natMX6-ins5
(Plasmid #195042)

• Purpose: pFA6a derived selection cassette flanked with transcription terminators (tDEG1/tCYC1), allows genome modification without disruption of insertion neighboring genes by transcription interference
• Depositing Lab: Gloria Brar
• Publication: Powers et al Elife. 2022 Dec 12;11:e81086. doi: 10.7554/eLife.81086.

Backbone

• Vector backbone: pFA6a
• Backbone size w/o insert (bp): 2458
• Total vector size (bp): 3973
• Vector type: yeast genomic targeting
• Selectable markers: Nourseothricin (Clonat)

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: NatR
• Alt name: Nourseothricin resistance cassette
• Alt name: confers resistance to Nourseothricin (Clonat)
• Species: S. cerevisiae (budding yeast); A. gossypii
• Insert Size (bp): 1515
• Tag / Fusion Protein:
  • S. cerevisiae DEG1 terminator, A. gossypii TEF promoter, NatR, A. gossypii TEF terminator, S. cerevisiae CYC1 terminator

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: SP6
• 3′ sequencing primer: T7

Resource Information

• Supplemental Documents:
  • natMX6-ins5.gb
• A portion of this plasmid was derived from a plasmid made by: Longtine et al., 1998

Terms and Licenses

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

Depositor Comments

This plasmid can be used in place of the original pFA6a series to introduce targeted mutation by selectable marker insertion in yeast while preventing off-target disruption of insertion neighboring genes caused by cassette promoter driven transcription interference.

For more information regarding these plasmids see our article (Powers et al., eLife 2022) "Bidirectional promoter activity from expression cassettes can drive off-target repression of neighboring gene translation"

The backbone and KanMX cassette was amplified from pFA6a-kanMX6 (Longtine et al., 1998), the tDEG1 and tCYC1 sequences were amplified from S. cerevisiae gDNA.