pKM495 (Flag-Das+4) Zeo
(Plasmid #109284)

• Purpose: ORBIT integrating plasmid for C-terminal tagging with degradation tag DAS+4, using zeocin as a selection marker.
• Depositing Lab: Kenan Murphy
• Publication: Murphy et al MBio. 2018 Dec 11;9(6). pii: mBio.01467-18. doi: 10.1128/mBio.01467-18.

Backbone

• Vector backbone: pKM446
• Backbone size w/o insert (bp): 3139
• Total vector size (bp): 3159
• Modifications to backbone: Replacement of Hyg-resistance gene with Zeo-resistance gene by recombineering; insertion of PGroEL to read downstream gene following chromosomal insertion.
• Vector type: ORBIT integrating plasmid
• Selectable markers: Zeocin

Growth in Bacteria

• Bacterial Resistance(s): Chloramphenicol, 25 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: attB-Flag-DAS+4
• Species: Synthetic
• Insert Size (bp): 109

Terms and Licenses

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

Depositor Comments

ORBIT plasmid for creating Flag-DAS4+ fusions to mycobacterial target genes, using zeocin as a resistance marker.

Strain should be grown in 20 ug/ml chloramphenicol for isolation of plasmid. Yields with Qiagen miniprep kits are typically in the range from 60-90 ug/ml from 5 ml culture.