pAD-GamS
(Plasmid #99246)

• Purpose: Expresses 6xHis-tagged GamS from T7lac promoter in the E.coli Tuner (DE3) strain. See notes below for important strain information.
• Depositing Lab: Jeff Hasty
• Publication: Didovyk et al ACS Synth Biol. 2017 Aug 10. doi: 10.1021/acssynbio.7b00253.

Backbone

• Vector backbone: pRSF-1b
• Backbone manufacturer: Novagen / EMD Millipore
• Total vector size (bp): 3795
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Mach1
• Growth instructions: This plasmid is now distributed in Mach1 cells as of August 2021 for amplification. The plasmid should be transformed into E.coli Tuner (DE3) for protein expression.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: gamS (with C-terminal 6xHis tag)
• Alt name: gamS fragment of phage lambda gam gene
• Species: phage lambda
• Insert Size (bp): 321
• Promoter: T7lac
• Tag / Fusion Protein:
  • 6xHis tag (C terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: TAA TAC GAC TCA CTA TAG GG

Resource Information

• Supplemental Documents:
  • pAD-GamS.gb
• A portion of this plasmid was derived from a plasmid made by: GamS gene was subcloned from a plasmid ParaBAD-gamS that was a kind gift from Zachary Z. Sun (see Sun et al. "Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system." ACS synthetic biology (2013)).

Terms and Licenses

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

Depositor Comments

The plasmid is for high level expression of 6xHis tagged GamS in E.coli Tuner (DE3) cells. IMPORTANT NOTE: the E. coli strain carrying this plasmid has changed since the original submission (as of August 2021). The plasmid is now distributed in a cloning strain E.coli Mach1 and must be subsequently transformed into E.coli Tuner (DE3) for protein expression.

This plasmid has been found to be prone to multimerization. Plasmid multimerization often does not impact plasmid function, but may reduce transformation efficiencies. You may need to screen multiple colonies to isolate the monomeric version of this plasmid. If you still have trouble isolating the monomeric version, you might consider linearizing, gel extracting, re-ligating, and transforming the plasmid.