Gene Synthesis and Mutagenesis Support - Getting Started

GeneArt™ Strings™ DNA Fragments are custom-made, uncloned, double-stranded linear DNA fragments, available in lengths from 150 to 3,000 base pairs (bp), assembled from synthetic oligonucleotides using the same process developed for GeneArt™ high-quality gene synthesis. GeneArt™ Strings™ DNA 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.

GeneArt™ Strings™ DNA Fragments are PCR amplicons of assembled oligonucleotides; an intermediate product of the gene synthesis production process. This process results in a pool of fragments, and cloning and screening need to be carried out to identify the correct clone. To ensure that correct fragments are present in the pool, GeneArt™ Strings™ DNA Fragments are bulk sequence-controlled before shipment.

GeneArt™ Strings™ DNA Fragments are shipped dried, ready for resuspension and cloning. Before using, we recommend spinning down the tube contents, adding some water to the bottom of the tube to get the desired concentration, and incubating for at least 1 hour at room temperature (alternatively, incubate at 4 degrees C overnight). Subsequently, we recommend resuspending the fragments carefully and using immediately. For longer storage, resuspended GeneArt™ Strings™ DNA Fragments should be dispensed into aliquots and frozen at –20 degrees C. Please avoid freeze-thaw cycles.

No. GeneArt™ Strings™ DNA Fragments from 150 to 1,000 bp are produced in 5 business days and GeneArt™ Strings™ DNA Fragments from 1,000 to 3,000 bp are produced in 8 business days. Depending on the nature of the sequence, production time can vary.

Yes. We recommend using the GeneArt™ web portal for ordering, where you can use the free gene editing and optimization functions to adjust the sequence to meet your needs. After editing and optimization, please check again that the final sequences of the fragments are exactly as needed for further processing in your lab (e.g., potential homologous overlaps of the fragments or restriction sites and buffer bases needed for cloning).

Yes, we offer GeneArt™ Strings™ DNA Libraries from 200 – 2000 bp, containing up to 3 blocks of degenerate nucleotides. Each block can consist of up to 30 bp using the full IUPAC code of DNA nucleotides. Blocks need to be separated by 30 bp of non-degenerate nucleotides.

No, GeneArt™ Strings™ DNA Fragments are limited to 150 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, theGeneArt™ 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 GeneArt™ 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™ DNA 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™ DNA Fragments are PCR amplicons of assembled oligonucleotides, ready for screening to identify the correct clone. GeneArt™ Strings™ DNA 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:

• GeneArt™ Strings™ DNA Fragments up to 1 kb: Sequence 2 to 4 full-length clones
• GeneArt™ Strings™ DNA Fragments 1–2 kb: Sequence 3 to 5 full-length clones
• GeneArt™ Strings™ DNA 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.

Yes, it is possible to directly assemble two GeneArt™ Strings™ DNA Fragments without pre-cloning. We offer several technologies for seamless assembly, e.g., GeneArt™ Type IIs Assembly or GeneArt™ Seamless Cloning and Assembly. Depending on the sequence length and the number of subfragments you want to assemble, the screening effort to find a correct clone can be high. Therefore, we generally recommend pre-cloning the subfragments to limit the screening effort required to find a correct clone after assembly.

We recommend this site: http://www.chick.manchester.ac.uk/SiteSeer/IUPAC_codes.htmL

The answer depends on the specifics of your project. In general, it is advantageous to keep the diversity of a library as low as possible, targeting only the regions of a gene/protein that are likely to be functionally important. The following information can help determine this: crystal structure, conserved motifs, presence of homologs, etc.

Conventional protocols for degenerated library creation (e.g., error-prone PCR) incorporate many unwanted mutations. Moreover, methods like DNA shuffling cannot typically cause recombination of directly adjacent mutations. Synthetic combinatorial libraries, on the other hand, limit the introduction of mutations to defined regions at the precise frequencies requested. In addition, adjacent mutations will be recombined (shuffled) independent of their proximity.

