I need to find some cDNA sequences for cloning into expression vectors. How do I go about doing it...the databases seem overwhelming. Please explain with example if you can.

Thank you.

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Open Biosystem distributes many EST and cDNA clones from the IMAGE consortium and other resources. you can search your gene by going to their website and use the gene search box or the blast tool on their website. The clones are very cheap, about $50
http://www.openbiosystems.com/

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Hi pcrman,

Thank you for the reply. After reading my question I realized that the question was not very clear about what I wanted to ask.

I want to find cDNA sequences for virtual cloning into expression vectors and then make the plasmid maps for my work. I tried looking on ncbi but it looks confusing to me. So my question is how do I find out cDNA sequences on ncbi or ensembl websites.

Thanks.

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You can use both NCBI or Ensembl, but it's better to check, that the sequences don't differ (sometimes they do, when it's not clear I stick to NCBI reference sequence, I think all human genes now have them).

So take a gene like human beta-actin.

In NCBI Entrez you go for a Gene search for "beta-actin" (it's better to add also "homo sapiens" because otherwise you'll spend long time looking through results). You need to know that you finded your gene exactly, in this case I know gene called ACTB is what I'm looking for.
You get to a gene page.

Now in the Genomic regions, transcripts, and products section you'll see all the transcripts the gene has, choose one you want cDNA from.
Mouseover the tiny link on the right of the transcript and title "mRNA links" should appear. Click it and choose FASTA format.
Now you have your RNA sequence and assuming that cDNA is a reverse complement of RNA, you need to reverse your sequence in program or web application.

In Ensembl it's quite as simple.
Again search for your gene in the organism you need (here "ACTB" search would be quicker). Usualy there comes two "variants" Vega gene encoding and Ensembl encoding. I don't know the diference, but if possible I always choose rather Ensembl encoding.
Again you get to a gene page.
Scroll to Transcripts section, choose a desired transcript and click the Transcript info link.
On the very end of transcript report page there is a complete sequence, with highlighted exons. Now you got your RNA sequence.

Hope it helped.

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Hi Trof...Thanks...it really helped......just one question.....do you need to reverse the sequence? because when i try to find out the ORF in mRNA sequence, It gives me right size sequence with open reading frame. Also the mRNA sequence does not contain Us in place of Ts.

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