Products FAQs – ORF cDNA clones

  

ORF clones

Answer: All you need to do is go to the ORF clones search page, search for your gene, and then choose the appropriate clones that will work for your system. If you have any custom requirements, then you will need to contact us and, after determining what you need, we will send you a custom quote.
Answer: We provide most ORF clones as 10 µg of purified plasmid. For our NextDay™ clones, you can choose between bacterial stock or transfection-ready DNA (10 µg or 50 µg). You can also request alternative delivery formats by contacting us at support@genecopoeia.com.
Answer: A NextDay™ ORF clone is a pre-made ORF clone. It is available at a competitive price and ships next day if the order is placed by Noon EST.
Answer: The turnaround time for ORF clones varies greatly, depending on many factors. If you order a NextDay™ clone in bacterial stock, the delivery time is normally 1-2 business days, if the order is placed by Noon EST. Most non-NextDay clones have a turnaround time of about 2-3 weeks. If we need to make any customizations, or if we encounter any unforseen technical difficulties, the turnaround time could be longer.
Answer: When you order an OmicsLink™ Expression-Ready ORF clone in one of our more than 200 vector types, you can order the same ORF clone in a different vector type for a reduced price.
Answer: Once the clone is constructed, GeneCopoeia follows a rigorous quality control process to ensure the correct clone is delivered to you. We first amplify the sequences by PCR and validate the size. Next, all ORFs are fully sequenced. Restriction enzyme digestion is used to check the integrity of the entire plasmid.
Answer: Yes. pShuttle™ cloning vectors are available for do-it-yourself (DIY) cloning using the EZShuttle™ BP Recombination Cloning System. (https://www.genecopoeia.com/category/product/cloning_products/).
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: Due to variations between naturally-occurring protein-coding sequences and the NCBI reference sequence, GeneCopoeia does not guarantee a 100% match of OmicsLinkTM ORF clone sequences with NCBI RefSeq. GeneCopoeia guarantees that our OmicsLinkTM ORF Expression clone sequences are free of artificial frame-shifting variations, including deletions and additions of bases, as well as translation termination mutations (non-stop codon to stop codon) resulting from point mutation. This policy does not cover point variations due to possible naturally occurring polymorphisms and/or mutations that may be introduced by PCR in very rare cases. If you want a specific version of a gene from different cDNA libraries, GeneCopoeia will make its best effort to obtain that particular version but without guarantee of success.
Answer: The expression system should be specified first. We recommend the lentiviral system if the mammalian target cell is hard to transfect. Next, tags should be chosen. GeneCopoeia provides fluorescent tags, solubility and purification tags, antibody immunoprecipitation tags and multifunctional tags.
Answer: Tags should be placed far from the active center and functional domains of the target protein. If the target protein has an N-terminal signal peptide, then the tag must be placed at the C terminus.
Answer: EZShuttle™ Gateway® PLUS ORF clones cannot express proteins in GeneCopoeia’s pShuttle™ vector. However protein-coding ORFs in these clones can be transferred into any compatible GeneCopoeia pEZ™ expression cloning or Gateway® destination expression vector simply and rapidly. These new vectors can then be used to express the desired protein in the targeted cell type.
Answer: This information can be found in the clone datasheet you receive.
Answer: GeneCopoeia provides a suite of pEZ™ expression cloning vectors (similar to pDEST destination vectors  from ThermoFisher Scientific) 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. EZShuttle™ Gateway® PLUS ORF clones can also be used to transfer ORFs to ThermoFisher Scientific’s pDEST destination vectors.
Answer: Currently, we offer pre-made human, mouse, and zebrafish ORF clones. We are in the process of producing full length ORF clones from other model animals such as rat. These full-length ORF clones will be released to market in the near future. If you are interested in a full length ORF of any genes of interest from other model organisms, please send us the information of these genes, such as GenBank Accession or Nucleotide sequences. They will be charged with the same price as our other catalog ORF clones. We also offer custom cloning services for cloning genes from other model organisms into any vector of your choice.
Answer: A correct DNA sequence with a promoter does not guarantee you will 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 membrane 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 its correct three-dimensional structure for proper function and/or immunological assays? 5) Have you checked the sequencing to see if you get an alternative splicing variant?
Answer: All expression clones in pReceiver-B01 and pReceiver-WG02/WG03/WG04/WG05/WG09/WG16 have been tested for expression in E. coli or the cell free protein production system made by Roche. Other expression clones with different features and for different host cell systems have not been tested for protein expression.
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: Even though we cannot provide replacement clones for this type of case, we do offer mutagenesis services to change any nucleotide composition as requested for a nominal fee.
Answer: We guarantee that there are no frame-shifting changes and no premature stop codons in our full length ORF inserts compared with reference sequences. We will replace defective clones or refund the charges if that occurs.
Answer: The differences can originate from two potential sources or a combination of the two: 1) Naturally occurring events including polymorphisms, alternative splicing, inconsistencies in multiple copies/versions of the same genes resulting from annotation updates, sequencing errors and/or ambiguities; 2) Artificially introduced changes caused by PCR cloning. 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 the vast majority of discrepancies can be clearly attributed to category 1), when comparing our sequence data with all transcripts and sequences in the corresponding gene locus including genomic sequence, ESTs, other cDNA/mRNA transcripts. Typically, we found that there are fewer discrepancies between the sequences of our ORF clones and the corresponding genomic sequence than the sequences of public domain gene transcripts and the 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 base nucleotide compositions.
Answer: We use PCR together with several patented technologies to maximize the fidelity of PCR to generate full length ORFs (more information can be found in the ORF White Paper). We used more than 70 cDNA libraries of human or mice tissues as templates to generate full length ORFs.
Answer: For EZShuttle™ Gateway® PLUS ORF clones, the forward sequencing primer (from upstream of the 5′ end of the ORF) is: 5′-CCCAGTCACGACGTTGTAAAACG, and the reverse sequencing primer (from downstream of the 3′ end of the ORF) is: 5′-ATGGTCATAGCTGTTTCCTG. For  OmicsLink™ clones, sequencing primers are indicated in the datasheets for each individual clone. To download the datasheet, please visit the Technical Resources section on our website. Please note that you will need the following information in order to download your data sheets: Login information for your account on our website, the catalog number(s) of your clone(s), and your sales order number (SO#). If you are unable to download the datasheets from our website, please contact Technical support.
Answer: Each full length ORF insert is fully sequenced. Before the delivery of each clone, we re-check the clone by PCR with gene-specific primers to ensure that the correct clone is shipped.
Answer: Yes, we will clone the gene for you. It is our mission and continuing effort to clone all full-length ORFs of human genes. It is likely that the gene 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 number, gene name, nucleotide or amino acid sequences, we will place the gene 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. You will be notified as soon as the clone becomes available. There will be no additional charge.
Answer: There are usually multiple gene transcripts in a gene locus, resulting from alternative splicing, updated information about ORFs, polymorphisms, or redundancies from duplication of cloning efforts 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 originate from changed annotations of ORF start and/or stop locations as the result of newly available sequence information and/or experimental evidence. We regularly check for information updates on ORF annotations in public databases such as NCBI and EMBL. The new ORF annotations will then be used in our recloning efforts to keep our ORF clones updated.
Answer: No, the sequence on the website is not the actual sequence but the reference sequence from NCBI. The actual sequence of the clone may differ due to genetic variation and alternative splicing. The actual sequence for any of the ORF cDNA clones can be requested at any time which can be sent by e-mail. However, please note that the actual sequence for clones in pre-production or clones offered via Custom Gene Synthesis may not yet be available.
Answer: The sequences are different depending on which full length ORF clone for both EZShuttle™ Gateway® PLUS ORF clones and OmicsLink™ ORF Expression Clones you purchased. First, check the datasheet that is shipped with the clone and use the figure to identify the sequence.

Fig1. Example: Multiple cloning site of Catalog No. GC-B0128

 
Answer: OmicsLink™ vector backbones may contain one to two selection markers, one for bacterial selection and one for mammalian stable selection. There are various options for the selections. Please check the selection options carefully when you order OmicsLink™ Expression Clones.(https://www.genecopoeia.com/product/orf-clone/).
Answer: KAN.
Answer: GeneCopoeia’s OmicsLink™ Expression Clones are generated by our patented RecJoin™ cloning technology. By using this technology to construct a 5′ tag-ORF fusion expression clone, there is no attB site between the 5′ tag and the start codon ATG of the full-length ORF. The extra 8 amino acids encoded by the attB site in ThermoFisher Scientific’s pDEST expression clones will not be present in the proteins produced by using OmicsLink™ Expression clones. The same principle applies to 3′ fusion protein production.
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 ATG start codon.
 
Answer: Each EZShuttle™ Gateway® OLUS ORF Clone has two multiple cloning sites (MCS) flanking the ORF. By choosing suitable restriction enzymes, you can cut out the entire ORF from EZShuttle™ Gateway® OLUS ORF Clones and insert the ORF into your vectors by classical sub-cloning methods.
Answer: You can make a point mutation to remove the stop codon. There are more than 35,000 full length ORFs that have been constructed with a tag at their 3′ ends, such as eGFP and HA. There are more than 10 types from which you can choose (https://www.genecopoeia.com/tech/omicslink/). You can ask us to remove the stop codon and clone it into the vector(s) of your interest for a small fee.
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 tags or C-terminal tags, such as His tag and eGFP. Tthere are more than 10 types of tags to meet your needs. Please check the OmicsLink™ product information page (https://www.genecopoeia.com/tech/omicslink/). Second, you may also use GeneCopoeia’s pEZ™ or ThermoFisher Scientific’s Gateway® expression vectors for expressing N-terminal or C-terminal fusion proteins. Because our EZShuttle™ Gateway® OLUS ORF Clones contain stop codons, you will need to remove the stop codons before transferring ORF inserts into GeneCopoeia’s pEZ™ or ThermoFisher Scientific’s pDEST expression vectors to produce C-terminal tagged fusion proteins.
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: GeneCopoeia provides a suite of pEZ™ expression cloning vectors (similar to pDEST destination vectors  from ThermoFisher Scientific) 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 vector for EZShuttle™ Gateway® OLUS ORF Clones manufactured by GeneCopoeia 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 GeneCopoeia’s EZShuttle™ Gateway® PLUS ORF Clones are mixed with Gateway® compatible pDEST expression vector(s), with optimal concentrations of enzymes and conditions, the ORFs in the EZShuttle™ Gateway® PLUS ORF 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: 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 impacts on the protein translation process. Please visit this page for further details on the comparison of these two types of clones: (https://www.genecopoeia.com/tech/application/).
Answer: We recommend that you refer to the product by its catalog number (eg: EX-V0379-M29) and refer to us as GeneCopoeia, Inc. (Rockville,MD).