What is the selection marker for the ORFEXPRESS™ Gateway® Shuttle Clones?
Answer: Kanamycin.
What is the difference between your OmicsLink™ Expression Clones and expression clones constructed with Gateway® pDEST destination vectors (expression vectors)?
Answer: GCI's OmicsLink™ Expression Clones are generated by its patented RecJoin™ cloning technology. By using this technology to construct a 5' tag-ORF fusion expression clone, there is no attB site between 5' tag and the start codon ATG of the full-length ORF. The extra 8 amino acids encoded by attB site in Invitrogen's pDEST expression clones will not be present in the proteins produced by using OmicsLink™ Expression clones. Same principle applies to 3' fusion protein production.
If I use Gateway® cloning system for 5′ fusion protein, the recombination site (attB) and the Multiple Cloning Site (MCS) in the ORFEXPRESS™ Shuttle Clone will become coding sequence. How many amino acids will be added?
Answer: There are fourteen amino acids between the fusion peptide (or N-terminal Tag) and the ORF encoded protein. These fourteen amino acids are, from N-terminus to C-terminus, Thr Ser Leu Tyr Lys Lys Ala Gly Leu Gly Gly Val Arg Thr. Eight amino acids (Thr Ser Leu Tyr Lys Lys Ala Gly) are encoded by the attB recombination site and six (Leu Gly Gly Val Arg Thr) are encoded by the sequences between attB and the start codon, ATG.
I would like to transfer the ORFs from ORFEXPRESS™ Gateway® Shuttle Clones into my own vectors that have specific features, but these vectors are not Gateway® compatible. How to do it?
Answer: Each ORFEXPRESS™ Shuttle Clone has two multiple cloning sites (MCS) flanking the ORF. By choosing suitable restriction enzymes, you can cut out the entire ORF from ORFEXPRESS™ Shuttle clones and insert the ORF into your vectors by classical sub-cloning methods.
How do I remove a stop codon of a full-length ORF that will be used to generate C-terminal tagged fusion protein?
Answer: You can make a point mutation to remove the stop codon. There are more than 35,000 full length ORFs have been constructed with a tag at their 3' ends, such as eGFP and HA, there are more than 10 types you can choose (https://www.genecopoeia.com/tech/omicslink/). You can request us to remove the stop codon and clone it into the vector(s) of your interest at a small fee.