 protein and immortalized BM cells.
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:HOXA9-MSCV-CD24 was a gift from Corey Largman (Addgene plasmid # 20975 ; http://n2t.net/addgene:20975 ; RRID:Addgene_20975)
For your References section:MicroRNA-126 regulates HOXA9 by binding to the homeobox. Shen WF, Hu YL, Uttarwar L, Passegue E, Largman C. Mol Cell Biol. 2008 Jul . 28(14):4609-19. 10.1128/MCB.01652-07 PubMed 18474618
Map uploaded by the depositor.