The use of TRIM technology in creating diversity allows for the replacement of complete codons instead of altering single nucleotides. You thus have complete control over which amino acids appear in a given position and in what ratios, while at the same time avoiding the undesired introduction of stop codons. Because complete codons are replaced, TRIM technology also results in fewer out-of-frame mutations than other mutagenesis technologies.

We routinely use optimized codons for the most common expression hosts (human, CHO and E. coli). All other codons are available upon request.

There are a number of reasons:

• Because you already know that certain amino acid substitutions disturb the function of your protein (e.g., cysteines in complementarity determining regions (CDRs)).
• Because the number of possible variants of a protein is astronomically high, exceeding the capacity of even the highest-throughput screening capabilities by many orders of magnitude. The fewer useless mutations, such as those occurring in less important regions of the protein or that cause frame shifts or stop codons, the better your chances of finding a variant that results in the desired phenotype.
• Some screening assays are cost and labor intensive; thus, screening fewer clones saves time and money.

• Combinatorial Libraries (up to 1,011 variants): Simultaneous randomization of multiple codons, TRIM technology optional
• Site-Saturation Mutagenesis (up to 20 variants): Randomization of a single codon with every possible non–wild type variant
• Sequential Permutation Libraries (# of codons x 20 variants): Successive site-saturation mutagenesis
• Controlled Randomization (up to 1,011 variants): Unbiased random substitutions with defined frequency
• Cloned cDNA of all known human SH3 domains
• Truncation Libraries: Customer-defined truncations without out-of-frame mutations
• GeneArt™ Strings™ DNA Libraries:  linear DNA fragments that can contain up to three regions of randomized nucleotides

If you require a degenerate library with an unusual design please send an email to geneartsupport@lifetech.com. We will consider any project and in the vast majority of cases will find a solution to fulfill your requirements.

Yes, GeneArt™ Combinatorial Libraries can be randomized using any nucleotide mix you require, down to single digit percentages of given nucleotides (“dirty bottle” approach).

No material is necessary. For library creation, all we need is the sequence file, submitted electronically, and information about the position and nature of the sites you want to randomize. We can provide a quote for your project through our online ordering system (https://www.thermofisher.com/order/geneartgenes/projectmgmt). If needed, you can then relate detailed information about your library request to our production scientists prior to starting the project.

Yes. For this purpose, please send in your own customized vector or a commercially available vector.

Two standard options are available:

• As a set of linear DNA fragments, ready for restriction enzyme digestion and subcloning.
• Subcloned into any vector and delivered as a glycerol stock and plasmid preparation.

• Increase or adjust promoter strength or specificity
• Enhance or modulate protein stability
• Modify or combine enzyme properties
• Increase binding affinities of receptors, ligands, and antibodies
• Optimize or alter signal peptide efficiencies
• Destroy protein function while retaining immunogenicity
• Combine and select natural polymorphisms
• Increase protein half life
• Adjust thermal stability

Three criteria are important for degenerate libraries and are quality-controlled in all GeneArt™ degenerate libraries (excluding the GeneArt™ Strings™ DNA Libraries): 

• Maximum sequence integrity of the non-degenerate parts
• Maximum sequence variation of the degenerate positions with the requested nucleotide distribution
• Maximum library diversity

A GeneArt™ Strings™ DNA Library is a linear DNA fragment with the following parameters:

• 200 bp to 2000 bp in length
• Up to 3 randomized regions of up to 30 bp each
• The randomized regions can contain all IUPAC defined ambiguous nucleotides (N, K, S, B, etc.); U is not optional
• Randomized regions need to be at least 30 bp from either end of the fragment and at least 30 bp from each other  

Note: GeneArt™ Strings™ DNA Libraries are not available with TRIM technology or custom nucleotide mixes. Please inquire about our other library products if you require this type of randomization.

