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iCre-FRT-neo-FRT
Codon improved Cre (iCre) with attached FRT flanked, Pro- and Eukaryotic Neomycin Selection Cassette
Manual [PDF]
Contact Support
Order
Sequences
Description
iCre-FRT-neo-FRT PCR template is designed to facilitate the insertion of such a functional cassette into targeting constructs by Red/ET recombination.
A codon-improved version of P1 bacteriophage derived Cre-recombinase is located upstream of the neomycin/kanamycin resistance gene (aminoglycoside phosphotransferase). Mammalian codon usage was applied for the altered Cre version (iCre). By introducing silent base mutations the high CpG content of the prokaryotic coding sequence was reduced, thereby reducing the chances of epigenetic silencing in mammals (Cohen-Tannoudji et al., 2000).
The iCre-FRT-neo-FRT template encodes the neomycin/kanamycin resistance gene which combines a prokaryotic promoter (gb2) for kanamycin resistance in E.coli with a eukaryotic promoter (PGK) for 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 normally used Tn5 promoter. The promoter of the mouse phosphoglycerate kinase gene (PGK) is used as eukaryotic promoter. A synthetic polyadenylation signal terminates the kanamycin/neomycin transcription. The cassette is flanked by FRT sites for later excision by Flp-recombinase.
Using the provided PCR template one can easily create an iCre-FRT-neo-FRT cassette flanked by homology arms to insert the cassette by Red/ET recombination into the vector of choice.
The iCre-FRT-neo-FRT template is not linear but plasmid based (3446bp in size). Due to its R6K origin it can’t replicate in most of the common 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 1µl per reaction as template.
Contents
- iCre-FRT-PGK-gb2-neo-FRT: PCR template (50 ng/ul, 20ul)
- Manual
Sequences
iCre-FRT-neo-FRT
ATGGTCTCCAACCTGCTGACTGTGCACCAAAACCTGCCTGCCCTCCCTGTGGATG
CCACCTCTGATGAAGTCAGGAAGAACCTGATGGACATGTTCAGGGACAGGCAGGC
CTTCTCTGAACACACCTGGAAGATGCTCCTGTCTGTGTGCAGATCCTGGGCTGCC
TGGTGCAAGCTGAACAACAGGAAATGGTTCCCTGCTGAACCTGAGGATGTGAGGG
ACTACCTCCTGTACCTGCAAGCCAGAGGCCTGGCTGTGAAGACCATCCAACAGCA
CCTGGGCCAGCTCAACATGCTGCACAGGAGATCTGGCCTGCCTCGCCCTTCTGAC
TCCAATGCTGTGTCCCTGGTGATGAGGAGAATCAGAAAGGAGAATGTGGATGCTG
GGGAGAGAGCCAAGCAGGCCCTGGCCTTTGAACGCACTGACTTTGACCAAGTCAG
ATCCCTGATGGAGAACTCTGACAGATGCCAGGACATCAGGAACCTGGCCTTCCTG
GGCATTGCCTACAACACCCTGCTGCGCATTGCCGAAATTGCCAGAATCAGAGTGA
AGGACATCTCCCGCACCGATGGTGGGAGAATGCTGATCCACATTGGCAGGACCAA
GACCCTGGTGTCCACAGCTGGTGTGGAGAAGGCCCTGTCCCTGGGGGTTACCAAG
CTGGTGGAGAGATGGATCTCTGTGTCTGGTGTGGCTGATGACCCCAACAACTACC
TGTTCTGCCGGGTCAGAAAGAATGGTGTGGCTGCCCCTTCTGCCACCTCCCAACT
GTCCACCCGGGCCCTGGAAGGGATCTTTGAGGCCACCCACCGCCTGATCTATGGT
GCCAAGGATGACTCTGGGCAGAGATACCTGGCCTGGTCTGGCCACTCTGCCAGAG
TGGGTGCTGCCAGGGACATGGCCAGGGCTGGTGTGTCCATCCCTGAAATCATGCA
GGCTGGTGGCTGGACCAATGTGAACATTGTGATGAACTACATCAGAAACCTGGAC
TCTGAGACTGGGGCCATGGTGAGGCTGCTCGAGGATGGGGACTGATGATGAAGAT
CTGAGCTCCCTGGCGGAATTCGGATCCAGATCTTATTAAAGCAGAACTTGTTTAT
TGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAA
GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTT
ATCATGTCTGGTCGATAATACGACTCACTATAGGGCTCGAGGAAGTTCCTATACT
TTCTAGAGAATAGGAACTTCCGCGCCGCACACAAAAACCAACACACAGATCATGA
AAATAAAGCTCTTTTATTGGTACCGAATTCGCCAGGGAGCTCTCAGACGTCGCTT
GGTCGGTCTTTATTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGC
GATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAA
GCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCT
ATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAA
AGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGAC
GAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGC
GCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCA
TCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGT
AGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTC
TCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATA
GCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAAC
GCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGG
GCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCC
GGAACACGGCGGCATCAGAGCAGCCGATCGTCTGTTGTGCCCAGTCATAGCCGAA
TAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATG
GCCGATCCCATGGTTTAGTTCCTCACCTTGTCGTATTATACTATGCCGATATACT
ATGCCGATGATTAATTGTCAACACGTGCTGCTGCAGGTCGAAAGGCCCGGAGATG
AGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCGG
GCCTCCGGAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCG
CCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGCAAAGCT
GCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGCTGTC
CATCTGCACGAGACTAGTGAGACGTGCTACTTCCATTTGTCACGTCCTGCACGAC
GCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGGGAGGAGTGGAAGGTGGCG
CGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTGGATGTGGAATG
TGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCTAAA
GCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAGAATGAAG
TTCCTATACTTTCTAGAGAATAGGAACTTCGCGGCGCCCTTTAGTGAGGGTTAATT