Answer: A full-length ORF (Open Reading Frame) clone is a plasmid that contains a protein coding DNA insert that encodes a full-length protein. The DNA insert contains only the protein coding sequence (from start codon to stop codon) of a full-length gene (or cDNA) without 5' and 3' end untranslated regions (UTRs), or introns.

Answer: The full-length ORF clones contain only the protein coding sequences, while other full-length whole transcript cDNA clones contain non-coding sequences, such as 5' and/or 3' UTRs, which have been known to have possible negative impact on the protein translation process. Please visit this page for further details on the comparison of these two types of clones: (http://www.genecopoeia.com/tech/omicslink/application.php).

Answer: The vector for ORFEXPRESS™ Gateway® Shuttle Clones manufactured by GeneCopoeia, Inc. (GCI) harbors full length ORFs flanked by attL1 and attL2 recombination sites that pair with the Gateway® pDEST expression vectors containing attR1 and attR2 recombination sites. When GCI's ORFEXPRESS™ Shuttle Clones are mixed with Gateway® compatible pDEST expression vector(s), with optimal concentrations of enzymes and conditions, the ORFs in the ORFEXPRESS™ Shuttle clones can be transferred into Gateway® pDEST expression vector(s). Because of the unique feature of recombination sites in the vectors, the transferred ORFs will be under the control of the promoter(s) in the destination expression vector(s).

Answer: There is a suite of pDEST expression vectors (destination vectors) available from Invitrogen Corporation that can produce various forms of proteins (such as native, N-terminal fusion or C-terminal fusion) in different host cell systems including bacterial, yeast, insect and mammalian cell systems.

Answer: The selection procedure is very stringent. It includes extraction, comparison and validation of gene sequences and their annotation information from multiple public and private sources. Clustering and manual curation are applied to reduce redundancy and eliminate erroneous genes.

Answer: Yes, and there are two choices. First, you can use the OmicsLink™ Expression Clones that we have developed. We have already constructed ORF clones in expression vectors with N-terminal His tag or C-terminal eGFP tag. Please check OmicsLink™ product information page (http://www.genecopoeia.com/tech/omicslik/). Second, you may also use Invitrogen's Gateway® expression vectors for expressing N-terminal or C-terminal fusion proteins. Because our ORFEXPRESS™ Gateway® Shuttle Clones contains stop codons, you will need to remove the stop codons before transferring ORF inserts into Invitrogen's pDEST expression vectors to produce C-terminal tagged fusion proteins.

Answer: You can make a point mutation to remove the stop codon. There are more than 12,000 full length ORFs have been constructed with a eGFP tag at their 3' ends (http://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.

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 ORFs from ORFEXPRESS™ Shuttle clones and insert the ORFs into your vectors by classical sub-cloning methods.

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.

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.

Answer: Kanamycin.

Answer: Ampicillin.

Answer: The sequences are different depending on which full length ORF clone for both ORFEXPRESS™ Gateway® Shuttle Clones and OmicsLink™ Expression Clones you purchased. First check the datasheet which is shipped with the clone and use the figure to identify the sequence.

Answer: No, the sequence you see is not the actual sequence of our clone. It is the sequence extracted from GenBank sequence record, with which we used as a reference to design 5' and 3' end primers and generate full length ORF of a gene.

Answer: There are usually multiple gene transcripts in a gene locus, resulting from alternative splicing, updated information about ORFs, polymorphism, or redundancies from duplication of cloning effort from different laboratory groups. During the process of selecting full-length genes for ORF cloning, we try to choose the most representative transcript from each gene locus, which may differ slightly or significantly from other transcripts in the same gene locus. This approach provides the broadest coverage of all gene loci. In many instances, we do obtain multiple variants or versions of full length ORFs from our cloning production pipeline. Customers are encouraged to send inquires to inquiry@genecopoeia.com if he/she is interested in obtaining a particular variant/version of a gene/ORF.

In some cases, discrepancies could be originated from changed annotation of ORF start and/or stop location as the result of newly available sequence information and/or experimental evidence. On regular basis, we check for information updates on ORF annotations. The new ORF annotations will then be used in our recloning effort to keep our ORF clones most updated.

Answer: Yes, we will clone the gene(s) for you. It is our mission and continuing effort to clone all full-length ORFs of human genes. It is likely that the gene(s) that you are interested in is already in our production pipeline or in the queue for future cloning. If you provide us with the GenBank accession, gene name, nucleotide or amino acid sequences, we will place the gene(s) in the list of priority genes to be cloned. Usually, it takes two weeks to complete the cloning, although we cannot guarantee that we will get the gene(s). You will be notified as soon as the clone(s) becomes available. There will be no additional charge.

