Do I need to provide restriction sites?

Yes. You can select specific 3′ and 5′ restriction enzymes from the drop down menu of the Gene Synthesis Ordering Wizard. The wizard will automatically add the appropriate DNA sequences to your gene. The complete gene sequence with restriction sites will be displayed for your review prior to moving your genes into the Shopping Cart.

What lengths of synthetic DNA can you synthesize?

We can synthesize sequences or complete genes from very short fragments up to several thousand base pairs in length. As each synthetic gene project is unique, we'll be able to provide more information when you contact us for a quote.

How are synthetic genes optimized?

Eurofins utilizes GENEius™ sequence optimization software, when requested, to optimize all aspects of your synthetic gene. You enter the amino acid or DNA sequence and GENEius software optimizes your construct in 4 dimensions:

• Codon optimization
• Secondary structure avoidance
• GC content optimization
• Bad motif avoidance

How does GENEius optimization differ from optimization by other software tools?

We believe that our optimization will result in better protein expression. But, to be absolutely certain we compared our optimized sequences with those optimized with other software packages. Download our White Paper to see the results.

What is the difference between a long oligo and a synthetic gene?

Due to limitations in oligo synthesis chemistry and other practical considerations, oligos are produced as mixture of single-stranded DNA strands and are limited in length to ~200 nucleotides. Synthetic genes, however, can extend to several thousand, double-stranded base pairs. Usually created as a sequence verified clone, these constructs may be the best tool for your research needs.

What is the difference between genes delivered cloned into a vector and delivered as linear PCR product?

A gene that has been cloned into a vector has been fully sequenced and confirmed to have 100% sequence identity to the expected sequence; a PCR product represents a mixture of correct and incorrect sequences due to errors incorporated during polymerase chain reaction. As a consequence of this property of polymerase, subclones created from the original confirmed clone may show variation from the expected sequence. Whenever you use cloning methods that incorporate PCR products for your cloning experiments, we recommend sequencing at least 6–12 colonies to identify one with the expected sequence.

When Eurofins Genomics delivers your cloned gene and any additional plasmid preps ordered by you, you will receive materials with the 100% correct sequence and a dried vector with the gene insert of a 100% correct clone.

Do you offer cloning into a vector of my choice?

Yes, Eurofins Genomics will create your gene in as many vectors you may choose. One popular example is for customers to order a standard clone, a second construct in a shuttle vector and also a third clone in an expression vector.

What is the difference between a cloning vector and an expression vector?

A vector is a plasmid or small strand of DNA into which a non-native DNA fragment (usually a synthetic gene) can be inserted via restriction enzyme treatment followed by ligation. Cloning vectors are utilized primarily for the replication of the gene insert. Expression vectors on the other hand are genetically engineered to enhance and promote the transcription of the gene insert to facilitate the expression of large amounts of its recombinant protein within the host cell. Besides the presence of expression signals most expression vectors can be distinguished from cloning vectors by the presence of protein tags. Most of these tags are used to help in the purification of the recombinant protein from the proteins of the host cell.

What are protein tags?

Protein tags are peptide sequences appended to recombinant proteins to modify them for various post-translational applications. There are numerous types of protein tags for different applications. Some protein tags can be used in tandem to confer dual functionality. The following are a few examples of the uses of protein tags.

• To aid protein folding in chaperone-deficient hosts (solubility tags)
• To facillitate protein purification by changing chemical characteristics of the recombinant protein (affinity tags)
• To label recombinant proteins (fluorescent tags)

Do your vectors have protein tags?

No. We do not provide protein-tagged vectors. If you would like a protein tag incorporated into your vector, you can either send Eurofins Genomics your protein-tagged vector and we will insert your synthetic gene into it or you can add the protein tag sequence to your gene sequence.

Can I optimize my gene but leave protein tags intact?

Yes. You can insert protein tag sequences into the 5′ and/or 3′ untranslated regions (UTRs) on the wizard. The UTRs are not optimized by GENEius software.

