Functional Genomics & RNAi

Multiple Knockouts

The TargeTron Vector pACD4K-C-loxP (T2826) is a linerized, E. coli expression vector to be used in conjunction with the TargeTron Gene Knockout System (TA0100). This vector differs from the original pACD4K-C vector in that it has loxP sites flanking each end of the kanamycin ORF allowing for multiple, site-specific knockouts in the same bacterial chromosome. After the initial knockout is generated, the kanamycin resistance marker used to select for insertions can be removed via Cre-loxP mediated recombination.1,2 After removal of the kanamycin resistance marker, sequential "targetron" insertions can be generated.3

pACD4K-C-loxP Vector Sequence

Technical Bulletin

Vector Map

Data

Ordering Information

TargeTron Vector

References

  1. Abremski, K. et al., J. Biol. Chem., 261 (1), 391-396 (1986)
  2. Hartung, M. et al., J. Biol. Chem., 273 (36), 22884-22891 (1998)
  3. Zhong, J. et al., Nucleic Acids Res., 31 (6), 1656-1664 (2003)

 


Related Products
TargeTron Gene Knockout System




TargeTron® Vector pACD4K-C-loxP Vector Map


Feature Position
p15A ori 585-1497
T7 promoter 1741-1762
5' exon (IBS) 1802-1826
Intron RNA 1827-4182
EBS2 (exon binding sequence 2) 2049-2053
EBS1d 2102-2111
5' loxP site 2523-2555
Kanamycin RAM 2556-3923
td group I intron RAM 2853-3245
3' loxP site 3924-3957
3' exon 4183-4192
LtrA ORF 4429-6228

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TargeTron® Vector pACD4K-C-loxP Data


PCR was performed using lacZ gene specific primers. Lanes 1-2 are duplicate reactions using wild-type BL21(DE3) as template. Lanes 3-4 are duplicate reactions using the lacZ1062a intron insertional mutant containing the kanamycin marker (i.e., before Cre recombination). Lanes 5-6 are duplicate reactions of the same lacZ1062a intron insertional mutant strain that was transformed with a plasmid expressing Cre recombinase (i.e., after Cre-loxP recombination). The removal of the kanamycin resistance marker (Lanes 5-6) is evident by the ~1.0 kb reduction in amplicon size after Cre-loxP recombination.




TargeTron is a registered trademark of InGex, LLC.



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TargeTron® Gene Knockout System