Gateway® HyPer-AS entry clone is a vector containing HyPer gene variant with codon usage optimized for high expression in Arabidopsis and Saccharomyces (see reporter description). HyPer 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 HyPer gene into a number of Gateway® destination vectors for expression in different experimental systems.

To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the HyPer 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.

LR site-specific recombination

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

  - Purified plasmid DNA of Gateway® HyPer-AS
  - 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.

Propagation in E. coli

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.