pstb-LAFR5
(Plasmid #86169)

• Purpose: (Empty Backbone) pLAFR5 with Smb21651 promoter. pLAFR5 is a broad-host range cosmid vector with double cos sites.
• Depositing Lab: Kay Jones
• Publication: Jones et al J Bacteriol. 2012 Aug;194(16):4322-31. doi: 10.1128/JB.00751-12. Epub 2012 Jun 8.

Backbone

• Vector backbone: pLAFR5
• Backbone manufacturer: Keen et al. (1988) Gene 70:191-197
• Backbone size (bp): 22000
• Modifications to backbone: added SMb21651 promoter
• Vector type: Bacterial Expression
• Promoter: SMb21651 promoter

Growth in Bacteria

• Bacterial Resistance(s): Tetracycline, 10 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH10B
• Copy number: Unknown

Cloning Information

• Cloning method: Restriction Enzyme

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Industry MTA

Depositor Comments

For expression in S. meliloti, this is the information on its construction and for cloning ORFs into it.
1) This is the sequence of the SMb21651 promoter fragment (reverse comp of 1683333-1682932) promoter fragment from pSymB of S. meliloti 1021. It spans the sequence at the beginning of the reference sequence of pSymB through the end of the pSymB plasmid:

Primers used to amplify the SMb21651 promoter:
SMb21651 promoter forward primer: 5’ GCaagcttGGCACGCGTCTGAATGTCCATTGT

SMb21651 promoter reverse primer: 5’ GCctgcagGTCGATGTCCGTGCCGCAACT
2) This fragment was amplified and cloned into HindIII/PstI sites of pLAFR5 (Keen et al. (1988) Gene 70:191-197).
The full mcs of pLAFR5 is HindIII—PstI—SalI—BamHI—SmaI (not unique)—EcoRI.

3) ORFs to be expressed in pstb-LAFR5 from the Smb21651 promoter can be cloned into PstI/BamHI

4) Important for cloning ORFs into pstb-LAFR5---Since there are uncharacterized ORFs in this promoter sequence, the forward ORF primer should have stop codons, followed by an rbs:
PstI ____stop codons__ ___rbs and spacer__
ctgcagTAACGTAACGTAAATGGAGTCACCTCT→followed by the ATG of the ORF

(The first use of this promoter [in a different construct] and this 3x stop codon/rbs/spacer design is from Zhang and Cheng. (2006) Applied and Environmental Microbiology 74: 2738-2748
The reverse ORF primer doesn't need anything except a BamHI site.