876 pJet1.2-Dr-Muts

PCR 3 fragments from genomic DNA
Fragment 1

874-Nde1-F2
CAGCGGCCTGGTGCCGCGCGGCAGC CATATGCGAGCAGTTTCCCCCACCAAAG

874-R1
TCGCCGAGCGTTTCCCGAATCTCAC
(GTGAGATTCGGGAAACGCTCGGCGA)

Fragment 2:
874-seq-F1: TGA GAT TCG GGA AAC GCT C  

847-R2
GGGCGCGGTCTTTGAGGGTCGCAATC
(GATTGCGACCCTCAAAGACCGCGCCC)

Fragment 3:
874-seq-F2: AGA TTG CGA CCC TCA AAG AC 
874-Sal1-R2
(CTGGGGGAGGGCCATGAATCCCGG GTCGACAAGCTTGCGGCCGCACTCGAGCACC)
GGTGCTCGAGTGCGGCCGCAAGCTTGTCGACCCGGGATTCATGGCCCTCCCCCAG

1st PCR

2nd PCR

mix 3 fragments, and gel purified, PCR using

874-Nde1-F2
CAGCGGCCTGGTGCCGCGCGGCAGC CATATGCGAGCAGTTTCCCCCACCAAAG

874-Sal1-R2
(CTGGGGGAGGGCCATGAATCCCGG GTCGACAAGCTTGCGGCCGCACTCGAGCACC)
GGTGCTCGAGTGCGGCCGCAAGCTTGTCGACCCGGGATTCATGGCCCTCCCCCAG

PCR screening using 

874-seq-F1: TGA GAT TCG GGA AAC GCT C  

847-R2
GGGCGCGGTCTTTGAGGGTCGCAATC
(GATTGCGACCCTCAAAGACCGCGCCC)

miniprep digest with Nde1+ Xho1