Understanding Flexi® Vectors for High-Fidelity Cloning
Abstract
This article describes how Flexi® Vector technology provides research options to explore protein function. We will answer the following questions: What are Flexi® Vectors? How do you interpret the Flexi® Vector nomenclature? How do I select a Flexi® Vector for my purposes? What publications demonstrate the use of the Flexi® Vector technology?
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
| Flexi® Vector Terminology. | ||
| Flexi® Vector Feature | Designation | Options |
| Plasmid | p | |
| Flexi® Vector Family | F | |
| Position of fusion partner | N | N-terminus position |
| C | C-terminus position | |
| Vector number | 1–24 | chronological development of the vectors |
| Antibiotic resistance | A | ampicillin resistance for positive selection in prokaryotic cells |
| K | kanamycin resistance for positive selection in prokaryotic cells | |
| neo | neomycin resistance for positive selection in mammalian cells | |
| Fusion tag or reporter gene | HaloTag® | HaloTag® reporter protein, a 34.1kDa modified hydrolase, provides a nonmammalian, soluble tag for purification, pull-downs and detection |
| HQ | histidine-glutamine tag for purification via immobilized metal affinity ligand | |
| GST | glutathione-S-transferase tag for purification via immobilized glutathione | |
| hRluc | synthetic Renilla Luciferase gene | |
| Promoter and regulatory elements | CMV | 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, and absence of the d indicates high expression levels |
| SP6 | SP6 RNA polymerase-driven protein expression in cell-free translation systems | |
| T7 | T7 RNA polymerase-driven protein expression in E. coli or cell-free translation systems | |
| BYDV | 5´ untranslated region (UTR) and 3´ translation enhancer (TE) sequences from the barley yellow dwarf virus to stimulate translation in wheat germ extracts | |
| RM | Twelve tandem repeats of the λ operator sequence followed by the CMV promoter for controlled protein expression in mammalian cells | |
| ICE | 5´ and 3´ UTR sequences from the baculovirus polyhedron gene for protein expression in cell-free insect systems | |
| Protein expression levels | No "d" included | High protein expression levels |
| d1 | Medium protein expression levels | |
| d2 | Low protein expression levels | |
| d3 | Ultralow protein expression levels | |
Learn more by visiting our blog article "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 ultralow). See detailed product categories under "Related Products".
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?
We maintain 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:
- Blommel, P.G. et al. (2006) High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector Cloning System. Protein Expr. Purif. 47, 562–70.
Find a comparison of the Flexi® Vector Systems to the Gateway® Cloning System. - Nagase, T., Yamakawa, H. and Ohara, O. (2009) Value-added human Flexi® ORF clones with HaloTag® labeling technology.
Learn how the HaloTag® Flexi® Vector System makes easy work of protein expression in both cell-free and mammalian expression systems.
