LOCUS pRF-HUE. 8709 bp DNA circular
DEFINITION Complementary copy of pRF-HUE.
COMMENT The vector can be used for ectopic over expression of genes in
The vector has been designed for the directional cloning of a single PCR
amplicon (typically a CDS plus terminator minus the start codon) after the
strong constitutive GAPDH promoter form Aspergillus nidulans, 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
The U2 primer contains a AUG (= ATG) which should be use as a start codon
in the gene you are trying to over express. Note that the vector does not
a 3'UTR or terminator, why these should be included in the PCR amplicon.
Digestion of vector prior to cloning:
1. Digest over night with PacI
2. Digest 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