TrueORF Clones and PrecisionShuttle System

Why should I use OriGene’s TrueORF clones?
Answer: All TrueORF Clones are derived from OriGene’s unique TrueClone Collection, and were isolated from high quality human cDNA libraries made from a variety of tissues.  TrueORFClones provide enough (10 µg) purified DNA to allow customers to directly apply these expression-ready, tagged ORF clones to experiments designed for protein expression, purification, protein-protein interaction and stable clone selection.  TrueORF clones also serve as the entry vector for OriGene’s PrecisionShuttle system and allow easy construction of variably tagged ORFs.  This saves valuable time by eliminating the need for subcloning, verification and amplification.  All TrueORF vectors share the same multiple cloning sites (MCS), which are compatible with many other commonly used vector systems, such as Promega’s Flexi system, Invitrogen’s Gateway system and Novagen’s PET system.  Customers can easily shuffle the cDNA of a TrueORF clone between multiple TrueORF destination vectors to generate clones with different epitope tags, or transfer the ORF to other expression systems designed for specific experimental purposes. 

What is the difference between OriGene’s entry vector and the destination vectors?
Answer: The major differences are the antibiotic selection marker and the epitope tags or markers.  The entry vector carries kanamycin resistance, while all destination vectors contain the ampicillin resistance gene.  This allows simple screening for successful subcloning products.  All of the vectors have a unique combination of N- and C-terminal epitope tags or a fluorescent marker, as described in Table I.

What restriction enzymes should I use if Sgf I or Mlu I sites are present in my ORF?
Answer: While 96% of all human and mouse ORFs can use the Sgf I - Mlu I combination, some ORFs do contain internal Mlu I site(s).  Most of those ORFs with an internal Mlu I site can be transferred using another rare cutter (Rsr II), whose restriction site is upstream of Mlu I, or Not I, whose site is immediately downstream of Mlu I. Using one of the four different subcloning combinations, any ORF can be transferred from one vector to another.  The recommended subcloning combination for every TrueORF cDNA is listed in the product information on our website.

Has OriGene fully sequenced all TrueORF clones?
Answer: Not always.  When transferring the cDNA into the TrueORF Entry Vector, OriGene  always uses fully sequenced plasmids as templates and Phusion High-Fidelity DNA Polymerase (New England Biolabs), which has a mutation rate less than 4 x 10^-7.  This ensures the highest fidelity of every TrueORF clone.  After cloning into the entry vector, each of OriGene’s TrueORF clones was sequenced at both the 5’ and 3’ ends, and the resulting sequence was matched to the corresponding reference sequence.  For many ORFs 1 Kb or less in length, the 5’ and 3’ sequencing reads have covered the full ORF.  For longer cDNAs, the ORF was not fully covered by sequencing reads.

Do TrueORF clones exactly match the reference gene sequence?
Answer: All TrueORF clones are guaranteed to match the ORF of the corresponding gene.  However, some clones may contain nucleotide changes compared to the published reference sequences.  This is due to SNPs (single nucleotide polymorphisms) reflecting the unique differences from genes expressed in different tissues and different individuals.  Published references may represent a different SNP than the OriGene transcript.  Should a specific SNP be required, this can be contracted from OriGene at cost.

Can I transfer large ORFs using this system?
Answer:  It has been reported that ORFs larger than 4 Kb are unstable in recombination-based systems; conversely, our restriction digest-based vector system has no real size limitation.  An ORF up to 18 Kb can be readily transferred from one vector to another.

What sites should I use to transfer a TrueORF clone into the Gateway system?
Answer: There are multiple sites in pCMV6-Entry than can be used to move the insert of a TrueORF clone into any of Gateway’s entry vectors (pENTR-1A, -2B, -3C, -4, and -11).  These sites are EcoR I, Sal I, BamH I and Kpn I at the 5' end, and Not I at the 3’ end.

What are the functional aspects of the pCMV6-AC-GFP vector?

