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Seamless Cloning Kit
The ZY Fusion One-Step Seamless Cloning Kit is based on one-step Gibson Assembly homologous recombination. Precise assembly of 2–3 fragments is completed in 20 minutes at 50°C.
It supports seamless directional cloning of 2–6 DNA fragments and inserts ranging from 50 bp to 10 kb.
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  • 20min

    Reaction time for 2–3 fragments

  • 2-6fragments

    Simultaneous multi-fragment assembly

  • 50bp-10kb

    Compatible insert range

  • Approx. 3.5h

    Complete workflow from PCR to plating

首页 > Products > Molecular Biology Reagents > Seamless Cloning Kit >
Product Overview
Ordering Information
Product Highlights
Product Specifications
Technical Principle
Procedure
Primer Design Guidelines
Applications
Frequently Asked Questions
Product Overview
Fast Seamless Assembly

ZY Fusion One-Step Seamless Cloning Kit

One-step homologous recombination, gene splicing in one step

Based on the principle of one-step homologous recombination (Gibson Assembly), the activities of DNA exonuclease, DNA polymerase, and DNA ligase are integrated into one system.

Simply mix the linearized vector with PCR fragments carrying 15–25 bp homologous arms at both ends, react at 50°C for 20 minutes, and precise single- or multi-fragment cloning can be completed without introducing extra bases at the junctions.

  • Compatible with insert fragments of 50 bp–10 kb
  • Supports simultaneous assembly of 2–6 DNA fragments
  • The entire process from PCR to plating takes approximately 3.5 hours
ZY Fusion Seamless Cloning

Fast Seamless Assembly

Eliminates tedious steps such as restriction digestion and ligation

2–3 fragments can be precisely assembled by incubating at 50°C for 20 minutes; for 4–6 fragments, extend to 60 minutes. The recombinant product does not introduce additional sequences and can be directly transformed into competent cells.

  • No restriction site limitations
  • No need for ligation, dephosphorylation, or other multi-step operations
  • Supports directional insertion into any vector at any site
  • Positive rate nearly 100%
Fast seamless assembly reaction

Ordering Information

Single-step seamless cloning of single or multiple fragments
Product Name Catalog No. Size Applicable scenarios Price
ZY Fusion one step
Seamless Cloning Kit		 ZY-Fus-20 20T Gene cloning, expression vector construction, reporter gene assembly,
Synthetic biology multi-fragment assembly;
Compatible with 50 bp–10 kb fragments
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Product Highlights

High efficiency and speed, extremely simple operation, wide compatibility, seamless precision, multi-fragment parallel
  • High efficiency and speed
    Reactions at 50°C complete precise assembly of 2–3 fragments in 20 minutes and splicing of 4–6 fragments in 60 minutes, with a positive rate close to 100%.
  • Extremely simple operation
    Eliminates multiple steps such as restriction digestion, ligation, dephosphorylation, and gel recovery, completing directional cloning in a single reaction.
  • Wide compatibility
    No restriction site requirements, compatible with common cloning vectors; efficiently clones insert fragments ranging from 50 bp to 10 kb.
  • Seamless and precise
    Recombinant products introduce no additional sequences, achieving truly scarless cloning.
  • Multiple fragment parallel
    Supports simultaneous assembly of 2–6 DNA fragments in a single reaction, meeting the needs of synthetic biology and complex vector construction.

Product Specifications

Product composition, storage and transportation, and reaction conditions
ItemContent
Product composition2× ZY-Fusion Master Mix（200 μL）
Positive linearized pUC Vector（250 ng，25 μL）
Positive Control Insert（500 ng，25 μL）
Storage and transportationStore at -20°C, transport with ice packs; avoid repeated freeze-thaw cycles
Validity period2 years
Reaction conditions50℃ incubation for 20 min (2–3 fragments) / 60 min (4–6 fragments)
Scope of ApplicationDirectional cloning and seamless assembly from a single fragment to 4–6 fragments
Statement of Use for Scientific ResearchThis product is for scientific research use only and may not be used for clinical diagnosis or treatment

Technical Principle

The three enzyme activities of Gibson Assembly work together under isothermal conditions at 50°C to complete seamless assembly.
  • DNA 5' exonuclease:Degrades the ends of linearized vectors and insert fragments to produce 3' single-stranded overhangs.
  • DNA polymerase:Fills the gaps between annealed fragments.
  • DNA ligase:Seals the nicks in the assembled DNA, forming a closed double-stranded circular plasmid.

Only ends carrying homologous sequences can undergo complementary pairing, effectively preventing non-specific recombination; the ligated products can be directly transformed into competent cells.

Procedure

From vector and fragment preparation to recombination, transformation, and clone identification, the whole process takes about 3.5 hours.
  • Preparation of Vectors and Fragments
    Prepare linearized vectors by restriction digestion or reverse PCR; introduce 15–25 bp homologous arms at both ends of PCR fragments.
  • Prepare the recombination reaction
    Mix the vector and insert fragment in proportion, add 2× ZY-Fusion Master Mix, and bring the volume up to 20 μL.
  • Incubate isothermally at 50℃
    Incubate 2–3 fragments for 20 min; 4–6 fragments for 60 min.
  • Transformation and identification
    Transform competent cells, plate and culture overnight, perform colony PCR or restriction digestion to identify positive clones.

