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LMBP plasmid details

Last data update: 28 November 2020 04:24 CET

Plasmid name: pMacBLAL2HI (LMBP 2316)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p2316.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Ampicillin resistance gene (amp)
Human immunoglobulin γ3 cDNA (hIG3); heavy chain hinge region
Promoter: -
Ribosome
binding site:
-
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: pMac254mSA; pBR325OMMBfmA
Further information: The plasmid was constructed by introducing the HinFI (nucleotide position 4828; filled in with Klenow) - RsaI (nucleotide position 5184) fragment from pBR325OMMBfmA anticlockwise in the SacI opened and blunted (with T4 DNA polymerase) pMac254mSA vector.
This phasmid leads to the expression of a fusion protein in which 10 additional amino acids (from the VH region of OMMB) are present between the β-lactamase gene and the first hinge region (H1) of the human γ3 heavy chain. The translation of this fusion protein terminates 2 amino acids behind the fourth hinge region (H4), caused by the introduced termination codon in the CH2 domain; hence, CH2f is no longer translated.
The sequence of the inserted hIG3 fragment was obtained from Genbank (Release 55; Locus : HUMIGHAF') and contains some non-identified nucleotides (indicated by N).
The fusion product still provides resistance to ampicillin.
The f1 part of the plasmid contains the F1-ori, but also the sequence around the gene II promoter; the two TRP terminators at the end of the cam gene are not present.
Name mentioned in De Sutter et al., (1992) is pMacBlaL2Hi.
Other name of the plasmid is pMac254BLAVHfHI.
EMBL Accession number: J00231, view at EMBL, GenBank, DDBJ
Latest sequence update: 22/01/1997
Sequence detail:
Nucleotide sequence at the start and at the end of the inserted hIG3 fragment:

   amp          linker (1)           H1-4            CH2 fragment
   ------- ------------------- ------------------- ---------------
5' CAT TGG GAG TCG GGC CCA ... GAG CTC ... TGC CCA GGG CCC TAA ...
           ^                   SacI            *1  ApaI    *2
            -----
            Blunted HinFI


-----------
... AAGTGGT  CTCAGGGCCC 3'
         *3  ^   ApaI (derived from vector)
         --
         Rest of RsaI


^:    Blunted SacI site.
*1:   End of the fourth hinge region H4.
*2:   Termination codon.
*3:   End of the inserted cE6Hy3 fragment.
(1):  The 10 amino acid linker segment, derived from the terminus of VH.
Authenticity test: The plasmid still needs to be subjected to the authenticity test.
Class: Recombinant plasmid
Type: Plasmid
History of deposit: This plasmid was deposited by Dr K. De Sutter(1).
(1) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: De Sutter et al., Mol. Microbiol. 6 (1992), 2201-2208 [PMID: 1406260]
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) + chloramphenicol (25 μg/ml)*
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: *: selection of transformants on chloramphenicol; subsequent cultivation of a single colony in liquid medium with ampicillin.
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.

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