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Cloning and Mutagenesis
Zhiyin Biotechnology provides customized gene cloning and site-directed mutagenesis services. With no restriction-site limitations, cloning into any vector and at any position is supported.
Services cover base substitutions, insertions, deletions, and combined mutations. Sanger sequencing verifies sequence accuracy, with delivery in as few as 1–7 calendar days.
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  • 1-7days

    Fastest delivery time

  • Any vector

    Flexible cloning position

  • 100%

    Sequence verification accuracy

  • 200+

    Complimentary in-stock vectors

首页 > Products > Gene Synthesis > Cloning and Mutagenesis >
Introduction
Service Selection Guide
Service Advantages
Service Details
Service Workflow
Information Required from Customers
Delivery Standards
Applications
FAQ
Gene Cloning
Site-Directed Mutagenesis
Cloning + Mutagenesis Package

Gene Cloning / Subcloning

Precise Cloning Independent of Restriction Sites

Traditional restriction-enzyme cloning is constrained by available sites in the vector and target fragment. We use homologous recombination cloning technology,without relying on vector restriction sites, enabling precise cloning of the target gene into a specified position in virtually any vector.

Target fragments can be amplified from plasmid DNA, genomic DNA, cDNA, or PCR products. Batch subcloning of the same gene into different vectors is also supported.

  • Precise insertion with flexible templates, vectors, and positions
  • High-throughput parallel subcloning of one fragment into multiple vectors
  • Use your own vector or select from 200+ vectors at no additional charge
Homologous Recombination Gene Cloning Diagram

Site-Directed Mutagenesis

Precise Modification to Reveal Gene Function

Precise base substitutions, insertions, or deletions in a target DNA sequence allow researchers to link specific amino acids to protein function, validate key domains, or optimize biochemical properties. We supportsingle-point, multi-point, insertion, and deletion mutationsand other mutation types.

  • Base Substitution:Single- or multi-point substitution
  • Insertion Mutation:Insert a tag, restriction site, or functional sequence
  • Deletion Mutation:Delete specified bases or a domain fragment
  • Combined Mutations:Flexible combination of multiple mutation types
Site-Directed Mutagenesis Diagram

Cloning + Mutagenesis Package

One-stop sequence construction and precise modification

For projects requiring both cloning and mutagenesis of a target gene, we provide a one-stop service covering sequence design, gene acquisition, vector cloning, targeted modification, and Sanger sequencing verification.

  • Reduce repeated deliveries and communication costs
  • Shorter overall turnaround and better total cost
  • Unified QC standards for consistent data
Combined Cloning and Mutagenesis Service Diagram

How to Choose: Cloning vs. Cloning + Mutagenesis Package

Select the service that best matches your project requirements
Cloning-Only Service

Customer provides template DNA + target vector:

  • Target fragment and vector are already available
  • Only amplification and cloning are required
  • No mutation needs to be introduced
  • Straightforward project with a shorter turnaround
  • Lowest cost option
  • Suitable for routine subcloning
Cloning + Mutagenesis Package

Provide the mutation requirements and receive one-stop delivery:

  • The target gene requires both cloning and mutagenesis
  • Seamless gene synthesis → cloning → mutagenesis workflow
  • Reduce communication and time costs from multiple deliveries
  • Shorter total turnaround and better overall cost
  • Unified QC standards and consistent data
  • Suitable for integrated projects beginning with sequence design

Service Advantages

Cloning and mutagenesis supported by a mature molecular cloning platform and extensive project experience under agreed project conditions
  • Broad Vector Compatibility
    Homologous recombination supports diverse vectors and insertion positions, with the final plan determined after vector and sequence assessment.
  • Customized Project Design
    For special vectors, toxic genes, large-fragment cloning, and other specific requirements, one-to-one technical planning is provided and the project plan is confirmed after assessment.
  • Comprehensive Sanger Sequencing
    All cloning and mutagenesis projects undergo comprehensive Sanger sequencing verification covering the insert or mutation region, key vector-backbone elements, and the agreed verification scope.
  • Rapid Delivery
    A mature standardized workflow supports delivery of conventional cloning projects (<2 kb) in as little as 1–7 calendar days. Expedited service may further shorten the timeline for urgent experiments.
  • Extensive Template-Handling Experience
    Projects can start from plasmid DNA, genomic DNA, cDNA, PCR products, bacterial cultures, colonies, and other materials, accommodating diverse sample conditions.
  • Strict Confidentiality
    Customer sequences, vector information, and project data are managed under strict confidentiality. A formal nondisclosure agreement (NDA) can be signed to protect intellectual property.