Like all OriGene vectors, the CMV promoter drives the heterologous expression of the specified open reading frame (ORF) which is in-frame with the Green Fluorescent Protein (GFP) on the C-terminus.  Such fluorescence permits the positive identification of mammalian cells positively transfected with plasmid.  The neomycin resistance gene is also expressed downstream of the SV40 promoter within the same vector and permits positive selection of transfected cells as well as stable cell line production.  For bacterial amplification, the ampicillin resistance gene is engineered on the opposite strand.

 OriGene’s GFP is listed as TurboGFP.  How is this different from other available GFPs?

TurboGFP is a fully licensed, 26kDA protein product from Evrogen JSC that works well in other standardized GFP assays as well.  Excitation max is 482nm and emission max is 502nm.  It yields 112% of the brightness compared to eGFP and has no known cell toxicity.  It is an isoform of the naturally occurring protein from Pnotillina plumata that is optimized for rapid labeling of cells/organelles and tracking of promoter activity.  It is a perfect choice for monitoring transient protein expression.

 What does it mean that this vector is compatible with OriGene’s other Precision Shuttle vectors?

The extremely rare cutting enzymes, SgfI and MluI, were used in the cloning of over 99% of OriGene’s TrueORFs, and these same sites are available in over 20 different destination vectors allowing for easy shuttling of ORFs to and from each without the requirement for full sequence confirmation.  Sequencing of the cloning sites is always encouraged for those rare cases when a cloning artifact occurs (usually a deletion due to base hydrolysis prior to ligation). 

 Why does my Certificate of Authorization (COA) indicate cloning sites other than SgfI and MluI?

Whenever one or both of these sites is present within the ORF of the transcript, the pCMV6-AC_GFP vector has other sites engineered to accommodate this, e.g. RsrII or NheI.

What restriction sites are available for subcloning into other vectors?

If you require a functionality that is not found in one of OriGene’s available destination vectors, the vector map is found on the OriGene website under TrueORF cDNA Clones; under sub-heading “Destination Vectors”. 

 How many amino acids are present in the linker between my protein and GFP?

To accommodate the MluI cloning site, which maintains the proper reading frame, this vector appends a threonine and arginine.  This is far fewer than other recombination based shuttling systems.

 Can I purchase the empty vector with out any open reading frame?

Yes, The catalog number is PS100010 for a lyophilized 10ug aliquot.

 Has OriGene fully sequenced all TrueORF clones in these Precision Shuttle vectors?

Not always.  When transferring the cDNA into the TrueORF Entry or GPF vector, OriGene always uses fully sequenced plasmids as templates and Phusion High-Fidelity DNA Polymerase (New England Biolabs), which has a mutation rate less than 4 x 10-7.  This ensures the highest fidelity of every TrueORF clone.  After, each of OriGene’s TrueORF clones was sequenced verified at both the 5’ and 3’ cloning sites. 

 I don’t see any GFP expression.  What should I do?

Call OriGene’s Technical Services group (301) 340-3188 or 1(888) 267-4436 for expert, Ph.D. level assistance.  If no solution is obvious, they will work with you to run the appropriate controls.  Sometimes, this means that new test constructs or replacement constructs will be offered free of charge.  OriGene is invested in your success and excellent post-sales support is available.

What does your disclaimer mean?
Answer: OriGene’s disclaimer for the TrueORF clones reads as follows: “Our molecular clone sequence data has been matched to the accession number below as a point of reference.  Note that the complete sequence of our molecular clones may differ from the sequence published for this corresponding accession number, e.g., by representing an alternative RNA splicing form or single nucleotide polymorphism (SNP).
The NCBI RefSeq human mRNA sequences are continuously being revised, as some may have been derived from aberrantly spliced transcripts or generated by incorrect prediction of intron-exon junctions in silico.  These sequences are therefore used only as a “reference” and not as a “standard”.  OriGene’s clones are isolated from full-length cDNA libraries and may differ from the reference sequence for this reason.

What is the TrueClone Guarantee?
Answer: OriGene warrants that the product will meet specifications listed.  At OriGene’s discretion, free replacement of any non-conforming product will be made if OriGene is notified within 30 days of product receipt.  If you experience any difficulty with any OriGene product, please contact our Technical Support Staff at 888-267-4436, or 301-340-3188 outside the US.