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How to Choose a SnapFast™ Expression Vector

With >1600 vectors to choose from, you have many options when planning your cloning constructs. Finding the perfect expression vector starts with understanding what you need, and what the options are.

Three Steps For Selecting A New SnapFast™ Vector

1. Determine the promoter you want to use, based on the host cell you will be expressing in, and how strong you want the expression to be.

Mammalian species typically use:

  • CMV – the most common constitutive promoter
  • SV40 – strong constitutive promoter
  • EF1α – very strong constitutive promoter
  • CAG – a constitutive  promoter made as a chimera of CMV early enhancer and chicken β actin (used in place of CMV that is occaisionally silenced by methylation)

Bacteria promoters include:

  • OXB – constitutive promoter series created by Oxford Genetics by random mutation of RecA
  • LacI – inducible promoter controlled by Lac operon (induced by IPTG)
  • T7 – inducible promoter controlled by Lac repressor

Yeast promoters include:

  • TEF1 – strong constitutive promoter
  • ADH1 – medium strength constitutive promoter
  • STE1 – weakest constitutive promoter
  • GAL1 – inducible promoter (induced by galactose)

2. Determine what cleavage sites, tags or reporter genes you need for your downstream applications.

Detection by immunoprecipitation (IP) and Western blot. They can also be used for protein purification. These tags have antibodies commercially available:

  • Histidine - 6 or 10 residues in a series, with a strong affinity for metal ion matrices, so they are commonly purified with IMAC
  • FLAG – 8 amino acid sequence usually folds to the outside of the protein for easy access
  • 3xFLAG – up to 200x stronger than FLAG system
  • C-myc – peptide derived from the myc regulator

Detection in-cell:

  • Luminescent reporters, based on luciferase enzyme catalyzing visible light
    • Firefly luciferase – cytosolic
    • Renilla luciferase – cytosolic
    • Ilumena luciferase – secreted
  • Fluorescent reporters (when excited by a fluorophore, emit light at a higher wavelength):
    • Green fluorescent protein (GFP)
    • Yello fluorescent protein (YFP)
    • Cyan fluorescent protein (CFP)

Cleavage of the tag will require selection of a vector with a cleavage tag:

  • Enterokinase (Ekt) – cleaves after a lysine residue
  • Tobacco Etch Virus (TEV) – cleaves between a Glu and Gly residues

3. Determine which selectable marker you prefer to work with. Your options will depend on which host cell type you are using.

Mammalian:

  • Neomycin, G418
  • Puromycin
  • Hygromycin
  • Zeocin

Bacterial:

  • Ampicillin
  • Kanamycin

Yeast:

  • Ura3
  • TRP1
  • Leu2
  • His3

Once you have decided on the attributes you would like to have in your vector, you can find it in the collection by either using the selection tables, checking the most popular vectors list, or using the enhanced search. For enhanced search, start with the list of all SnapFast™ vectors, and use the facets on the left to narrow down your options based on the characteristics you find most important in your desired vector.

Materials

     
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