Service Details

Gene cloning and site-directed mutagenesis options with standard turnaround times
Gene Cloning:Target fragments can be amplified from plasmid DNA, genomic DNA, cDNA, or PCR products. Batch subcloning of the same gene into different vectors is also supported.
Sequence Type Length Turnaround Time (Calendar Days) Application
Conventional Sequence
 0 - 2 kb 1 - 7 Standard gene cloning with rapid delivery
2 kb - 3 kb 7 - 12 Cloning of larger gene fragments
3 kb - 4.5 kb 12 - 15 Long-fragment cloning with segmented assembly and verification
4.5 kb - 5 kb 15 - 20 Large-fragment cloning requiring multiple steps
> 5 kb
Contact Us
 Ultra-large fragments assessed according to the specific sequence
Challenging Sequence
 1 kb - 5 kb
Contact Us
 High GC content, repeats, toxic genes, and similar features
Site-Directed Mutagenesis:Supports base substitutions, insertion mutations, deletion mutations, and combined mutations.
Number of Mutations Length Turnaround Time (Calendar Days) Notes
1 Mutation
 0 - 2 kb 1 - 7 Rapid mutagenesis with the shortest turnaround
2 kb - 3 kb 7 - 12 Medium-size plasmid mutagenesis
3 kb - 4.5 kb 12 - 15 Larger plasmid mutagenesis
4.5 kb - 5 kb 15 - 20 Large plasmid mutagenesis
>5 kb or challenging sequence
Contact Us
 Assessed according to the sequence and mutation position
2 Mutations
 0 - 2 kb 1 - 10 Two-site mutagenesis requiring two rounds of work
2 kb - 3 kb 10 - 15 Two-site mutagenesis in a medium-size plasmid
3 kb - 4.5 kb 15 - 20 Two-site mutagenesis in a larger plasmid
4.5 kb - 5 kb 20 - 25 Two-site mutagenesis in a large plasmid
>5 kb or challenging sequence
Contact Us
 Site spacing and complexity require assessment
Multiple Mutations
Contact Us
 Three or more mutations, assessed according to the total number

The above timelines are based on standard commercial vectors and conventional E. coli hosts. Timelines for special vectors or hosts are assessed for each project.

Service Workflow

Transparent, traceable workflow from requirement submission to plasmid delivery
  • Submit Requirements
    Provide the template sequence, vector information, and cloning or mutation requirements.
  • Project Assessment
    Complete feasibility analysis, project design, and quotation within 1 business day.
  • Experimental Execution
    PCR amplification or mutagenesis, vector construction, transformation, positive-clone screening, and sequencing verification.
  • Delivery and After-Sales Support
    Deliver plasmid DNA, glycerol stocks, sequencing reports, and QC documents with technical support.

Information Required from Customers

Complete information helps the project start promptly
  • For Cloning Projects
    · Complete target-gene sequence;
    · Template material (plasmid DNA ≥1 μg, glycerol stock, PCR product, or genomic DNA);
    · Target vector name, map, and resistance marker;
    · Desired cloning site or insertion position. When using your own vector, provide sequencing results or mail the plasmid.
  • For Mutagenesis Projects
    · Complete template-plasmid sequence;
    · Plasmid DNA ≥2 μg (recommended) or a glycerol stock;
    · Mutation position, original base, and target base;
    · For insertion mutations, provide the insert sequence; for deletions, specify the start and end positions;
    · Confirm the vector resistance marker.

Delivery Standards

Every cloning and mutagenesis project is delivered to defined standards for prompt downstream use
  • Plasmid DNA
    Deliver 5 μg of high-purity plasmid DNA containing the target fragment, available as lyophilized powder for storage and shipping or as a liquid.
  • Glycerol Stock (Optional)
    Provide a glycerol stock containing the correct plasmid (E. coli DH5α or a specified host), stored at −80°C for recovery when needed.
  • Standard Sequence Files
    Provide the final plasmid sequence in FASTA/GenBank format with complete annotation of the insert, mutation sites, and key vector elements.
  • Sequencing Results
    Provide raw Sanger sequencing chromatograms (.ab1) and a complete sequence alignment report, including verification of the mutated region.
  • Plasmid Map
    Provide a fully annotated plasmid map showing the origin of replication, resistance gene, promoter, multiple cloning site, insert, and mutation sites.
  • COA Quality Report
    Provide a Certificate of Analysis recording key parameters such as plasmid concentration, purity (A260/A280 ratio), and delivery amount.