Answer: Each full length ORF clone goes through the following quality control steps: 1) restriction enzyme digestion check; 2) PCR with its specific primers; 3) 5' and 3' end sequencing, which verifies at least 400-500 bp of ORF inserts at each end. Furthermore, before the delivery of each clone, we re-check the clone by PCR with gene specific primers to ensure that the right clone is shipped.

Answer: As one important step of our standard QC process, all clones are sequenced at both ends of ORF inserts, which covers about 1100 to 1200 bases of ORF inserts. For those clones with ORF lengths shorter than 1200 base pairs, our sequencing has covered the full length ORF inserts. We offer services to sequence the full length of any clones at a low fee (which will be used to cover only the cost for materials). Since sequencing the full length ORF inserts of all clones is still relatively costly and time-consuming, doing so will inevitably increase the delivery time and cost of production which will result in increased prices of clones.

Answer: For the ORFEXPRESS™ Shuttle clones, the forwarding sequencing primer (from upstream of 5' end of ORF) is: 5'-CCCAGTCACGACGTTGTAAAACG, and the reverse sequencing primer (from downstream of 3' end of ORF) is: 5'-ATGGTCATAGCTGTTTCCTG. For the OmicsLink™ clones: you can find the primer sequences in our website.

Answer: We use PCR together with several patented technologies to maximize the fidelity of PCR to generate full length ORF (more information in ORF White Paper). We used more than 70 cDNA libraries of human tissues as templates to generate full length ORFs.

Answer: The differences can originate from two potential sources or combination of the two: 1) naturally occurring events including polymorphisms, alternative splicings, inconsistencies in multiple copies/versions of the same genes resulting from annotation updates, sequencing errors and/or ambiguities; 2) artificially introduced changes by PCR cloning process. Very often, potential users of our clones compare our sequencing data with the sequence of only one reference transcript, and attribute any discrepancies to PCR introduced mutations. Through analysis of multiple clones of close to 20,000 human genes, we found that vast majority of discrepancies can be clearly attributed to category 1), when comparing our sequence data with all transcripts and sequences in corresponding gene locus including genomic sequence, ESTs, other cDNA/mRNA transcripts. On average, we found that there are fewer discrepancies between the sequences of our ORF clones and corresponding genomic sequence than the sequences of public domain gene transcripts and corresponding genomic sequences. We certainly cannot entirely eliminate the possibility of mutations introduced by our PCR cloning process, but we believe they are rare occurrences. We offer services at a small fee for changing single base nucleotide compositions using a proprietary non-PCR based mutagenesis kit.

Answer: Our system incorporated a new high fidelity PCR composition that improves fidelity in generating full length ORFs by 10 fold. It is accomplished by reducing the non-specific binding of primers with template DNA at lower reaction temperature and by reducing the cycles needed for amplifications. We guarantee that there are no frame-shifting changes and no pre-mature stop codon in our full length ORF inserts compared with reference sequences. We will replace the defect clones or refund the charges if that occurs.

Answer: Even though we cannot provide replacement clones for this type of cases, we do offer mutagenesis services to change any nucleotide compositions as requested for a nominal fee.

Answer: We will make the best effort to clone the original splicing form and provide you with the replacement clone. However, we cannot guarantee that we will get the clone of a specific splicing variant.

Answer: All expression clones in pReceiever-B01 have been tested for expression in E. coli. Other expression clones with different features and for different host cell systems have not been tested for protein expression.

Answer: A correct DNA sequence with a promoter does not guarantee you to get protein expressed. Many factors contribute to the success or failure of protein expression 1) Did you use the right type of expression vector for your expression host system, for instance our pReceiver-M01 vector is for mammalian cell expression and pReceiver-B01 is for bacterial expression? 2) If the protein is a membranous protein, does it need target sequence for proper localization? 3) Will the over-expressed protein be toxic to the host? 4) Will the protein be folded into a right three-dimensional structure for its proper function and/or immunological-assays? 5) Have you done a full-length sequencing to see if you get an alternative splicing variant?

Answer: Currently, we only offer pre-made Homo Sapiens ORF clones. We are in the process of producing full length ORF clones from model animals such as rat and mouse. These full length ORF clones will be released to market in the near future. If you have full length ORF of any genes of interest from these model animals, please send us the information of these genes such as GenBank Accession or Nucleotide sequences. We will place them on the list of high priority Full length ORFs of genes for cloning into the vectors we offer. They will be charged with the same price as our other catalog ORF clones. We also offer custom cloning services for cloning genes from these two model animals into any vector(s) of your choice.