pPEE23
(Plasmid #128366)

• Purpose: (Empty Backbone) Dominant marker in Cryptococcus neoformans. Targets DNA constructs to a Safe Haven Site 7, a small gene-free region. Contains the amdS2 counterselectable marker.
• Depositing Lab: James Fraser
• Publication: Erpf et al Fungal Genet Biol. 2019 Jun 17;131:103241. doi: 10.1016/j.fgb.2019.103241.

Backbone

• Vector backbone: pBlueScript II SK(-)
• Backbone manufacturer: Stratagene
• Vector type: Unspecified
• Selectable markers: Acetamide, Fluoroacetamide

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Acetamidase (amdS)
• Species: Synthetic
• Promoter: TEF1

Cloning Information

• Cloning method: Gibson Cloning

Resource Information

• Supplemental Documents:
  • pPEE23.gb

Terms and Licenses

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

Depositor Comments

Contains the Safe Haven Site 7 sequence (CNAG_05557 - CNAG_05558) 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.