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URL: http://www.addgene.org/86169

Proper Citation: RRID:Addgene_86169

Bacterial Resistance: Tetracycline

Defining Citation: PMID:22685282

Vector Backbone Description: Backbone Marker:Keen et al. (1988) Gene 70:191-197; Backbone Size:22000; Vector Backbone:pLAFR5; Vector Types:Bacterial Expression; Bacterial Resistance:Tetracycline

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.

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