Last data update: 24 January 2024 16:39 CET
Plasmid name: pOmIL2 (LMBP 3074)
| New search | Print data sheet |
| Price category: | Cat. 1 (cf. price list) |
| Status: | GeneCorner core plasmid |
| GeneCorner sequence: |
p3074.gb
(View with Genome Compiler) p3074.txt p3074.pdf |
| Sequence analysis results Genecorner: |
- |
| Cloned DNA: |
Escherichia coli alkaline phosphatase A gene (phoA); signal sequence 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 alkaline phosphatase A gene (phoA) Ribosome binding site (RBS) of the Escherichia coli lac Z gene (lacZ) Ribosome binding site (RBS) of the Escherichia coli outer membrane protein II gene (ompA) |
| Terminator: | Phage fd terminator |
| Selection marker: | Chloramphenicol (cam) |
| Replicon: | Escherichia coli plasmid pMB1 origin Phage f1 origin |
| Host range: | Escherichia coli |
| Parental clone: | pMc519; pMaHB2Mm3s; pSP64mIL2m |
| Further information: | The plasmid was derived from pMc519 by ligation of two fragments into the SmaI-XbaI vector fragment: 1) the DdeI fragment (filled in with T4 DNA polymerase) from pMaHB2Mm3s, containing the E. coli ompA signal sequence and 2) the FspI (nucleotide position 75) - SpeI fragment from pSP64mIL2m, containing the mature mouse IL2 cDNA. The plasmid was not designed for expression. The ompA signal sequence is fused to the mouse IL2 mature sequence. The fusion gene still contains an additional serine codon between the two subunits. The E. coli phoA signal sequence starts with ATG, while in the wild-type sequence the start codon is GTG. Only 13 nucleotides upstream the ATG start codon of phoA are derived from the original phoA ribosome binding site. The plasmid provides resistance to chloramphenicol, but is sensitive to ampicillin in sup(-) strains. This plasmid 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. When cloning a fragment downstream from the tac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide. 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 the mouse IL2 cDNA corresponds with the EMBL Nucleotide Sequence Database accession number X01772.1. Other name of the plasmid is pMc519sompAmIL2. |
| EMBL Accession number: | X01772.1, view at EMBL, GenBank, DDBJ |
| Latest sequence update: | 28/05/1997 |
| Sequence detail: | Nucleotide sequence at the sompA-mIL2 fusion:
filled in DdeI-FspI
---------- pMaHB2Mm3s --------->|-------- pSP64mIL2m ------->
---------- 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: HindII/HindIII, PvuII/XmnI, ScaI, SspI, StuI and XbaI. |
| 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: | PhD thesis Johan Robbens (1994) |
| Restricted distribution: | - BCCM MTA |
| Distributed as: | H/P active culture or plasmid DNA |
| Host for distribution: | Escherichia coli K12 RR1ZΔM15 |
| 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: |
pOmIL2 (LMBP 3074) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr E. Remaut and was published in Johan Robbens, 1994. |
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.