Sequence design guidelines

Direct Cloning

Vectors indicated by are available for direct cloning which eliminates the need for subcloning and saves up to 6 business days for production.

To qualify for direct cloning, the provided insert sequence must meet the following criteria

• For standard, non-complex sequences up to 5kb
  • GC content between 20% and 80% in 41bp sliding window
  • Direct/inverted repeats, secondary structure identity-score ≤18 in 20bp sliding window
  • Free of homopolymers ≥8bp
• For complex sequences the accepted insert length will be shorter and depends on the length of the complex region
• Type IIs enzyme recognition sites are not present in the insert
  • Primarily AarI[CACCTGC] or BsaI[GGTCTC]
  • Absence of BbsI[GAAGAC], BsmBI[CGTCTC] is also beneficial

Note: Optimizing the insert sequence using the GeneOptimizer algorithm may help to enable direct cloning in cases where these criteria are not initially met.

If any of the criteria above are not met, standard subcloning will be used to ensure delivery in your chosen vector.

Usage and cloning guidelines

For more information on how to use a vector, you can hover the cursor over the next to the selected vector.

For more information on the vector, you can also click on the “View vector info” below the dropdown of the selected vector.

Please note detailed descriptions on our standard GeneArt cloning vector (pMX) below.

Insertion Points

Some vectors allow you flexibility in choosing where your gene of interest is inserted into the vector. In these cases, a dropdown with “Select insertion points” will appear where you can define the 5’- and 3’-flanking cloning sites for your insert sequence.

Please note that these enzyme recognition sites must not be present in your gene of interest.

Standard GeneArt cloning vector – pMX series

These are our standard production vectors serving for downstream cloning purposes. Your gene of interest will be delivered in that vector with options to choose between resistances. No addition on turnaround time for subcloning at all!

• pMA – Ampicillin resistance
• pMK – Kanamycin resistance

pMX selection will synthesize the insert in any of our standard cloning vectors depending on the most suitable manufacturing pathway.