|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||40999||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Modifications to backboneVenus-3xFlag added (C-terminal), converted to Gateway destination vector (see notes below)
Vector typeMammalian Expression
- Promoter CMV
/ Fusion Proteins
- Venus YFP (C terminal on insert)
- 3xFLAG (DYKDHDGDYKDHDIDYKDDDDK) (C terminal on insert)
Growth in Bacteria
Bacterial Resistance(s)Chloramphenicol and Ampicillin
Growth Strain(s)ccdB Survival
- Cloning method Restriction Enzyme
- 5′ sequencing primer LNCX
- 3′ sequencing primer EGFP-N, BGH-rev (Common Sequencing Primers)
Venus YFP-3xFlag was inserted via BstXI-XhoI sites (to create pcDNA5/FRT/TO-Venus-Flag, Addgene Plasmid# 40998).
The plasmid was then converted to a Gateway destination vector by inserting Gateway cassette C1, containing recombination sites attR1 and attR2, CmR, and ccdB, into the Afl II site (blunt ended).
This plasmid can be used to place genes into the pcDNA5/FRT/TO-Venus-Flag construct by Gateway reaction, to make tetracycline-inducible stable cell lines.
For subcloning, the following restriction sites are present:
Between the CMV promoter and the Gateway cassette: PmeI (non-unique)
Between the Gateway cassette and Venus: HindIII, Asp718I, KpnI, BamHI (non-unique, also in GW casette), and BstXI
Between Venus and BGHpA: XhoI, Eco0109I, ApaI, and PmeI (non-unique)
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:pcDNA5/FRT/TO-Venus-Flag-Gateway (1124) was a gift from Jonathon Pines (Addgene plasmid # 40999 ; http://n2t.net/addgene:40999 ; RRID:Addgene_40999)