Understanding Flexi® Vectors for High-Fidelity Cloning
Learn how Flexi® Vector technology provides research options to explore protein function.
What are Flexi® Vectors?
The Flexi® Vectors provide a simple method for directional cloning of protein-coding DNA sequences. The method is based on the rare-cutting restriction enzymes, SgfI and PmeI, to insert DNA fragments and provide a rapid, high-fidelity way to transfer protein-coding regions between vectors without the need to resequence.
Use Flexi® Vectors to:
- study protein function/control with in vivo expression systems
- express fusion proteins in cell-free or in vivo expression systems
- study protein interactions using expression tags for pull-down studies
- create stable or transient clones expressing the protein of interest
How do you interpret the Flexi® Vector nomenclature?
As a first step in selecting the Flexi® Vector system for your research purposes, learn how the product name describes the vector characteristics. Interpret this Flexi® vector name using the table below:
Example: pFC15A HaloTag® CMVd1 Flexi® Vector
p = plasmid
F = Flexi® Vector Family
C = C-terminus position of HaloTag fusion protein
A = ampicillin-resistance gene
HaloTag® = modified hydrolase fusion tag
CMVd1 = cytomegalovirus promoter with medium expression level
Table: Flexi® Vector Terminology
|Flexi® Vector Feature
|Flexi ®Vector Family
|Position of fusion partner
||1–24 indicates chronological development of the vectors|
||ampicillin resistance for positive selection in prokaryotic cells|
||kanamycin resistance for positive selection in prokaryotic cells|
||neomycin resistance for positive selection in mammalian cells|
|Fusion tag or reporter gene
||HaloTag® reporter protein, a 34.1kDa modified hydrolase, provides a nonmammalian, soluble tag for purification, pull-downs and detection |
||histidine-glutamine tag for purification via immobilized metal affinity ligand|
||glutathione-S-transferase tag for purification via immobilized glutathione |
||synthetic Renilla Luciferase gene|
|Promoter and regulatory elements
||human cytomegalovirus for constitutive expression in most mammalian cells; the d1–d3 designation following CMV describes the deletion variant that controls expression levels where d1 is medium, d2 is low, d3 is ultralow. Absence of the d indicates high expression levels|
||SP6 RNA polymerase-driven protein expression in cell-free translation systems|
||T7 RNA polymerase-driven protein expression in E. coli or cell-free translation systems|
||5´ untranslated region (UTR) and 3’ translation enhancer (TE) sequences from the barley yellow dwarf virus to stimulate translation in wheat germ extracts|
||Twelve tandem repeats of the λ operator sequence followed by the CMV promoter for controlled protein expression in mammalian cells|
||5´ and 3´ UTR sequences from the baclovirus polyhedron gene for protein expression in cell-free insect systems |
|Protein expression levels
||No "d" included
||High protein expression levels
||Medium protein expression levels
||Low protein expression levels
||Ultra-low protein expression levels
Learn more by visiting our blog article entitled "Understanding the Flexi® Vector Terminology."
How do I select a Flexi® Vector for my purposes?
There are over 40 Flexi® Vector Systems from which to choose. When selecting a vector consider the following outcomes; 1) improving protein expression for a specific expression system (e.g., mammalian, bacterial, cell-free), 2) creating a fusion protein with a reporter enzyme or tag, 3) designating the position (C- or N-terminus) of a tag or reporter, 4) modulating protein expression levels (high to ultra-low). See detailed product categories under "RELATED PRODUCTS" above right.
Find a more detailed overview on "How to Use Flexi® Vectors Part 1 and Part 2" at the Promega Connections blog. Gain a better understanding of the strategy behind vector selection.
What publications demonstrate the use of the Flexi® Vector technology?
Promega maintains a citations database of peer-reviewed articles at the Promega Technical Resources web page where you can search parameters relevant to your field of study.
Here are just a few key citations demonstrating the advantages of the Flexi® Vector Systems: