I have the complete sequence of cloning vector pBR322 (GenBank: J01749.1) and want to know if it is from 5` to 3` or 3` to 5`. I also have a sequence of a gene on this sequence from GeneBank but it is a complement to the sequence on the vector. I do not know which one is the reverse and which one is the forward sequence. I want a way to recognize the reverse and the forward sequence (the orientation).
Thanks,
Hi,
Sequences at the NCBI are from 5' to 3'. All sequences are always written in this direction!
You could use transeq (e.g. here: http://www.ebi.ac.uk/Tools/st/emboss_transeq/) to get the encoded peptide sequence of your DNA sequence. DNA sequence in the 'wrong' direction should not encode meaningful peptides due to a high frequency of stop codons. Always use a 'reverse complement' function if you would like to change the direction of DNA sequences. Producing the complement sequence is usually a very bad idea!
In NCBI/GenBank, there is a "show reverse complement" feature built in. And it also changes the annotation to give the annotation for the inverted view too. You can download the annotated sequence in either orientation.
Thank you, Boas and Brian. That helps. I have found out by getting the encoded peptide sequence. I wanted to know which strand is the plus strand and which one is the minus, and I found the complete genome of the vector is the minus and the gene is the plus.
https://www.researchgate.net/profile/Boas_Pucker I just did not understand what do you mean by "Producing the complement sequence is usually a very bad idea!".
Thanks
Hi again,
You probably already know that you should always use the reverse complement. However, you could produce the complement instead of the reverse complement of a sequence. It is just a conversion of characters into other characters. Some people do this to design primers, because they are not aware of the 5' to 3' orientation of nucleic acids. The resulting sequence is something totally different! The complement of a sequence is only a common reason for mistakes in the context of cloning. Therefore, you should avoid it and instead use the reverse complement if you intend to change the direction of your sequence.
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