pMYO127TAG-SepT
(Plasmid #34625)

• Depositing Labs: Jesse Rinehart, Dieter Söll
• Publication: Park et al Science. 2011 Aug 26;333(6046):1151-4.

Backbone

• Vector backbone: pSepT
• Modifications to backbone: To construct plasmid pSepT, the full-length gene encoding tRNA^Sep was constructed from overlapping oligonucleotides and ligated immediately downstream of the lpp promoter in pTECH (Bunjun et al., 2000) using EcoRI and BamHI restriction sites.
• Vector type: Bacterial Expression

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: Myoglobin-127TAG
• Species: Sperm Whale: Physeter macrocephalus
• Insert Size (bp): 492
• Mutation: codon-optimized + an amber stop codon was introduced to the myoglobin gene at position Asp127
• Promoter: Ipp
• Tag / Fusion Protein:
  • 6xHIS (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NotI (not destroyed)
• 3′ cloning site: BglII (not destroyed)
• 5′ sequencing primer: pJR_F1
• 3′ sequencing primer: M13 forward

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest

Terms and Licenses

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

Depositor Comments

To prevent possible enzymatic dephosphorylation of O-phospho-L-serine (Sep) in vivo, the gene encoding phosphoserine phosphatase (serB), which is catalyzing the last step in serine biosynthesis, was deleted from Escherichia coli strains Top10 (Top10∆serB - Addgene #34928) and BL21 (BL21∆serB - Addgene #34929). These strains are required hosts when using this plasmid.