I am currently conducting molecular cloning.
I want to put a new restriction enzyme site in a given peptide sequence, and I want to preserve the peptide sequence as much as possible. More specifically, I would like to check any restriction enzyme list that can be put in the N-terminal sequence of antibody CL, "TVAAPSVFIFPP". Is there any web tool or data base that can check the restriction enzyme site that can be put for a given peptide sequence?
Thank you for reading it!
Hi there,
You should try the silent mutator:
https://molbiotools.com/silentmutator.html
Of course you need to input a DNA sequence to Dominique's linked program, and this can come from a reverse translation program that you load with a codon usage table for your organism like this one https://www.bioinformatics.org/sms2/rev_trans.html
(or use the DNA sequence you've already cloned!)
Dear Daoseong
theoretically you ca certainly do it.
It depends to which is the dna sequence that is codifying for your peptide, but if you will synthetize the peptide choosing the opportune codons is it possible that you can add restriction enzymes also with out any modification.
However, why you would like to use restriction enzymes? Actually there are several enzyme free cloning approaches that may allow to you to cut and paste different DNA fragment from one to one other vector or template with out any modification to the sequence.
For example i'm using a lot the PIPE cloning approach (see attached paper)
Recently i used it to insert mouse and human CL-k and human igG1 CH chains in pcdna3.4 to create vectors for cloning and expression of chimeric antibodies and for the rapid replacement of signal peptides for mab secretion in the surnatant.
As you can found in the video of the following link of my blog PRoteoCool
https://www.blogger.com/video.g?token=AD6v5dymTB4mmANTKeTOyzXQq7QSUurTLQFPDsGxi4FqyNDpu3YZutcuN0VfLTfDTPRD8edEIDPD6bJZIe_VWirwCNZHkJ1A34BATcDIzXZEbV1UUtRLR6Nc19tU2_t6-Or5Sag2dwU
the only limit of this approach that to my knowledge, the best strain to perform it are the Hk100 (they work well also with home made calcium competent cells) which unfortunately are not commercially available, but it work quite well also with commercial chemical competent MACH1 (but not homemade) ot Thermo fisher.
good luck
Manuele
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