Last data update: 24 November 2020 04:18 CET
Plasmid name: pMa58SAm3 (LMBP 1997)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Streptomyces avidinii streptavidin gene (SA); mutated sequence|
|Terminator:||Phage fd terminator|
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Phage f1 origin
|Host range:||Escherichia coli|
|Parental clone:||pMa58; pMa58SA|
|Further information:||The plasmid was derived from pMc58SA by introducing two BamHI sites, according to the site-specific mutagenesis protocol of Stanssens et al. (1989): the first site is located at the position of the first mature codon of the Streptomyces avidinii streptavidin gene (SA); the second site is located at the position of the termination codon of the streptavidin gene.
This phasmid provides resistance to ampicillin but is sensitive to chloramphenicol.
More than one mutagenesis round is possible by switching from ampicillin resistance to chloramphenicol resistance.
pMa58SAm3 contains the replication origin of the single-stranded DNA phage f1, so that it can be rapidly switched between the plasmid and the phage mode (ssDNA) of replication. The latter requires infection with a helper phage M13KO7.
The sequence of the streptavidin gene was taken from Genbank (Release 55; Locus: STMSTRAVG) and shows, as compared to Dr Lieberman's sequence map of the parental clone pSA307, a few differences in the 5' and 3' untranslated regions of the gene.
After mutagenesis, use mutS strains for primary transformation (e.g. sup(-) strain WK6mutS, sup(+) strain BMH71-18mutS); for segregation of possible mutants: sup(-) strains (e.g. WK6).
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727617.1.
Other name of the plasmid is pSAB12.
|EMBL Accession number:||LT727617.1, view at EMBL, GenBank, DDBJ|
|Latest sequence update:||12/07/1990|
Difference between the wt streptavidin gene and the mutated gene: 1) At the position of the first mature codon: - wild type gene: GCT.TCG.GCA.GAC.CCC.TCC.AAG.G ^ * - mutated gene, making use of the following mutator oligonucleotide: GCT.TCG.GCG.GAT.CCC.TCC.AAG.G ^ * BamHI ^: Last codon of the signal sequence of the streptavidin gene. *: First codon of the mature sequence of the streptavidin gene. 2) At the position of the termination codon: - wild type gene: C.GTT.CAG.CAG.TAG.TCGCGTCC * - mutated gene, making use of the following mutator oligonucleotide: C.GTT.CAG.CAG.GAT.CCGCGTCCC ^ BamHI *: Termination codon of the streptavidin gene. ^: Last codon of the mature sequence of the mutated gene. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: AflIII, BamHI, EcoRI and EcoRI/HindIII. The plasmid seems to be 200 à 300 bp larger as compared to the compiled nucleotide sequence, the extra nucleotides being located in the vector part of the plasmid.|
|History of deposit:||This plasmid was deposited by J. Viaene(1) and Prof. Dr E. Remaut(1).
(1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||Stanssens et al., Nucleic Acids Res. 17 (1989), 4441-4454 [PMID: 2501754]
|Restricted distribution:||- BCCM MTA|
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 MC1061|
|Host reference:||Casadaban et al., J. Mol. Biol. 138 (1980), 179-207 [PMID: 6997493]
|Related host reference:||Brigé et al., Biochem. J. 394 (2006), 335-344 [PMID: 16293111]
|Cultivation medium:||LB-Lennox + ampicillin (100 μg/ml)|
|Other culture collection numbers:||-|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.