Gene Synthesis and Mutagenesis Support - Getting Started
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GeneArt® Strings™ DNA Fragments
GeneArt® Strings™ DNA fragments are custom-made, uncloned, double-stranded linear DNA fragments, available in lengths from 100 to 3,000 base pairs (bp), assembled from synthetic oligonucleotides using the same process developed for GeneArt® high-quality gene synthesis. GeneArt® Strings™ fragments can be used to quickly clone your gene of interest. At least 200 ng of GeneArt® Strings™ DNA fragments are produced within 5 business days (up to 1,000 bp) or 8 business days (from 1,000 to 3,000 bp). GeneArt® Strings™ DNA fragments can be designed, sequence-optimized, and ordered online and are a cost-effective and smart alternative to clone your expression plasmid.
No, GeneArt® Strings™ DNA fragments are limited to 100 to 3,000 bp. If your sequence is longer, you need to order it as gene synthesis or assemble your gene from two or more GeneArt® Strings™ DNA fragments. In addition, the GeneArt® Strings™ DNA fragments manufacturing process is streamlined to quickly and cost-effectively provide you with your gene product. If your sequence is complex, you may receive a message that it cannot be produced as a Strings™ DNA fragment due to high complexity. In that case, we recommend modifying your sequence to match the production criteria (given below) or ordering your gene as gene synthesis. In many cases, optimizing your sequence using the portal function enables manufacturing with the GeneArt® Strings™ fragments process. If it is still not able to be produced, you can manually edit the sequence to enable production.
Important production criteria are:
- GC content from 20 to 80% (in peaks, not overall content)
- No long secondary structures or sequence repetitions
- No A/T stretches >25 bp nor G/C stretches >20 bp
GeneArt® Strings™ fragments are PCR amplicons of assembled oligonucleotides, ready for screening to identify the correct clone. Strings™ fragments are bulk sequence-controlled as part of the QC process to help ensure that your desired sequence is present in the fragment pool.
To identify a correct clone >90% of the time, we recommend using these screening guidelines:
- Strings™ fragments up to 1 kb: Sequence 2 to 4 full-length clones
- Strings™ fragments 1–2 kb: Sequence 3 to 5 full-length clones
- Strings™ fragments 2–3 kb: Sequence 4 to 8 full-length clones
No, subcloning and assembly services are not available for GeneArt® Strings™ DNA fragments, as they are designed to offer you the fastest and most affordable way to get access to your genes. If you do not want to clone the gene yourself, we recommend that you order full gene synthesis service.
GeneArt® Gene Synthesis: Ordering
- Start by entering in your gene sequence, adding the attB1 and attB2 sites flanking your gene of interest.
- Save and close. Add “Subcloning” to your gene synthesis project.
- Subclone into pDONR221 or pDONR/Zeo. Scroll to the bottom of the page and check “Subcloning” under “Add service”.
- Save and close. Your process should now look like this:
- Choose the pDEST vector you wish to shuttle your gene of interest into, then save and close.
- Click on the Optimize Sequence tab, then choose either Protect Restriction Sites or Protect Sequence Parts.
- Choose sites you would like to protect, or enter nucleotide(s) you would like to protect. Protected sites will appear at the bottom of the screen. Click on Optimization tab, then “Optimize”.
- Create a gene synthesis project.
- Click on Proceed to Summary.
- 3. Choose Delivery Speed, then Add to Cart. (*Please note, if Add to Cart is greyed out and only “Send for Review” is active, the project must be reviewed by a scientist. Click on “Send for Review” and your project will be analyzed.)
- Pricing for the project, as well as estimated delivery will be shown for your project.
GeneArt® Gene Synthesis
A project can have multiple processes, but can be ordered as a single project. In this way shipping/handling are paid only once.
PLoS ONE 6(3): e17doi:10.1371/journal.pone.0017596
GeneArt® Strings™ DNA Fragments are uncloned double-stranded DNA fragments that are limited to length of 3,000 bp. GeneArt® Gene Synthesis products are double-stranded DNA fragments that have been cloned into a standard GeneArt® vector (pMx). Please note, a sequence may be too complex to be produced as a Strings™ DNA Fragment due to repetitions or high GC content. In this case, our scientists will recommend that you order your gene via gene synthesis. You could try optimizing your sequence through the GeneOptimizer® algorithm which, in some cases, could optimize the sequence and allow the DNA Fragments to be ordered.
Genes are typically cloned into a pMx standard vector. These vectors contain:
- a multiple cloning site
- an ori (colE1)
- an antibiotic resistance marker (ampicillin, kanamycin, streptomycin, chloramphenicol); please note standard orders do not guarantee any specific standard vector
Please note that standard orders do not guarantee any specific standard vector.
We do not offer free choices of restriction sites in our standard production vectors. For our production process, restriction enzymes are added that generally eliminate most of the standard vectors' MCS. The reason the MCS contains more sites is to allow for fallback scenarios in case certain sites cannot be used due to insert sequence intrinsic restrictions.
No, and this cannot be requested. You would need to subclone into a vector of your choice. The only thing you can request as part of the “Geneart® vector” is the antibiotic resistance (for a small markup fee).
GeneArt® synthesis adds standard restriction sites for automated cloning into GeneArt® standard vectors in addition to your selected restriction sites.
Yes, we keep a sample of every GeneArt® synthesized product.
In theory, there is no limit. However, very large DNA fragments (greater than 10 kb) need to be cloned into a low-copy vector to ensure genetic stability. Therefore, increasing length leads to increased production time.
Please provide 5-10µg of vector DNA for your GeneArt® custom service. We prefer the DNA dissolved in TE buffer or water, or as a pellet.
There are different compliance regulations/rules for synthesis, including the use of different labs. With Biosafety 2, it takes a longer time to synthesize genes, therefore, these projects may be more expensive. Please note, regardless of your selection, Life Technologies™ Corporation and/or its affiliates will perform a biosafety evaluation. In the case that you select Biosafety Level 1 but our evaluation requires the DNA to be a Biosafety Level 2, you will be sent a modified quotation according to the Biosafety Level 2 requirements for review.
TSE stands for Transmissible Spongiform Encephalopathy, meaning the media used is soy-based for TSE-free plasmid preps.
You typically would receive the following with your order: vector map, alignment maps, sequencing traces. Extended documentation can include:
- Data storage certificate – guaranteed storage of all production specific data
- Material documentation – includes all information on all used chemicals including providers and catalog numbers
- GLP source documentation – includes the granularity of the material documentation to lot number level of all used reagents
You can download your optimized sequence in FASTA format. It can be read in a txt file/format. The FASTA file will give a simple description of the gene, and then give a non-annotated version of the sequence. You can align this file with their original sequence using a free online tool, or if you have Vector NTI® software, you can use this to align the files.
Partial deliveries can occur when upon reaching the estimated completion date not all order items have been finalized. Partial shipments of incomplete orders are initiated on a weekly basis. Separate shipping of items does not incur an extra cost.
Generally, we ship immediately upon completion of the project item with the longest estimated turnaround time. In rare cases, completely finalized projects are not shipped until the estimated completion date has been reached. Please keep in mind, if a customer orders gene synthesis and subcloning as a process, they will most likely receive the gene synthesis in a Geneart® vector before they receive their gene in their subcloned vector.
No, your custom vector has been banked and sequenced and is available to you in your individual short list “My Portal Vectors” as soon as they have been ordered/used once.
Yes, all pMx vectors contain an M13 – 20 primer binding site and a M13-R primer binding site. The sequences are as follows:
- M13 -20 primer binding site: 5'-TTGTAAAACGACGGCCAG-3'
- M13 –R primer binding site: 5'-GGAAACAGCTATGACCAT-3'
Single variants and variant bundles (more than one variant process) are variants of a master gene. The variant criteria are as follows:
Variants may contain up to four of the following modifications:
- 5'/3' deletion of any length with additional 52 nt of new sequence on each side
- 5'/3' modification of a maximum of 52 nt on each side
- 5'/3' extension of a maximum of 52 nt on each side
- Internal modifications of up to 40 nt (a maximum of 3 of these internal modification blocks are allowed)
- Internal deletions of any length
Modifications (whether internal or 5´/3´) must be separated by at least 100 bp.
If a gene falls outside of these criteria, it must be created as a separate gene synthesis instead of as a variant.
Yes. Add “single variant” as a new process, then choose an “upstream service” ("gene synthesis", "already at Geneart®", or "I will provide this master gene"). If you are providing your own gene, you will need to fill in the field for the complete sequence of the vector + insert. If it is a plasmid only, you will enter the mutagenized area as the “insert” and the rest of the plasmid as the “vector”. The “insert” can be up to 4 kb long.