1. Start by entering in your gene sequence, adding the attB1 and attB2 sites flanking your gene of interest.
   
   
   
2. Save and close. Add “Subcloning” to your gene synthesis project.
   
   
   
3. Subclone into pDONR221 or pDONR/Zeo. Scroll to the bottom of the page and check “Subcloning” under “Add service”.
   
   
   
4. Save and close. Your process should now look like this:
   
   
   
5. Choose the pDEST vector you wish to shuttle your gene of interest into, then save and close.

1. Click on the Optimize Sequence tab, then choose either Protect Restriction Sites or Protect Sequence Parts.
   
   
   
2. 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”.
   

1. Create a gene synthesis project.
2. Click on Proceed to Summary.
   
   
   
3. 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.)
   
   
   
4. Pricing for the project, as well as estimated delivery will be shown for your project.

A project can have multiple processes, but can be ordered as a single project. In this way shipping/handling are paid only once.

Yes. We can subclone your gene into a vector you provide. You will receive 5 µg each of your gene in a GeneArt™ cloning vector and in your vector (plus the quality control information for both constructs).

GeneArt™ Gene Synthesis Service includes gene optimization (if requested), synthesis of the DNA, cloning into our standard vector, and delivery of 5 µg of lyophilized DNA plus a compact disc containing the in silico analysis and quality control information.

Our proprietary GeneOptimizer™ software calculates the optimal DNA sequence needed to encode the protein of interest (gene optimization). Adapting the codon usage of the gene to the codon preferences of the expression organism (codon optimization) is just one of the parameters addressed. The GeneOptimizer™ software currently evaluates up to 50 factors that can compromise mRNA stability, such as extreme GC content, ribosomal binding sites, consensus and cryptic splice sites, repeats, and secondary structures. Increased numbers of stable mRNA molecules often lead to higher yields of protein.

The GeneOptimizer™ process takes a multi-parameter approach to analyzing the gene sequence. It takes codon usage, GC content, cryptic splice sites, direct repeats, RNA secondary structures, and instability sequences into account when optimizing a gene sequence.

Yes, you can protect specified regions or restriction enzymes sites that you would like to not be optimized by the the GeneOptimizer™ process. In the portal, under "Optimize sequence", choose "Protect Restriction Sites" and/or "Protect Sequence Parts" from optimization.

Fath et al. (2011) A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression.
PLoS ONE 6(3): e17doi:10.1371/journal.pone.0017596

GeneObserver shows the manufacturing status of your web and non-web orders, as well as the completed date so that you can track your gene synthesis project.

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 GeneArt™ 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.

Production time is based on gene length and choice of shipment speed. Please see this link for the differences between our shipping conditions.

The kit and custom services offer different advantages. Please click here to view a selection guide comparing the technologies, including deliverables and production time, to determine what is best for you.

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.

GeneArt™ Standard vector (pMx) zero cutter restriction enzymes table

GeneArt™ Standard vector (pMx) map

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.

You will receive both vectors – your gene in the basic cloning vector, and your gene in the subcloned vector of your choice.

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:

1. Data storage certificate – guaranteed storage of all production specific data
2. Material documentation – includes all information on all used chemicals including providers and catalog numbers
3. 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, we will optimize the protein based on the species you choose, to provide you with the optimized gene sequence for your studies.

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.

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).

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.

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.

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.

For GeneArt™ mutagenesis services, we will need the DNA or amino acid sequence of the gene you would like to mutagenize, the host organism you plan to use (this is important for gene optimization if you choose to include it with your request), the restriction sites you need at the 5’/3’ ends and/or to avoid internally, and whether or not you want any other added motifs (e.g., Kozak sequence, stop codons, etc.).

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 maximum 52 nt on each side
• 5'/3' extension of maximum 52 nt on each side
• 5'/3' extension of maximum 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

Note: Modifications (whether internal or 5´/3´) have to be separated by at least 100 bp