Cloning strategy for the simultaneous generation of entry clones in open and closed configuration. A: Sequences of entry clones 3' of the ORF either containing or not containing a stop codon. The sequences correspond to the reverse primer sequences of 2-step PCR. In presence of an A at the degenerated position, a stop codon is created and the Bam HI site (underlined) destroyed. In contrast, the inclusion of a G generates a Bam HI site and results in a translational read-through. B: Schematic presentation of the entry clone map. 'for' and 'rev' indicate the binding sites of the colony PCR primers. The degenerated position is indicated by the arrow. C: Bam HI colony-PCR restriction digest of eight independent colonies resulting from BP cloning of four different ORFs amplified using degenerated reverse primers. The arrows mark the additional band which appears in presence of the Bam HI recognition sequence, indicating that the ORF does not contain a stop codon. ORF 4 contains an internal BamHI site indicated by the appearance of a band of about 100 bp. 'M' indicates the molecular weight marker lanes.