LOCUS pRF-HU2E. 8696 bp DNA circular
DEFINITION Complementary copy of pRF-HU2E.
COMMENT The vector has been designed for targeted (in locus) over expression
of gene in
filamentous fungi. Compared to pRF-HU2 this vector contains the GAPDH
promoter from Aspergillus nidulans.
Like pRF-HU2 this vector allows for the simultaneous directional cloning of
PCR amplicons into two unique sites of the vector, located on either site of
hph (hygromycin B resistance gene) and pGAPDH, utilizing the USER friendly
cloning technology from NEB.
Add the following 5' overhangs to the primers for amplification of the two
specific homologous recombination flanks:
Gene-U1 (LB forward) 5´- GGGTTTAAU - Forward primer sequence
Gene-U2 (LB reverse) 5'- GGACTTAAUG - Reverse primer sequence
Gene-U3 (RB forward) 5'- GGCATTAAU - Forward primer sequence
Gene-U4 (RB reverse) 5'- GGTCTTAAU - Reverse primer sequence
The U2 primer contains a AUG (= ATG) which can be used as start codon for
the gene you want to over express.
Digestion of vector prior to cloning:
1. Digest over night with PacI
2. Diges for 2 hours with Nt.Bbv.C IB (nicking enzyme)
Transformation of the vector into the fungi:
The vector is originate designed for Agrobacterium tumefaciens mediated
transformation, but can also be used for protoplast transformation