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Understanding Flexi® Vectors for High-Fidelity Cloning


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? 

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

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 d1d3 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: