Gene Synthesis for Site-Directed Mutagenesis

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Creative Enzymes provides high-fidelity Gene Synthesis Services tailored for Site-Directed Mutagenesis (SDM) projects. As part of our comprehensive upstream services for enzyme engineering, we deliver error-free, fully optimized DNA templates that form the essential foundation for precise genetic manipulation. Our advanced synthesis technologies ensure codon optimization, sequence verification, and seamless cloning into your preferred vectors—accelerating downstream mutagenesis, expression, and characterization workflows.

Gene Synthesis: Core Process of SDM

Accurate and stable DNA templates are essential for successful site-directed mutagenesis. Even minor sequence errors, poor codon usage, or unwanted secondary structures can compromise cloning efficiency and distort experimental results. Synthetic gene preparation provides a precise, reproducible foundation for reliable enzyme engineering. The core process includes sequence design, oligonucleotide synthesis, assembly, and cloning with sequence verification. Together, these steps ensure complete control over gene content and accuracy.

Gene synthesis also enables advanced mutagenesis strategies. By allowing DNA to be "written" from scratch, researchers can perform codon optimization for improved expression in hosts such as E. coli or mammalian cells, or build saturation mutagenesis libraries covering every amino acid variation at key positions.

A simplified workflow of a site-directed mutagenesis strategy: the step that follows template DNA sequencing is gene synthesis Figure 1. A site-directed mutagenesis strategy. After preparing the template DNA, the gene for mutagenesis is synthesized. (Allemandou et al., 2003)

Gene Synthesis for SDM: Services & Capacities

At Creative Enzymes, we combine high-precision synthesis with advanced bioinformatics to deliver mutation-ready, codon-optimized templates. Our verified constructs eliminate the need for error-prone PCR or manual cloning, ensuring every mutagenesis project begins with maximum accuracy and expression efficiency. Our service includes:

Custom gene synthesis (up to 10 kb) with guaranteed sequence accuracy
Codon optimization for any expression host, improving translation efficiency
Error-free sequence verification through sanger sequencing and alignment reports
Cloning into standard or custom vectors suitable for SDM workflows
Fragment assembly and modular design for complex mutagenesis targets
Optional addition of tags and restriction sites for downstream manipulation

Service Workflow

Service workflow of gene synthesis for site-directed mutagenesis

Service Details

Parameter	Specification
Gene Length	Up to 10 kb (longer sequences available upon request)
Host Optimization Options	E. coli, Bacillus, Pichia, insect, mammalian
Turnaround Time	5–15 business days, depending on complexity
Verification Method	Sanger sequencing and restriction digestion
Deliverables	Plasmid DNA (≥2 µg), sequence report, vector map
Optional Add-ons	Cloning, expression testing, mutagenesis-ready verification

Connecting Services

Our Gene Synthesis for Site-Directed Mutagenesis service works seamlessly with the other two preparatory offerings in our Upstream Services for Site-Directed Mutagenesis suite:

Template DNA Sequencing for Site-Directed Mutagenesis: Ensuring template integrity before mutagenesis.
Cloning into Expression Vectors for Site-Directed Mutagenesis: Delivering high-efficiency vector systems for precise genetic manipulation.

Together, these services establish a robust and error-free foundation for successful enzyme engineering workflows.

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Why Partner With Us

High Accuracy and Full Sequence Guarantee

100% error-free gene synthesis confirmed by Sanger sequencing.

Mutagenesis-Ready Templates

Seamlessly compatible with our SDM and expression services.

Host-Specific Optimization

Custom codon usage tailored to expression system requirements.

Flexible Cloning Options

Broad vector compatibility with multiple selectable markers.

Expert Bioinformatics Support

Advanced sequence analysis, secondary structure prediction, and annotation.

Fast and Reliable Turnaround

Scalable synthesis and parallel processing minimize delivery time.

Case Studies and Success Stories

Case 1: Codon Optimization Enhancing Expression Efficiency

Client Need:

A university research group sought to perform multi-site mutagenesis on a thermophilic xylanase gene to improve its catalytic efficiency and thermostability. However, the native gene sequence exhibited low expression in E. coli due to unfavorable codon usage and mRNA secondary structures, resulting in poor soluble yield and unreliable kinetic data.

Our Approach:

We employed its proprietary codon optimization algorithm to redesign the gene for efficient expression in E. coli. The optimized sequence was synthesized de novo, assembled using polymerase cycle assembly, verified by full-length sequencing, and cloned into a pET expression vector. This ensured a mutation-ready template suitable for site-directed mutagenesis.

Outcome:

The codon-optimized gene produced a 5.2-fold increase in soluble protein yield and exhibited proper folding with consistent enzymatic activity. Using the new construct, the client successfully introduced three targeted mutations that enhanced catalytic turnover, validating the critical role of pre-synthesis optimization in mutagenesis workflows.

Case 2: Synthetic Modular Gene Assembly for Multi-Site Mutagenesis

Client Need:

An industrial enzyme developer aimed to perform simultaneous site-directed mutagenesis on several catalytic residues of an oxidoreductase to fine-tune substrate selectivity. However, their existing gene architecture was rigid, making parallel substitutions time-consuming and error-prone. They required a flexible and reusable system for rapid mutagenic customization.

Our Approach:

We designed a modular synthetic gene construct, dividing the oxidoreductase into structural domains and flanking each with unique restriction sites to enable easy recombination. Each module was synthesized, sequence-verified, and cloned into an expression-ready plasmid. This design allowed quick swapping, insertion, or combination of fragments without re-synthesizing the entire gene.

Outcome:

The modular construct enabled the generation of over 40 combinatorial variants within half the usual development time. The streamlined workflow accelerated structure–function screening and reduced cloning complexity. The client continues to use the modular template as a versatile backbone for ongoing enzyme engineering projects, demonstrating its long-term value and adaptability.

Common Questions About Gene Synthesis for SDM

Q: What is the recommended sequence format for submission?

A: We accept sequences in FASTA, GenBank, or plain text format. Please indicate restriction sites and any specific cloning requirements.
Q: Can you synthesize genes with complex secondary structures or repeats?

A: Yes. Our synthesis platform incorporates sequence optimization algorithms that minimize GC-rich regions and repetitive motifs to ensure stable assembly.
Q: Do you offer codon optimization for rare or unconventional hosts?

A: Absolutely. We can perform custom optimization for less common hosts, including Bacillus, Pichia pastoris, and insect cell systems.
Q: What quality control measures are included?

A: Each synthesized gene is verified by full-length Sanger sequencing and restriction digestion analysis prior to delivery.
Q: Can the synthesized gene be directly used for site-directed mutagenesis?

A: Yes. Our constructs are delivered as mutation-ready templates, fully compatible with our SDM workflows.
Q: What if I need multiple variants synthesized simultaneously?

A: We offer parallel synthesis and combinatorial design options for high-throughput mutagenesis projects, allowing efficient production of variant libraries.

References:

Allemandou F, Nussberger J, Brunner HR, Brakch N. Rapid site‐directed mutagenesis using two‐PCR‐generated DNA fragments reproducing the plasmid template. BioMed Research International. 2003;2003(3):202-207. doi:10.1155/S1110724303209141

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Project Description:

For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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