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GeneCorner plasmid details

Last data update: 19 October 2020 04:30 CEST

Plasmid name: pAIL2 (LMBP 3076)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner core plasmid
GeneCorner sequence: p3076.gb (View with Genome Compiler)
p3076.txt
Sequence
analysis results
Genecorner:

-

Cloned DNA: Escherichia coli outer membrane protein II gene (ompA); 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 lac Z gene (lacZ)
Ribosome binding site (RBS) of the Escherichia coli outer membrane protein II gene (ompA)
Terminator: Phage fd terminator
Selection marker: Ampicillin (amp)
Chloramphenicol (cam)
Replicon: Escherichia coli plasmid pMB1 origin
Phage f1 origin
Host range: Escherichia coli
Parental clone: pSIL2
Further information: pAIL2 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 lacks the 3' terminal alanine residue of the OmpA signal peptide and contains an additional serine residue between the two subunits. The signal peptide cleavage occurs at the serine, resulting in mouse IL2 with an amino-terminal alanine.
As compared to pSIL2 and pNIL2, the ompA-mIL2 fusion conformation results in an intermediate mouse IL2 secretion level.
pAIL2 provides resistance to ampicillin and to chloramphenicol also in sup(-) strains. This has been experimentally verified at LMBP.
pAIL2 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).
This plasmid was derived from pSIL2 via site-specific mutagenesis. Consequently, the 3' terminal alanine-encoding GCC-triplet of the Escherichia coli ompA signal sequence was removed. The mutation was confirmed by DNA sequence analysis.
Other names of the plasmid are pMa519KSompAmIL2Ala(-) and pOmIL2A.
EMBL Accession number: X01772, view at EMBL, GenBank, DDBJ
Latest sequence update: 24/10/2003
Sequence detail:
Primer used for site-specific mutagenesis:

5' GAAGTGGGTGCTGACTGCGCTACGGTAGCG 3'

Nucleotide sequence at the ompA-mIL2 fusion:
                                
              - ompA signal sequence->     ---------- mIL2 ---------->
pAIL2:  5' ... TTC.GCT.ACC.GTA.GCG.CAG.TCA.GCA.CCC.ACT.TCA.AGC.TCC.ACT ... 3'
               Phe Ala Thr Val Ala Gln Ser Ala Pro Thr Ser Ser Ser Thr


                                filled in DdeI-FspI fusion
              --- ompA signal sequence -->     ----------- mIL2 ---------->
pSIL2:  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: PhD thesis Johan Robbens (1994)
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 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 chloramphenicol (mutated chloramphenicol resistance gene present on the plasmid).
Other culture collection numbers: -

Refer in your Materials and Methods:

pAIL2 (LMBP 3076) 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.

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