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Constructing a Gateway Destination vector

Cloning

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#1 allsorts

allsorts

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Posted 08 August 2013 - 01:28 AM

Hi everyone! I want to construct an N-terminally 6xHis-TEV-3xFlag-tagged Gateway Destination vector based on pcDNA5/FRT/TOPO using overlap PCR and a set of 4 oligos. I dont have the cassettes or the ccdb strain, only the pcDNA5/FRT/TOPO vector - I havent done this before so im not sure if it will work. So basically what I want to make is 5'-Kozak-6xHis-TEV-3xFlag-AttR1-AttR2-3' and Topo clone this into pcDNA5/FRT. What I have for the oligos is

 

1. 5'-Kozak-6xHis-TEV-3xFlag-3' Forward 60 bp, GC 45%

5'- AAC ATG GCG CAT CAT CAC CAT CAC CAC GAG AAT CTT TAT TTT CAG GGC GAC TAC AAA GAC -3'

 

2. 5'-TEV-3xFlag-3' reverse 66 bp, GC 42.4%

5'- GTC ATC CTT GTA GTC GAT GTC ATG ATC TTT ATA ATC ACC GTC ATG GTC TTT GTA GTC GCC CTG AAA -3'

 

3. 5'-3xFlag-AttR1-AttR2-3' forward 48 bp, GC 39.6%

5’-ATC GAT GAC AAG ACA AGT TTG TAC AAA AAA GCT GAA CAC GTC GTT CAG-3’

 

4. 5'-AttR1-AttR2-3' reverse 37 bp, GC 45.9%

5'- ACC ACT TTG TAC AAG AAA GCT GAA CGA CGT GTT CAG C -3’

 

The overlapping regions between oligos have Tms of 53.3-54.9C and are about 20 bp in length.

 

I was wondering whether I have the reading frame correct and also how many nucleotides do I need between the AttR1 and AttR2 sites? Do you think this strategy will work or is there another better way to do this?

 

Thanks!







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