Applications

Broad applications of gene cloning and site-directed mutagenesis in molecular biology
Expression Vector Construction

Expression Vector Construction

Clone the target gene into an appropriate expression vector for protein expression, mammalian-cell expression, purification, or functional experiments.

Vector System Conversion

Vector System Conversion

Subclone the same target gene between different vector systems, such as from a cloning vector to an expression vector or from a prokaryotic vector to a eukaryotic vector.

Tag Fusion

Tag Fusion

Add fluorescent proteins such as GFP/mCherry or purification tags such as His/Flag/HA/GST to the N- or C-terminus of the target gene for detection and purification.

Functional-Site Validation

Functional-Site Validation

Use site-directed mutagenesis to alter key amino acids, such as catalytic triads, phosphorylation sites, or binding sites, and evaluate their effects on protein function.

Activity Optimization

Activity Optimization

Introduce specific mutations to alter enzyme substrate specificity, catalytic efficiency, thermal stability, or pH tolerance for protein engineering.

Truncation Construct Design

Truncation Construct Design

Create protein truncations through deletion mutations for domain mapping, minimum functional-region identification, and crystallization optimization.

Promoter / Regulatory Element Analysis

Promoter / Regulatory Element Analysis

Clone promoter fragments of different lengths into reporter vectors or alter transcription-factor binding sites by mutagenesis to study gene-expression regulation.

Viral Vector Construction

Viral Vector Construction

Clone the target gene into lentiviral, AAV, adenoviral, or other viral expression vectors for gene delivery and gene-therapy research.

FAQ

Common questions about cloning technology, templates, mutation design, and plasmid use
How does your cloning technology differ from restriction-enzyme cloning?
Traditional restriction-enzyme cloning requires matching restriction sites in the target fragment and vector and usually involves multiple digestion, ligation, and transformation steps. We use homologous recombination cloning:

· Independent of vector restriction sites, allowing insertion at a selected position
· Completed through a single recombination step without multi-step ligation
· Particularly suitable for mismatched restriction sites or large-fragment cloning
· High positive-clone rate, reducing screening work
Can I use my own vector, and what information is required?
Yes. Please provide:

· Complete vector sequence (a sequence verified by sequencing is recommended)
· Vector map or plasmid information (resistance marker, copy number, etc.)
· Physical plasmid or glycerol stock if sequence information is incomplete
· Desired insertion position

If you do not have a suitable vector, you may select from our library of 200+ vectors at no additional charge.
What is the success rate of site-directed mutagenesis?
For conventional sequences with normal GC content and no complex secondary structure, single-point mutagenesis has a success rate close to 100%. Multi-point mutations or complex templates are assessed in advance and an appropriate plan is discussed. All mutation results are verified by Sanger sequencing before delivery. If a correct clone cannot be obtained under normal experimental conditions, we will discuss an alternative solution.
How is one mutation unit defined?
One mutation unit is defined as any number of base changes within the coverage of a single primer, approximately a 30 bp window. For example:

· Single-base substitution = 1 mutation
· Three consecutive base substitutions (changing one codon) = 1 mutation
· Insertion of a 10 bp tag sequence = 1 insertion mutation
· Deletion of a 20 bp domain fragment = 1 deletion mutation

If two mutation sites are far apart and require two independently designed primer pairs, they count as two mutations.
What should I do if my template sequence is uncertain or has not been sequenced?
We recommend whole-plasmid sequencing of the template before cloning or mutagenesis. Whole-plasmid sequencing is available as an upstream service. Performing PCR cloning or site-directed mutagenesis with an uncertain template sequence risks errors caused by pre-existing template mutations.
Can multiple mutations be introduced in the same project?
Yes. We support single-point, double-point, and multiple combined mutations. For three or more mutations, two strategies are available:

· Sequential mutagenesis: introduce mutations one at a time and verify each step before proceeding. Suitable for widely separated sites; the process is longer but each step is controlled.
· Gene synthesis + mutagenesis: when many mutations are required, directly synthesize a new gene fragment containing all mutation sites to replace the original sequence, which may shorten turnaround and improve cost efficiency.

Contact technical support for a recommendation based on your project requirements.
Can the delivered plasmid be used directly for transfection or transformation?
Yes. The delivered plasmid DNA meets molecular biology–grade purity requirements (A260/A280 ≈ 1.8–1.9) and can be used directly for:

· E. coli transformation
· Mammalian-cell transfection (standard grade)
· PCR amplification (as a template)
· Restriction digestion analysis
· In vitro transcription (IVT)

For endotoxin-free (transfection-grade) or in vivo injection-grade plasmids, contact us to select anupgraded preparation service。

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