Last data update: 21 October 2020 04:26 CEST
Plasmid name: pCAhIFNGSTm (LMBP 4482)
|Price category:||Cat. 1 (cf. price list)|
|Status:||GeneCorner core plasmid|
(View with Genome Compiler)
|Cloned DNA:||Human interferon γ gene (IFNG); signal sequence
Human α2,6-sialyltransferase gene (ST6GalI); mutated mature sequence
|Promoter:||Chicken β-actin/rabbit β-globin hybrid promoter (AG)
Human cytomegalovirus immediate early promoter (CMV-IE); enhancer only
Escherichia coli lac operon promoter
|Terminator:||Rabbit β-globin polyadenylation signal (β-globin polyA)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)|
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Parental clone:||pSCGALST; pATHIL6HIFNG; pCAGGS|
|Further information:||The plasmid was constructed as follows: 1) pATHIL6HIFNG was NdeI opened (filled in with Klenow DNA polymerase); 2) The EaeI fragment (filled in with Klenow DNA polymerase) from pSCGALST, containing the mature human α2,6-sialyltransferase gene (hST6GalI), was inserted, resulting in the intermediate construct pATHIL6HIFNG-ST10; 3) pCAGGS was XhoI/MscI opened and the mature hST6GalI sequence, preceded by the signal sequence of the human interferon γ gene (hIFNG), was cloned as a HindIII (filled in with Klenow DNA polymerase)/XhoI fragment from pATHIL6HIFNG-ST10, leading to pCAIFNSTstop; 4) Finally, the termination codon at the 5' end of the hST6GalI coding sequence was deleted and a unique NotI site was introduced via site-specific mutagenesis using the synthetic oligonucleotide 5'-GCCAGGACCCAGCGGCCGCCATGGGG-3', resulting in pCAhIFNGSTm.
pCAhIFNGSTm is useful for highly efficient expression of human α2,6-sialyltransferase under the control of the chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.
When cloning a fragment downstream from the lac promoter it may be advisable to use lacI(q) strains in order to prevent fortuitous expression of a possibly noxious polypeptide.
The nucleotide sequence of this plasmid corresponds with the EMBL Nucleotide Sequence Database accession number LT727221.1.
The nucleotide sequence of hST6GalI corresponds with the Genbank accession number X62822.1.
Other name of the plasmid is pCAIFNSTΔstop.
|EMBL Accession number:||X62822.1, view at EMBL, GenBank, DDBJ
LT727221.1, view at EMBL, GenBank, DDBJ
|Latest sequence update:||25/08/2000|
Nucleotide sequence at the fusion between the hIFNG signal sequence and the mutated mature hST6GalI gene: -> hIFNG signal seq. hST6GalI --> -------------------- ----------------- Leu Gly Cys Tyr Cys Gln Asp Pro Ala Ala Ala Met Gly 5' … CTT.GGC.TGT.TAC.TGC.CAG.GAC.CCA.GCG.GCC.GCC.ATG.GGG … 3' ^^ *** NotI ^: Mutated nucleotide. *: Nucleotide inserted. Punctuation indicates reading frame.
|Authenticity test:||Restriction enzyme pattern analysed at GeneCorner: EcoRI, NotI, NotI/PstI and PstI.|
|History of deposit:||This plasmid was deposited by Dr W. Laroy(1) (2) and Prof. Dr R. Contreras(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Plasmid reference:||Laroy et al., Glycobiology 11 (2001), 175-182 [PMID: 11320056]
|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)|
|Other culture collection numbers:||-|
Refer in your Materials and Methods:
|pCAhIFNGSTm (LMBP 4482) is available at BCCM/GeneCorner. This plasmid was deposited by Dr W. Laroy and Prof. Dr R. Contreras and was published in Laroy et al., 2001.|
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.