What are the quality standards for your Gene Synthesis services?

We verify each synthetic gene using DNA sequencing of both strands and ensure 100% sequence accuracy for every synthetic gene. Our gene synthesis lab maintains the highest quality standards throughout the complete synthesis process. The quality management system for our Louisville, Kentucky facility has been certified to ISO 9001:2008 and ISO 13485:2003 standards.

How long will it take before I receive my gene?

The standard delivery times are:

For Standard Genes (160–1,000 bp):	6 working days
For every additional ~950 bp:	+5 working days
For Complex Genes:	Please inquire
Additional subcloning into customer vector:	5–10 working days

How do I order your Gene Synthesis service?

Click on the Order Now button to bring up the Gene Sythesis Ordering Wizard. This wizard will guide you through the process of entering your gene information, optimizing your gene sequencing, and selecting the proper vector and preparation scale. Upon completion of the wizard, your gene is placed in your Shopping Cart. After you have entered and optimized all of your genes, a simple checkout process will place the order with our lab.

In which applications can I use synthetic genes?

Among the many uses for synthetic genes are adapting codon usage for optimizing gene expression, for protein over-expression and/or protein engineering; as standards for Real Time PCR and standard PCR, mutagenesis studies, to construct hybrid genes, and the production of DNA vaccines.

How should a gene be designed?

The formation of potential hairpin structures of 4 or more bases should be avoided within your synthetic gene sequence. Also, sequences that are commonly associated with poor oligo quality such as long stretches of single or di-nucleotide repeats, or high GC content should be avoided. To achieve high levels of gene expression (protein production), we also recommend that you avoid sequences that introduce rarely used codons into a gene.

Our proprietary GENEius sequence optimzation software will optimize your gene, even if your original sequence contains some of the problem sequences listed above. Even many native gene sequences benefit from optimization in addition to codon optimization for a non-native host.

What are some of the different techniques for assembling synthetic genes?

There are three different approaches for the assembly of synthetic genes. In the approach developed by Khorana (Gupta et al., 1968), a series of sequentially overlapping oligonucleotides are synthesized. As the complementary sequences of the oligos anneal, double-stranded DNA fragments containing nicks on both strands are formed. The nicks are repaired with DNA ligase, an enzyme that catalyzes the formation of a phosphodiester bond between the 5′-phosphate of one double-stranded oligo fragment and the 3′-hydroxyl terminus on an adjacent double-stranded oligo fragment.

Another broadly used approach is the one developed by Narang (Scarpulla et al., 1982) making use of the template-directed and primer-dependent 5′ to 3′-synthesis capability of the large subunit of the enzyme DNA-Polymerase I (Klenow fragment). After end-to-end annealing of the oligos, Klenow uses deoxynucleosidetriphosphates to fill the gaps. Treatment with DNA ligase repairs any nicks in the resulting, double-stranded DNA.

An alternative strategy has been developed, using very long oligonucleotide chains. In this approach (Rossi et al., 1982), two long oligos are synthesized and upon annealing, their 3′-ends will overlap. The construct is completed to a full length double-strand using a DNA polymerase to fill in missing bases. After treatment with the polymerase, overhanging ends are generated on the double-stranded fragment by digestion with the appropriate restriction enzyme.

Typically all of these methods are followed by the molecular cloning of the gene into an appropriate vector, so technical limitations of subsequent cloning steps must be considered while developing the assembly strategy. For example, assemblies for larger genes may be divided into sub-assemblies which are sequenced to confirm 100% identity and then brought together to complete the full construct which receives a second round of sequencing to verify the full assembly.

Is de novo synthesis or site-directed mutagenesis preferable if I need to create variant constructs for my experiment?

When you need the mutations to be distributed across the whole gene, we recommend de novo synthesis. De novo synthesis allows you an opportunity to also optimize features of the gene such as codon usage, GC content, restriction sites, etc. Site-directed mutagenesis is best used when the modifications are few, or are clustered in a small area of the gene.