GeneArt® Subcloning and Express Cloning Service
Using an alternative production procedure with pre-prepared expression vectors, our GeneArt® custom service team can clone your synthesized genes directly into selected Life Technologies™ expression plasmids, saving 4-5 business days compared to the standard workflow with cloning into a pMx series vector first. Please visit this page for the differences between Express cloning and Classical cloning.
You should receive your synthesized gene in the selected expression vector. Please note, no additional copy in a pMx cloning vector will be provided as with traditional GeneArt® Cloning Services.
The following vectors are currently available:
- pcDNA 3.1(+)vector
- pcDNA3.3-TOPO® vector
- pcDNA3.4-TOPO® vector
- pFastBac1 vector
- pET100/D-TOPO® vector
- pET151/D-TOPO® vector
- pRSET A vector
- pYes2.1V5-His TOPO® vector
Genes qualifying for Express cloning must be <4 kb and not complex (optimization of your sequence can reduce complexity).
Additional Custom Services
Please use the following link to explore our synthetic biology services which include: vector construction, cDNA library construction, protein expression services, directed evolution services, plasmid services, and cell line & protein services.
TALs are transcription activator-like effector proteins that are naturally occurring transcriptional activators. GeneArt® Precision TALs allow researchers to determine the exact DNA sequence they would like to have their functionality delivered to and have specific TAL genes built to perform the function.
GeneArt® Site-Directed Mutagenesis Kits
The kits are almost the same, but the PLUS kit contains an improved 2X GeneArt® Enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites in plasmids up to 14kb). The PLUS kit can also be used for single-site mutations of up to 25 nucleotides, while the original kit can be used for single substitution/deletion/insertions up to 12 nucleotides in plasmids up to 14kb. A web tool is available for primer design, that can also be used for the original kit.
- The GeneArt® kit combines the methylation and DNA amplification steps saving 1 hour in the protocol
- The enzyme mix from the GeneArt® Seamless Cloning and Assembly Kit is part of the protocol, boosting the colony output without the need to do many PCR cycles (another time saving step)
- The new kit also contains an enhancer used during the PCR step that increases mutagenesis efficiency for a wide range of plasmid sizes.
We do not offer the DNA methylase from GeneArt® Site-Directed Mutagenesis kits as a separate product.
The concentration is 32 mM.
The primer should be 30-45 nts in length with the mutation centrally located. The mutation should be flanked by at least 15-20 nts on either side, depending on the size of the mutation, with the primer length no longer than 45 nts. We recommend using our online GeneArt® primer design tool.
During the first cycle of PCR on the circular template, the polymerase will generate two linear products that are able to anneal and undergo primer extension in the next cycle (see diagram below). PCR products that incorporate the mutation at both ends will accumulate in subsequent cycles. After PCR, the recombination reaction results in circularization of the PCR product which enhances the mutagenesis efficiency.
In general, the annealing temperature used will be between 55-64 degrees C, which is what we recommend for PCR with AccuPrime™ Pfx. The Tm of the primers will be about 5 degrees C above the annealing temperature. For example, the Tm of the top strand control primer that comes with the kit is 65 degrees C (nearest neighbor method).
We recommend shortening the primer length for GC-rich sequences, since a very high Tm may result in nonspecific bands. A previous customer reported having success using 21-nt primers with a Tm of ~80 degrees C.
The polymerase should not have a problem reading through AT-rich regions. Only GC-rich regions can be difficult for polymerases because of the higher Tm.
The primer design guidelines in the GeneTailor™ kit (discontinued) should work as long as the primers have an overlapping region of 15-20 nucleotides. However, to make primer design straightforward we recommend that users switch and use the GeneArt® guidelines only.
This is most likely due to rearrangement caused by hairpin formation of the primer during PCR. You can try and increase the annealing temperature of the PCR step to relax secondary structure formation. Screening more colonies should also help.
In general, the PCR enhancer increases the yield of PCR product. It has nothing to do with DNA methylation. The 10x enhancer is optimized for Accuprime™ Pfx and it may not work with other polymerases. If you need to optimize the PCR (for annealing temp, extension time, etc.) you should do standard optimization with AccuPrime™ Pfx but without the enhancer.
We currently do not supply this kit with a DNA polymerase. We do recommend purchasing AccuPrime™ Pfx DNA polymerase for your site-directed mutagenesis experiments with this kit, as we have optimized the conditions and protocol using this enzyme.
For Research Use Only. Not for use in diagnostic procedures.