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Promega Corporation

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 Designation Options
Plasmid p
Flexi® Vector Family F
Position of fusion partner N N-terminus position
C C-terminus position
Vector number 1–24 1–24 indicates 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. 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 baclovirus 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 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:

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