pSDMA25
(Plasmid #67940)

• Purpose: (Empty Backbone) Complementation in Cryptococcus neoformans. Targets DNA constructs to a Safe Haven Site, a small gene-free region. Contains nourseothricin resistance marker (NAT)
• Depositing Lab: James Fraser
• Publication: Arras et al PLoS One. 2015 Apr 9;10(4):e0122916. doi: 10.1371/journal.pone.0122916. eCollection 2015.

Backbone

• Vector backbone: pBluescript II -SK
• Backbone manufacturer: Stratagene
• Modifications to backbone: Added Safe Haven gene free region (intergenic region between H99 ORFs CNAG_00777 and CNAG_00778) and NAT resistance marker and removed F1 origin
• Vector type: Cryptococcal complementation vector
• Promoter: ACT1 for Cryptococcal marker
• Selectable markers: Nourseothricin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: M13F
• 3′ sequencing primer: M13 Reverse

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Contains the safe haven site sequence in the backbone of pBluescript II SK- to enable integration into the C. neoformans genome via homologous recombination, in addition to a linker sequence containing multiple rare-cutting restriction enzyme cut sites to facilitate linearization prior to biolistic transformation. Importantly, as the safe haven and marker fragments were inserted into the pBluescript II SK- backbone in place of the f1 origin of replication, each plasmid still retains the original multicloning site and blue/white screening capabilities. Linearization of the safe haven constructs is made possible by the inclusion of a linker containing cut sites for three rare cutting restriction enzymes—BaeI, AscI and PacI. To use the vectors, the gene of interest is subcloned into the standard pBluescript II SK- multicloning site.
If possible BaeI should be used, as it integrates into the safe haven site approximately 60% of the time, while AscI and PacI only 10-16%.