* 2019 이후 취급 안함*

For fast and simple integration of loxP-sites into large vector plasmids at any intended position (for generation of transgenic mouse models).

Applications

This kit is designed to integrate loxP sites in a vector plasmid at any intended position without the necessity to use restriction enzymes within 2-4 weeks

Generation of a conditional knockout construct based on loxP sites.

1. Clone containing gene of interest
A gene of interest (orange) was subcloned from e.g. a BAC into a minimal vector by Red/ET recombination using the BAC Subcloning Kit.

2. Red/ET Recombination
To insert a single loxP site a loxP-PGK-gb2-neo-loxP cassette containing 50 bp homology arms added by PCR amplification is introduced at the position of choice by Red/ET recombination with the Quick & Easy Conditional Knockout Kit loxP.

3. Cre-mediated excision
Cre-mediated excision to leave a single loxP site behind. (See also our loxP selection cassettes and Cre expression plasmids & strains).

4. Red/ET Recombinationn
A second loxP-PGK-gb2-neo-loxP cassette containing 50 bp homology arms added by PCR amplification is introduced at the position of choice by Red/ET recombination using the Quick & Easy Conditional Knockout Kit loxP to yield the desired targeting vector.

Description

The included loxP-PGK-gb2-neo-loxP cassette is designed to allow neomycin / kanamycin selection in prokaryotic and eukaryotic cells. It combines a prokaryotic promoter (gb2) for expression of kanamycin resistance in E.coli with a eukaryotic promoter (PGK) for expression of neomycin resistance in mammalian cells. The included 706-Cre expression plasmid allows Cre recombination in E.coli. This kit can also be used to work on bacterial artificial chromosomes. High Red/ET efficiency plus convenient removal of the Red/ET Recombination protein expression plasmid pRedET after recombination.

Red/ET Recombination protein expression plasmid pRedET. Any E. coli strain can be made Red/ET proficient by transformation with this plasmid
loxP-PGK-gb2-neo-loxP template to be used for your own experiments
Expression plasmid for Cre recombinase 706-Cre
Positive controls to introduce a loxP site in a 18 kb high copy plasmid
Detailed protocols, descriptions of plasmids, maps and sequences

Sequences

loxP-PGK-gb2-neo-loxP

loxP

Promoter

neo^R

Terminator

AATTAACCCTCACTAAAGGGCGGCCGCATAACTTCGTATAGCATACATTATACGA AGTTATATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGC GCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCA CACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCGT CGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTC GCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGA TGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATA GCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGG GGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAG GCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTC CTCATCTCCGGGCCTTTCGACCTGCAGCAGCACGTGTTGACAATTAATCATCGGC ATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGGATCG GCCATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGC TATTCGGCTATGACTGGGCACAACAGACGATCGGCTGCTCTGATGCCGCCGTGTT CCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGT GCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCC GAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCG GATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTC GCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATC TCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGAC ATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACC GCTTCCTCGTGCTTTACGGTATCGCCGCCCCCGATTCGCAGCGCATCGCCTTCTA TCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAATAAAGACCGA CCAAGCGACGTCTGAGAGCTCCCTGGCGAATTCGGTACCAATAAAAGAGCTTTAT TTTCATGATCTGTGTGTTGGTTTTTGTGTGCGGCGCGATAACTTCGTATAGCATA CATTATACGAAGTTATCTCGAGCCCTATAGTGAGTCGTATTA

K005_Conditional_Knockout_Kit_loxP.pdf (3,768,553kb)

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