Last data update: 24 January 2024 16:39 CET
Plasmid name: pSIL2 (LMBP 2525)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p2525.gb
(View with Genome Compiler) p2525.txt p2525.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Escherichia coli outer membrane protein A cDNA (ompA, tolG, GeneID 945571); signal sequence Mouse interleukin 2 cDNA (IL2); mature sequence |
| Promoter: | Escherichia coli hybrid tryptophan/lacUV5 promoter (tac) |
| Ribosome binding site: |
Ribosome binding site (RBS) of the Escherichia coli outer membrane protein II gene (ompA) Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) |
| Terminator: | Phage fd terminator |
| Selection marker: | Chloramphenicol (cam) |
| Replicon: | Escherichia coli plasmid pMB1 origin Phage f1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pOmIL2 |
| Further information: | This plasmid was derived from pOmIL2 after digestion with BamHI (nucleotide position 3857) and XbaI, and ligating the filled-in (with T4 DNA polymerase) ends. As a result, the 600 bp fragment between the tac promoter and the ompA signal sequence was eliminated. pSIL2 is an expression vector containing the ompA signal sequence fused to the mature mouse IL2 cDNA downstream from the IPTG-inducible tac promoter. The fusion product still contains an additional serine residue between the two subunits. After cleavage of the OmpA signal peptide, the serine remains attached to mouse IL2 but without effect on the biological activity of the protein. As compared to pNIL2, pSIL2 gives rise to an approximate 40.000 fold increase of the mouse IL2 secretion upon induction at 28°C. pSIL2 provides resistance to chloramphenicol, but is sensitive to ampicillin in sup(-) strains. pSIL2 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, e.g. M13KO7. The presence of the lacI(q) repressor gene is necessary to control the activity of the tac promoter. 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 LT727522.1. The nucleotide sequence of the mouse IL2 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number X01772.1. Name mentioned in Robbens et al. (1995) is pOmIL2S. Other name of the plasmid is pMc519KSompAmIL2. |
| EMBL Accession number: | X01772.1, view at EMBL, GenBank, DDBJ LT727522.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 29/05/1997 |
| Sequence detail: | Nucleotide sequence at the sompA-mIL2 fusion:
filled in DdeI-FspI fusion
---------- sompA ----------> ----------- mIL2 ---------->
5' ... TTC.GCT.ACC.GTA.GCG.CAG.GCC.TCA.GCA.CCC.ACT.TCA.AGC.TCC.ACT ... 3'
Phe Ala Thr Val Ala Gln Ala Ser Ala Pro Thr Ser Ser Ser Thr
StuI DdeI
HaeIII
Punctuation indicates reading frame. |
| Authenticity test: | Restriction enzyme pattern analysed at GeneCorner: BamHI, HindII, PvuII/XmnI, ScaI and StuI. |
| Class: | Recombinant plasmid |
| Type: | Plasmid |
| History of deposit: | This plasmid was deposited by Prof. Dr E. Remaut(1) (2). It was constructed by J. Robbens(1) (2). (1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium (2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium |
| Plasmid reference: | Robbens et al., Protein Expr. Purif. 6 (1995), 481-486 [PMID: 8527934] |
| Related plasmid reference: | PhD thesis Johan Robbens (1994) Robbens et al., Process Biochem. 41 (2006), 1343-1346 [DOI: 10.1016/j.procbio.2006.01.009] |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 RR1ΔM15 |
| Host reference: | Altieri et al., J. Bacteriol. 168 (1986), 648-654 [PMID: 2946661] |
| Cultivation medium: | LB-Lennox + ampicillin (100 μg/ml) + chloramphenicol (25 μg/ml)* |
| Cultivation temperature: | 28°C |
| Biosafety level: | L1 |
| Cultivation remark: | *: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin. E. coli RR1ΔM15 is characterized by a suppressor mutation (supE44). As such, this host/plasmid combination also provides resistance to ampicillin (mutated ampicillin resistance gene present on the plasmid). |
| Other culture collection numbers: | - |
Refer in your Materials and Methods: |
pSIL2 (LMBP 2525) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr E. Remaut and was published in Robbens et al., 1995. |
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.