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View additional product information for ULTIMATEHORF CLONE - FAQs (HORF01)
4 product FAQs found
The backbone vector that is used is pENTR221, which is a Gateway Entry vector. The insert (an open reading frame that begins with ATG and ends with a TAG stop codon) is flanked by attL1 and attL2 sites. The construct is ready for Gateway LR recombination with a DEST vector containing attR1 and attR2 sites. It is compatible with N-terminal tagged DEST vectors but not with C-terminal tagged DEST vectors. To express the C-terminal tag, the Tag-On-Demand technology would have to be used or the TAG stop codon would have to be mutated. In place of the Tag-On-Demand adenovirus product we sold previously, we offer the vector expressing the tRNA suppressor as a custom service. If you are interested in this service, please e-mail custom.services@lifetech.com.
The cDNA clones we offer are Ultimate ORF clones, Full-Length human cDNA clones, MGC clones, and GeneStorm Expression-Tested clones. The Ultimate ORF clones, Full-length human cDNA clones, and the GeneStorm Expression-Ready clones are made in-house, whereas the MGC clones are derived from external sources and distributed by Thermo Fisher Scientific. The Ultimate ORF clones are fully-sequenced and guaranteed to match GenBank sequence information 100% at the amino acid level, whereas the sequence of the other cDNA clones we offer is not guaranteed.
We recommend choosing the Ultimate ORF clones if quality and sequence are your main priority. Further, these clones are provided in a Gateway vector backbone, so it is easy to shuttle the insert into multiple host systems. The Full-length human cDNA clones and MGC clones are only 5´ and 3´ end-sequenced, but Full-length human cDNA clones offer the advantage of being in a Gateway Entry vector backbone. MGC clones are available for human, mouse, and rat species; Ultimate ORF clones are available for human and mouse species and Full-Length human cDNA clones are available only for human species.
These clones are not fully Gateway compatible. While the inserts are flanked by attB sites and can be transferred via BP reaction into Donor vectors, they are less than suitable for subsequent transfer into destination (DEST) vectors for protein expression, for the following reasons:
1. The inserts generally include a stop codon; hence C-terminal tagged DEST vectors are useless unless the intent is to express the insert in its native form (untagged).
2. The clones contain 5' and 3' untranslated regions of undetermined length, i.e. inserts were not specifically cloned into the Gateway reading frame. Even if the gene is in frame with a tag, the untranslated region would result in potentially several extra amino acids between the tag and the insert.
3. The 5' untranslated regions of the inserts may not contain a ribosome binding sequence (RBS) located 5-13 bases upstream of the ATG (a requirement for bacterial expression). While this does not apply to N-terminal tagged DEST vectors, which contain an RBS and ATG, it does apply to the use of C-terminal tagged vectors or vectors for native protein expression since they have no provision for an RBS upstream of the ATG.
In short, cDNA clones generated in pCMVSport6 can be transferred into mammalian DEST vectors for untagged native expression, or N-terminal tagged expression if the insert is found to already be in the Gateway reading frame (caveat - untranslated region will code for several amino acids). But for most other purposes, inserts should be amplified by PCR with primers directed at the ATG and stop codon regions and cloned into the appropriate pENTR/D-TOPO vector, or restriction-cloned in frame into the appropriate supercoiled pENTR vector.