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 Welcome  
 Vector PPAC4 Information/Map    

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.

Reference: Frengen E. et al. (2000) Modular Bacterial Artificial Chromosome Vectors for Transfer of Large Inserts into Mammalian Cells. Genomics 58:118-126.

pPAC4 clones have Kanamycin antibiotic resistance.  Clones should be grown in LB containing 25ug Kanamycin/ml/

 

 

 

 


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