Gateway® TurboGFP-C entry clone is a vector containing green fluorescent protein TurboGFP gene variant with codon usage optimized for high expression in mammalian cells (humanized) [Haas et al., 1996] (see reporter description). TurboGFP coding sequence is flanked by attL1 and attL2 sites allowing easy site-specific recombination. The Invitrogen Gateway® Technology provides a rapid and highly efficient way to transfer the TurboGFP gene into a number of Gateway® destination vectors for expression in different experimental systems. Multiple cloning site (MCS) located at the 3'-end of TurboGFP gene allows to generate fusions to the TurboGFP C-terminus for expression, localization and cellular dynamics studies.

To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TurboGFP coding sequence [Kozak, 1987].

The vector backbone contains pUC origin of replication and kanamycin resistance gene (Kan^r) for propagation and selection in E. coli.

A localization signal or a gene of interest can be cloned into MCS of the vector both before and after site-specific recombination with a destination vector. It will be expressed as a fusion to the TurboGFP C-terminus when inserted in the same reading frame as TurboGFP and no in-frame stop codons are present.

Alternatively, TurboGFP gene can be fused to the 3'-end of a gene of interest by LR recombination of the Gateway® TurboGFP-C with a destination vector containing this gene in a correct reading frame. In this case, the protein of interest will be expressed as a fusion to the TurboGFP N-terminus.

TurboGFP-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo.

Note: The plasmid DNA was isolated from dam⁺-methylated E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam^- host and make fresh DNA.

Please refer to Invitrogen Gateway® Technology description for detailed instructions regarding LR site-specific recombination reaction. In general, to transfer TurboGFP gene or TurboGFP-fusion construct into the destination vector you will need:

  - Purified plasmid DNA of Gateway® TurboGFP-C
  - A destination vector of choice
  - Invitrogen LR Clonase^TM II enzyme mix (Invitrogen Cat.# 11791-020)
  - Proteinase K solution (supplied with the LR Clonase^TM II enzyme mix)
  - TE-Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
  - Appropriate chemically competent E. coli host and growth media for expression
  - Appropriate selective plates.

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.