The pPAC4 vector was
constructed by Eirik Frengen at Roswell Park Cancer Institute,
in the laboratory of Pieter J. de Jong. The Vector contains
a number of potentially very useful elements.
1.
|
The low copy number replication of bacteriophage
P1 ("plasmid replicon"), which can stably
propagate most foreign DNA sequences in E.coli. |
2.
|
The kanamycin resistance gene (only with
E.coli promotor, thus no G418 resistance). |
3. |
The BamHI cloning site (insert sequences
will replace the small 2.7 kb BamHI fragment). |
4. |
Flanking T7 and SP6 promotor sequences. |
5. |
Two Not1 restriction sites immediately
flanking the T7-BamHI-SP6 segment. |
6. |
The yeast intron-encoded nuclease recognition
site (PI-SceI), which permits linearization of all recombination
clones irrespective of insert size or content (useful
for optical mapping procedures). |
7. |
The Epstein Barr Virus replicon (EBV
oriP), for replication in human cells (require presence
of EBV EBNA-1 protein to be provided in trans). |
8. |
The 34 bp site-specific recombination
site loxG (recombines with wildtype loxP sites catalyzed
by the cre recombinase, and has a CMV promotor + ATG
start codon in the vicinity). In vivo site-specific
recombination with a complementary 1ox site in the mammalian
genome (coupled to truncated, promotor-less neo gene)
allows activation of G418 resistance under control of
the CMV promotor and ATG startcodon. |
9. |
The mutated loxP511 site (recombination
proficient relative to similar loxP511 sites but less
proficient with respect to the wildtype loxP site. |
10. |
The Blasticidin-deaminase gene under
SV40 promotor control, providing resistance to blasticidin
in mammalian cell culture. The file ppac4.txt
has a GenBank compatible The Blasticidin-deaminase gene
under SV40 promotor control, providing resistance to
blasticidin in mammalian cell culture. The file
ppac4.txt has a GenBank compatible sequences from other
elements already present in GenBank. |
pPAC4 clones have Kanamycin antibiotic resistance.
Clones should be grown in LB containing 25ug Kanamycin/ml/