Quick & Easy Conditional Knock Out Kit - FRT

* 2019  ÀÌÈÄ Ãë±Þ ¾ÈÇÔ*

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

Applications

  • This kit is designed to integrate FRT 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 FRT 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 FRT site an FRT-PGK-gb2-neo-FRT 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 FRT.
3. Flp-mediated excision
Flp-mediated excision to leave a single FRT site behind. (See also our FRT selection cassettes and Flp expression plasmids & strains).
4. Red/ET Recombinationn
A second FRT-PGK-gb2-neo-FRT 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 FRT to yield the desired targeting vector.

Description

The included FRT-PGK-gb2-neo-FRT 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 pCI-FLPe expression plasmid is coding for an improved FLP recombinase which shows a four to tenfold higher recombinational activity compared to the wildtype FLP enzyme at temperatures between 37¡ÆC (optimal growth temperature for E.coli) and 40¡ÆC (mouse body temperature). 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.

Contents

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

Sequences

FRT-PGK-gb2-neo-FRT
 
FRT
Promoter
neoR
Terminator
 
 
 
 
 
AATTAACCCTCACTAAAGGGCGGCCGCGAAGTTCCTATTCTCTAGAAAGTATAGG AACTTCATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGC GCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCA CACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTT CGCGCCACCTTCCACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTC GCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGA TGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATA GCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGG GGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAG GCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTC CTCATCTCCGGGCCTTTCGACCTGCAGCAGCACGTGTTGACAATTAATCATCGGC ATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGGATCG GCCATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGC TATTCGGCTATGACTGGGCACAACAGACGATCGGCTGCTCTGATGCCGCCGTGTT CCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGT GCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGG GCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCT GCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCC GAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGG CTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCG GATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTC GCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATC TCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCG CTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGAC ATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACC GCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTA TCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAATAAAGACCGA CCAAGCGACGTCTGAGAGCTCCCTGGCGAATTCGGTACCAATAAAAGAGCTTTAT TTTCATGATCTGTGTGTTGGTTTTTGTGTGCGGCGCGGAAGTTCCTATTCTCTAG AAAGTATAGGAACTTCCTCGAGCCCTATAGTGAGTCGTATTA
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