I have a fasta file containing the amino acid sequence of glycogenin-1: https://www.rcsb.org/fasta/entry/6EQJ

I want to create a plasmid that produces glycogenin-1.

Is it possible to use the glycogenin-1 amino acid sequence to produce said plasmid? Or would one need the nucleic acid sequence to do so?

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  • $\begingroup$ Companies like Origene sell pre-made plasmids in a variety of backbones, with or without epitope tags, for a relatively small amount of money. For example, human glycogenin-1 for US$420. $\endgroup$ – MattDMo Aug 1 at 15:32
  • $\begingroup$ Thanks @MattDMo. However I don’t need to purchase a plasmid, I need to make one. If I had the nucleic acid sequence I would just use that, but all I have is the amino acid sequence, so I need to know if I can use the amino acid sequence to make the plasmid. $\endgroup$ – user1454024 Aug 2 at 0:31
  • $\begingroup$ You just need to reverse-translate the protein sequence (e.g. on the free online IDT codon optimization tool) and include it in a common plasmid backbone. Basically in order to answer we need to know exactly what you are trying to build. $\endgroup$ – Mowgli Aug 2 at 15:45
  • $\begingroup$ What happened to the answer to this question and the comments on that answer? Someone decided to just delete it? $\endgroup$ – user1454024 Aug 2 at 22:41
  • $\begingroup$ @user1454024 The answer was deleted by the author. Users with high enough reputation (like me) can still see it and the comments, though, and I have two responses. First, you don't need to optimize the codons like you suggested, because once the transcription machinery is in place on the open reading frame, it skips ahead 3 bases (1 codon) at a time. So, unless you expressly design the plasmid to have two overlapping ORFs, you don't need to worry about that. Second, the nucleic acid sequence of your protein is available online, so you don't have to reverse-engineer your protein sequence. [...] $\endgroup$ – MattDMo Aug 3 at 2:52

I lack the knowledge to fully answer your question but I can give some potentially useful pieces of information:

  • In theory you can easily in-silico reverse translate a peptide sequence to an unambiguous nucleotide sequence using the GMOs optimized codon usage. I can share a Python script for that.

  • Also you could easily search for the 'real' nucleotide sequence in databases. For example use 'Blast' to find the gene of your peptide sequence.

  • But attention! There are known cases of codon-optimized sequences causing bad protein folding or even (paradoxically) worse expression levels than the native sequence.

  • There is software that helps with the design of plasmids, also in light of use of unique restriction sights, helping to decide which of your restriction enzymes are applicable.

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