PHUSER

PHUSER automates primer design for USER fusion cloning to produce PCR-optimized primers that enable directional assembly of multiple DNA fragments into plasmids using customizable USER cassettes.

Key Features:

• Optimized Primer Design: Automated design of PCR-optimized primers that target directional fusion into plasmids containing customizable USER cassettes and aim for similar annealing temperatures (Tm) across primers.
• Avoidance of Assembly Errors: Prevents creation of identical overhangs among primers to ensure correct sequential assembly of DNA fragments during USER fusion.
• Comprehensive Primer Analysis: Performs per-primer analyses including GC content, 3'-end GC clamp, primer-dimer risk assessment, intra-primer complementarity, and detection of polyN stretches.
• Flexible Design Options: Supports insertion of linkers between DNA fragments and configurable USER cassette arrangements.
• Experimental Validation: Has been experimentally validated for primer design in USER fusion applications.

Scientific Applications:

• USER fusion primer design: Design of primers for USER fusion assembly of multiple DNA fragments into plasmids.
• Genetic engineering and synthetic biology: Assembly of genetic constructs in genetic engineering and synthetic biology projects.
• Molecular cloning workflows: Molecular cloning workflows requiring directional assembly, linker insertion, or precise overhang specification.

Methodology:

Automated primer generation with Tm optimization, overhang uniqueness checks, and computational analyses of GC content, 3'-end GC clamp, primer-dimer risk, intra-primer complementarity, and polyN stretches, with optional linker insertion and customizable USER cassette specification.