The restriction site for NdeI enzyme was added before the start codon of confirmed gene sequence and complete open reading frame was ligated into pET22b (+) expression vector using NdeI and EcoRI restriction sites for both the gene and the vector.
Artificial transposable elements pTn5cat1 and pTn5Spcat endowed with restriction sites for PacI, PmeI and SwaI (unpublished) were used to obtain a collection of insertional mutants, each with an extra restriction site for these endonucleases.
1) was produced using oligonucleotide primers p903SalI (5'GTCGACATAAG TAGGAAATTAAAGTCCAGTAAGGTTACTGGCATTTCT [This oligonucleotide primer was appended with a SalI restriction site (underlined) and the introduced late gene polyhedrin core promoter sequence, ATAAG (bold)]), and p738 (5'ACGAGCT GTGAACT CACCAAGAAT CCAACGTT) with cDNA being generated using oligonucleotide primer p738.
The PCR product which contained the D2EDIII coding sequence preceded by DNA sequence coding for 6 histidine residues and flanked by the AatII restriction sites at both the 5' and 3' termini was then cloned into the corresponding AatII restriction site of pBS-HP-D2D (Fig.
Oligonucleotides used to amplify CLCuKoV promoter were P1 (5' GTTGACTAAAATTGAATCACC-3') as forward with the addition of SacI restriction site and P2 (5'- CAAACGCATACTTAGCAACG-3') as reverse primer with the addition of SalI restriction site.