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?

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 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

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).

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: