Gene Designer is easy and intuitive to learn. It has a graphically rich molecular viewer for displaying and manipulating genetic constructs using simple drag-and-drop manipulations, coupled with a hierarchical data structure for storing, managing and accessing sequence objects. Gene Designer is a stand-alone secure software that provides an efficient integrated solution for gene design projects.

New sequence objects in Gene Designer can be entered as AA (amino acid sequence), DNA (nucleotide sequence object) or ORF (amino acid sequence linked to a nucleotide sequence). Each object can be imported directly in FASTA format or manually imported by cut-and-paste into a data entry window. Once loaded, each object can be displayed in icon, sequence or notes (annotation) view.

A set of commonly used genetic objects are provided in a tree structured Design Toolbox. The list includes prokaryotic and eukaryotic transcriptional and translational regulatory elements, purification and solubility tags, protease cleavage sites, secretion signals, restriction sites and recombinase cloning elements. The toolbox is not a complete and final list of genetic elements, but rather a convenient starting point for each user to assemble their own custom set of genetic objects. The software is built to enable the user to add and edit new custom objects and make notes associated with each object. These objects can be saved in the toolbox and can be shared between users. For detailed and up to date information of each existing building block, or to create new building blocks, we recommend the user searching NCBI databases and the World Wide Web.

The Icon View provides an immediate overview of the entire design project. Each genetic object is shown as a differently colored arrow indicating the orientation of the object. Objects can be moved in this view by drag-and-drop. This is particularly convenient when moving affinity tags from the N to the C-terminal of a protein, creating chimeric proteins and editing restriction sites at ends of a construct.

The Sequence View provides a detailed display of the nucleotide and/or amino acid sequences of each object below a single nucleic acid sequence corresponding to the entire construct. For AA objects, each amino acid (single letter code) is shown immediately above its corresponding codons. Codons are shown in descending order of their frequency in the corresponding codon usage table.

The Notes View provides a convenient way for the user to annotate the sequence elements for future reference. There is also a feature in the Notes View for reports on the entire project.

The genetic code uses 64 nucleotide triplets (codons) to encode 20 amino acids and stop. Each amino acid is encoded by on average 3 codons that are read during translation by tRNAs charged with the cognate amino acid. The degeneracy of the genetic code enables many alternative nucleotide sequences to encode the same protein. The frequencies with which different codons are used by different organisms and different types of genes vary significantly11 and are correlated to the concentration of the corresponding tRNA population in the cell12. Rare codons are not only strongly associated with low levels of protein expression due to ribosome stalling and abortive translation13, but also implicated in frameshift and amino acid misincorporation1415. Codon usage has been identified as the single most important factor in prokaryotic gene expression16.

The simplest way to design a DNA sequence from an amino acid sequence is to assign the most abundant codon to all instances of that amino acid in the sequence. Codon usage preference in a gene is often measured by Codon Adaptation Index (CAI score). The CAI score for such a construct is 1.0, i.e. in each case only the most abundant codon is used. This 'one amino acid – one codon' or 'CAI = 1.0' approach has several drawbacks. First, a strongly transcribed mRNA from such a gene will generate high codon concentrations for a subset of the tRNA populations, resulting in imbalanced tRNA pool, skewed codon usage pattern and increased translational error17. Heterologously expressed proteins may be produced at levels as high as 60% of total cell mass, making an imbalance tRNA pool a significant problem resulting in reduced growth due to tRNA depletion18 and increased frameshift due to translational pausing at the ribosomal A-site19. Second, with no flexibility in codon selection, it is impossible to avoid repetitive elements and mRNA secondary structures in the gene. Severe repetitive elements can affect the genetic stability of a gene and may lead to excision through recombination. Third, it is often desirable to incorporate or exclude sequence elements such as restriction sites from the sequence to facilitate subsequent manipulations. These modifications are impossible to accommodate if the codon usage is rigidly fixed. Fourth, in the literature there are many and sometimes conflicting data suggesting sequence elements that decrease protein expression levels. Such elements can not be avoided if the codon usage is fixed. Gene Designer users who wish to use the CAI = 1 optimization approach can either increase the threshold for codons used or use a modified codon usage table.

In contrast to the 'CAI = 1.0' method, Gene Designer optimizes genes for expression by using a codon usage table in which each codon is given a probability score based on the frequency distribution of the codons in the genome normalized for every amino acid. The codon usage tables for 25 common protein expression hosts are included with the download, and new codon usage tables can be imported from the Codon Usage Database http://www.kazusa.or.jp/codon20 or manually edited as required. The codon usage table created by one user is automatically imported when another user shares the project. For E. coli expression we recommend the user to use the EColi_CII table that is derived from a collection of highly expressed E. coli genes21. Candidate sequences are generated in silico using a Monte Carlo algorithm by selecting codons based on the probabilities obtained from the codon usage table, with codons below the threshold value (default is 10%) excluded from consideration. Each designed sequence is then passed through subsequent iterations to ensure a match with additional design criteria such as filtering out mRNA secondary structures and DNA repeats, eliminating or incorporating restriction sites and avoiding methylation sites that overlap methylation sensitive restriction sites4. A pseudo code for the algorithm in Gene Designer can be found in appendix A.

