Last data update: 24 January 2024 16:39 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: | - |
Refer in your Materials and Methods: |
pMacBLAL2HI (LMBP 2316) is available at BCCM/GeneCorner. This plasmid was deposited by Dr K. De Sutter and was published in De Sutter et al., 1992. |
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.