DNA Fragment Splitter for Gibson Assembly

Split large DNA inserts into overlapping fragments for seamless Gibson assembly

How It Works

This tool splits large DNA inserts into smaller overlapping fragments to overcome synthesis size limitations and complexity constraints. Split your insert to match your synthesis provider's capabilities, then seamlessly reassemble using Gibson assembly.

Inputs: Insert DNA (required) + optional vector for circular constructs
Split Modes: By target fragment size (bp) OR by number of fragments
Outputs: FASTA file (fragments to order) + GenBank file (final assembled construct)
Features: GC-optimized junction placement • Overlap validation • Self-circularization option

Disclaimer: This tool assists with fragment design but does not guarantee assembly success. Actual assembly efficiency depends on fragment quality, handling, and reaction conditions.

Construct name

Used in FASTA record IDs and download filenames (e.g., PLASMID01_1, PLASMID01_2...)

Insert DNA sequence (IUPAC accepted: A T G C R Y S W K M B D H V N)

Self-circularize insert (adds 3′→5′ wrap-around overlap)

Automatically disabled when a vector sequence is provided below.

Optional vector DNA sequence (5'→3')

Vector creates a circular construct. The tool adds overhangs: 5′ end gets vector's last overlap bp, 3′ end gets vector's first overlap bp.

💡 Synthesis Vendor Guidelines: Typical DNA synthesis constraints are ~200 bp (lower limit) to ~3-5 kb (upper limit), with ~400-1000 bp being the optimal range for yield, cost, quality, and turnaround time. Fidelity is highly sequence-depedent. Note that splitting fragments in different ways can result in lower or higher sequence complexity—try a few different designs in your vendor's portal to test complexity and find what works best.

Choose split mode

Fragment size (bp)

Overlap size (bp)

FASTA Output

Fragment sequences will appear here after generation...

Questions about fragment design or ordering?