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Sequence-Validated and Expression-Tested Human cDNA in a Dual Expression,,,Vector

(Figure 5). The GFP was biologically active during protein purification and cleavage (data not shown).

Fig.5

Unique Site for Nucleotide Sequence Insertion

The unique Not I site between the cDNA insert and thrombin cleavage site in the pDual GC vector allows any desired nucleotide sequence to be inserted (Figure 1). Not I was chosen as it is estimated to occur only once in 100,000 bases in the human genome and is, therefore, unlikely to be present in most cDNA. For example, inserting nucleotide sequences that encode GFP permits visualization of cellular proteins in mammalian cells. Alternatively, sequences encoding a translation stop codon would result in translational termination at the inserted sequence.

Fast and Easy Subcloning

Unique Pme I sites flanking the cloned cDNA can be used to subclone the RBS/Kozak and cDNA sequences into other vectors. Pme I is estimated to occur only once in 70,000 bases in the human genome and, hence, is unlikely to be present in most cDNA. Digestion with Pme I creates blunt ends that can either be directly ligated to other blunt ends or to adaptors containing the desired ends. Being able to directly subclone the sequence-verified cloned cDNA enables the known nucleotide sequence to be preserved.

Conclusions

The GeneConnection expression-tested clones offer sequence-validated and mammalian expression-tested human cDNA in a dual prokaryotic and eukaryotic expression vector. Using sequence-validated clones makes it possible to draw valid conclusions based on the function of a protein. The dual expression vector eliminates the need to obtain and validate separate expression vectors. The vector contains a hybrid bacterial promoter fo
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