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loxP-FRT-PGK-gb2-neo-FRT

Pro- and Eukaryotic Neomycin Selection Cassette flanked by FRT-sites and one additional upstream loxP site

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Description

The loxP-FRT-PGK-gb2-neo-FRT template is designed to allow kanamycin/neomycin selection in prokaryotic and eukaryotic cells, respectively. 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 prokaryotic promoter gb2 is a slightly modified version of the Em7 promoter. It mediates higher transcription efficiency than the generally used Tn5 promoter. The promoter of the mouse Phosphoglucokinase gene (PGK) is used as the eukaryotic promoter. A synthetic polyadenylation signal terminates the kanamycin/neomycin expression. The cassette is flanked by FRT sites for later excision by Flprecombinase. An additional single loxP site is located at the 5’ end of the cassette. Unique NotI and XhoI sites flank the cassette for convenient cloning with restriction sites. Using the provided PCR template one can easily create a loxP-FRT-PGK-gb2-neo-FRT cassette flanked by any other restriction sites to clone the cassette into the vector of choice. The restriction sites can be introduced by adding the corresponding sequence in the PCR primer. The template can easily be used to generate targeting constructs mediated by Red/ET Recombination. The loxP-FRT-PGK-gb2-neo-FRT template is not linear but plasmid based (3485 bp in size). Due to its R6K origin the plasmid cannot replicate in most E.coli strains. The PCR product can therefore be used directly for downstream applications without any further purification. At least 20 PCR reactions can be performed using 1 ul per reaction as template.

Contents

  • loxP-FRT-PGK-gb2-neo-FRT: PCR template (50 ng/ul, 20ul)
  • Manual

Sequences

loxP-FRT-PGK-gb2-neo-FRT

loxP
FRT
Promoter
neoR
Terminator
AATTAACCCTCACTAAAGGGCGGCCGCATAACTTCGTATAATGTATGCTATACGA AGTTATAAGCTTGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATTCTACCG GGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCG CTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCA CCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCCAC TCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACG TGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTG AGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTT CGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGC GGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCA CGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCT TTCGACCTGCAGCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCAT AGTATAATACGACAAGGTGAGGAACTAAACCATGGGATCGGCCATTGAACAAGAT GGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACT GGGCACAACAGACGATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCA GGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTG CAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAG CTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGT GCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATC ATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCG ACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCT TGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTG TTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATG GCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCAT CGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACC CGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTT ACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGA GTTCTTCTGAGCGGGACTCTGGGGTTCGAATAAAGACCGACCAAGCGACGTCTGA GAGCTCCCTGGCGAATTCGGTACCAATAAAAGAGCTTTATTTTCATGATCTGTGT GTTGGTTTTTGTGTGCGGCGCGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTT CCTCGAGCCCTATAGTGAGTCGTATTA



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