Detailed operational steps

Complete vector construction in 6 steps
Steps Key points of operation
1. Prepare the linearized vector Digest with restriction endonucleases (dual digestion + gel recovery recommended) or use inverse PCR; for PCR, it is recommended to digest the methylated template plasmid with Dpn I
2. Prepare the insert fragment Introduce 15–25 bp homologous sequences at the 5' end of the primers, amplify using high-fidelity polymerase, and purify
3. Set up the recombination reaction Mix the linearized vector and insert fragment proportionally, add 2× ZY-Fusion Master Mix, and adjust with ultrapure water to 20 μL
4. Incubate isothermally 50℃ for 20 min (2–3 fragments) or 60 min (4–6 fragments); if cannot convert in time, temporarily store at -20℃
5. Transformation culture Transform competent cells, plate on selective media, incubate at 37℃ overnight
6. Clone identification Colony PCR or restriction enzyme digestion for identification; it is recommended to set up a negative control to avoid false positives

Recommended reaction system

Suggested molar ratio of insert fragment to linearized vector is 2:1–3:1; if insert fragment is <200 bp, ≥5:1 is recommended
Components 2–3 fragment assembly 4–6 fragment assembly Positive control
PCR product + linearized vector plasmid X μL（0.02–0.5 pmol） X μL（0.2–1 pmol） 10 μL
2× ZY-Fusion Master Mix 10 μL 10 μL 10 μL
Ultra-pure H₂O Bring to 20 μL Bring to 20 μL
Total volume 20 μL 20 μL 20 μL

Primer Design Guidelines

Reasonably design homologous arms and amplification primers to improve recombination efficiency and specificity.
  • Homologous arm length
    Recommended 15–25 bp, with a suggested Tm value ≥48℃; can be estimated using A-T=2℃, G-C=4℃.
  • 3' end of primers
    Avoid three or more consecutive identical bases, avoid the degenerate third position of codons, and prevent primer dimers.
  • Primer overall
    The Tm difference between forward and reverse primers should be ≤2℃, avoiding the formation of more than 4 consecutive pairings within the primer or between primers, and avoiding hairpin structures.
  • Double-digestion vector
    Restriction sites can be retained at the ends of homologous arms, so the recombinant plasmid still retains these sites, facilitating subsequent subcloning.
  • Design tools
    It is recommended to use tools such as SnapGene, Benchling, or NEB NEBuilder to assist in designing overlapping sequences.

Applications

Covers conventional cloning, expression vectors, reporter systems, multi-fragment assembly, and broad fragment compatibility
Gene Cloning

Gene Cloning

Precisely inserts the target DNA fragment into any vector site without relying on restriction sites and without introducing extra sequences.

Expression Vector Construction

Quickly completes seamless assembly of ORFs and expression vectors, providing efficient tools for protein expression and functional research.

Reporter Gene Vector Assembly

Reporter Gene Vector Assembly

Precisely splice promoters, reporter genes, and regulatory elements to construct reporter systems and functional validation vectors.

Multi-fragment splicing

Synthetic biology multi-fragment splicing

Supports splicing 2–6 DNA fragments in a single reaction, suitable for complex vectors and synthetic biology assembly.

Wide fragment compatibility

Compatible with small fragments of 50 bp to large fragments of 10 kb, allowing insertion of target fragments into any position of common vectors.

Frequently Asked Questions

Common questions regarding cloning methods, homologous arms, false positives, DNA quality, storage, and multi-fragment assembly
What is the difference between seamless cloning and traditional restriction enzyme ligation methods?
Seamless cloning does not require restriction sites or ligase. Directed cloning is completed in a single reaction through homologous recombination without introducing extra sequences. It allows parallel multi-fragment assembly, has a higher positive rate, and requires less operation time.
How long should the homologous arms be?
It is recommended to be 15–25 bp with Tm ≥48°C. If the arms are too short, recombination efficiency will decrease; if too long, it may affect PCR amplification specificity.
The false positive rate is very high. What should I do if I can't pick colonies containing the target gene?
Check whether the vector is fully linearized. You can increase the amount of enzyme, extend the digestion time, and purify by gel recovery; check whether the antibiotic on the plate has lost potency, and use freshly prepared antibiotic plates; for single-enzyme digestion, it is recommended to perform dephosphorylation.
Why does colony PCR show a band, but no band is detected after plasmid extraction?
It is possible that non-specific bands appeared during PCR amplification of the target gene, or the vector/fragment contains homologous repeat sequences causing recombination disorder. It is recommended to purify the product by gel recovery, optimize PCR conditions, and pick multiple colonies for plasmid extraction verification.
Do the quality of the linearized vector and the insert fragment affect recombination?
Yes. Insufficient purity or concentration can significantly reduce efficiency. It is recommended that A260/A280 = 1.6–1.9, A260/230 ≈ 2, and concentration > 50 ng/μL; perform gel recovery purification if necessary.
How long can the reaction product be stored?
If the reaction at 50°C is completed but cannot be transformed immediately, the reaction tube can be temporarily stored at -20°C to avoid repeated freeze-thawing.
Can the ZY Fusion kit be used for multi-fragment assembly?
Yes. This product supports the assembly of 2–6 fragments in one reaction; the more fragments, the more it is recommended to increase the total amount of DNA appropriately and extend the reaction time to 60 minutes.

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