Motifs such as internal Shine-Dalgarno sequences have been shown to decrease gene expression22. Gene Designer allows the user to filter out Shine-Dalgarno sequences, splice donor and acceptor sequences as well as any other sequence motif defined by the user. The user can also maximize or minimize the similarity of the designed sequence to a reference sequence, for example to make RNAi-resistant genes5 or to maximize the probability of recombination between two variants. Since the algorithm is a Monte Carlo based algorithm where each codon choice is an independent probabilistic event, the software can iterate the optimization each time finding a new and equally good solution.

Gene Designer does not utilize advanced RNA folding calculation software such as the popular mFold23 as these types of software are designed to calculate RNA secondary structures for naked RNA. The translated mRNA within an ORF is in fact densely covered by ribosomes. Chemical footprinting of mRNA-ribosome complexes show that up to 20 codons (60 bases) are covered by a single translating ribosome24, and the ribosomes are translating at ~18 codons (54 bp)/sec with one ribosome initiating translation every ~2 second25 leaving only ~50 mRNA bases available between translating ribosomes for folding an mRNA secondary structure. During translation, a stem-loop structure in the coding part of the mRNA does not hinder the progress of the translational machinery, and actively translating ribosomes can break up such structures, either by the energy driven translation process itself or by the support of RNA helicases 262728.

Gene Designer filters out (or flags, if it can not be avoided) any mRNA structure with double-stranded RNA stem of 12 bp or more. This feature is included because it is very often requested by users and also because it ensures that oligonucleotides used in the gene synthesis process will not predominantly self-anneal during gene assembly.

The codons immediately 3' of the initiation ATG codon have a strong influence on gene expression22293031. Accordingly, the codon optimization module in Gene Designer gives the user the option to treat the 5' end of the ORF separately. The default is conservatively set to include the first 15 codons of the ORF as 5' end, but can be changed as needed. Gene Designer will filter out NGG codons in the 5' region32 and predominantly use A/T in the wobble position3334. The 5' end is also set to filter out repeats of 8 bases or more and filter out mRNA secondary structures of 8 bp or more.

The local context of a codon can influence the protein expression levels. Back in the early 1980s it was shown that the efficiency of the UAG stop codon in E. coli is typically decreased in the presence of a 3' adenine and increased in the presence of a 3' cytidine3536. Since then, a multitude of experimentally validated codon contexts have been shown to affect ribosomal frameshift, missense and nonsense incorporations and translational efficiency 37383940. Gene Designer avoids known codon context issues by omitting the use of rare codons and filtering out runs of C's and G's. We also recommend the addition of two stop codons at the end of an ORF to ensure proper translational termination.

Aside from the experimentally validated cases of codon context effect on protein expression levels, there are several publications where in which codon context effects have been proposed based on in silico analysis of genomes 414243. The absence or low level of certain codon contexts in the analysis of entire genomes does not necessarily reflect that the identified sequences affect protein expression of a recombinant gene when grown in rich media, but more likely is a consequence of other evolutionary pressures such as facilitating DNA replication, mutational bias, expression during starvation, intrinsic metabolic regulation etc.. 44 In at least one case45, the predicted codon pair bias effect on protein expression could not be experimentally validated46. The current version of Gene Designer only includes pre-set sequence constraints that have been experimentally validated. The individual user may add to these any sequence elements they wish to eliminate.

Any object can be split into two or more daughters by selecting a part of the sequence and using the Split function. Users can thus easily divide proteins into domains for easy drag-and-drop construction of chimeras or gene variants. Objects can also be linked within and between projects; changes in linked objects then propagate throughout all open projects. All changes, such as editing an object's sequence, changing codon table or codon threshold are incorporated into the final sequence in real time.

The Gene Designer can also be used to design oligonucleotides. To assist with this, a real-time Tm calculator can be positioned in the Sequence View and dragged until a preferred location, length and melting temperature is found. The DNA melting temperature calculation is performed using the nearest neighbor method4748. The software can also design sequencing primers for a specified region or spanning the entire construct through an integrated 'Actions' module.

Once a sequence has been designed, sequences can be saved with all the graphical elements and captured relationships as Gene Design files (.gd suffix), saved as a graphic image (.jpeg) or as plain text (.txt). Reports can be generated that contain the complete nucleotide sequence, the nucleotide sequence of each object, notes, translation map of each object, a restriction site summary, codon usage frequencies and GC content. Finally, by clicking the 'Get quotation' or 'Order gene' icon, the designed synthetic DNA fragment can be priced or placed in the gene synthesis pipeline of DNA 2.0.