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GeneCorner plasmid details

Last data update: 27 October 2020 04:27 CET

Plasmid name: pAG25-AOX1-MFhIL6 (LMBP 5074)

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Price category: Cat. 1 (cf. price list)
Status: GeneCorner non-core plasmid
Depositor's sequence: p5074.gb
Sequence
analysis results
Genecorner:

-

Cloned DNA: Saccharomyces cerevisiae α-mating factor 1 gene (MFα1); prepro secretion signal sequence (ppMF)
Human interleukin 6 gene (IL6); mature sequence
Promoter: Eremothecium gossypii translation elongation factor 1α promoter (TEF1α)
Pichia pastoris alcohol oxidase 1 promoter (AOX1)
Ribosome
binding site:
-
Terminator: Eremothecium gossypii translation elongation factor 1α terminator (TEF1α)
Pichia pastoris alcohol oxidase 1 terminator (AOX1)
Selection marker: Ampicillin (amp)
Nourseothricin (ntc)
Replicon: Escherichia coli plasmid pMB1 origin
Host range: Escherichia coli
Pichia pastoris; integrative
Parental clone: pAG25; pPIC9(GARA)hIL6
Further information: It has been speculated for Pichia pastoris that monoarginilyc residues, especially within the sequence [Leu-Ile-Val-Met]-Xaa-Yaa-Arg can be recognized and cleaved by Kex2 or Kex2-like proteases. In the constructed vector, Arg lies within the sequence Leu-Gly-Ala-Arg-Ala and as such fullfils the theoretic requirements for Kex2- or Kex2-like cleavage. Indeed, upon analysis of Pichia clones, transformed with the vector, hIL6 seems to be secreted without the presence of the N-terminal MF.
Vector pPIC9 was cut XhoI/EcoRI, blunted using Klenow and dephosphorylated using Cip, hence removing the coding sequence for the Kex2 recognition site. A phosphorylated blunt linker, containing an AscI site and a MluI site, was ligated into the opened pPIC9 vector resulting into vector pPIC9(-Kex2). The MluI site was used to check the integration of the linker sequence. Vector pPIC9(-Kex2) was cut with AscI and EcoRI, blunted using Klenow and dephosphorylated using Cip. The vector fragment was ligated to a fragment encoding human IL6, the latter which was generated via a BsmI (T4 treated)/StuI digest on vector pFGA6HIL6T1.
The vector was called pPIC9(-Kex2)abi. Sequencing showed the vector contains mistakes. Due to the loss of the EcoRI site, the blunted human IL6 fragment was introduced into the vector pPIC9(-Kex2)abi which was only linearized and blunted at the unique AscI site. Furthermore, due to the loss of one 'A', the human IL6 sequence was no longer ligated in frame to the MF sequence (without the Kex2 recognition site). The corresponding vector was called pPIC9(-Kex2)abihIL6. To get the IL6 sequence in frame to the MF, the vector was cut at the AscI site, lying in between both coding sequences, blunted using Klenow and religated. The corresponding vector was called pPIC9(GARA)hIL6 and contains the IL6 sequence in frame to the MF, but lacking the original Kex2 cleavage site (EKR). In pPIC9(GARA)hIL6 the linker sequence between the MF signal and IL-6 is Gly-Ala-Arg-Ala (GARA) instead of Glu-Gly-Ala (EGA) in the theoretical plasmid pPIC9(-Kex2)hIL6. Finally the human IL6 expression cassette (containing AOX1 promotor and terminator) was isolated via a BglII/EcoRV digest and integrated via a sticky-blunt ligation into the BglII/PvuII opened pAG25 vector, containing the resistance marker against nourseothricin. The corresponding plasmid was called pAG25-AOX1-MF(GARA)hIL6.
Other name of the plasmid is pAG25-AOX1-MF(GARA)hIL6.
EMBL Accession number: -
Latest sequence update: 20/09/2005
Sequence detail:
Linker sequence:
5' GGGCGCGCCACGCGTG 3'

Theoretical sequence around linker:
   Lane 1: ...TATCTCTCGAGGGCGCGCCACGCGTGAATTC…

Actual sequence around linker:
   Lane 2: ...TATCTCTCG_GGGCGCGCCACGCGTG___TTC...

The nucleotide sequence at the fusion between ppMF and hIL6:

      ------- ppMF ------>  L   G   A   R   A  ------- hIL6 ------>
5' ... GAA.GAA.GGG.GTA.TCT.CTC.GGG.GCG.CGC.GCG.CCT.GTA.CCC.CCA.GGA ... 3'
       Glu Glu Gly Val Ser Leu Gly Ala Arg Ala Pro Val Pro Pro Gly
                           AvaI    BssHII
                                        BssHII

Punctuation indicates reading frame.
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 Prof. Dr N. Callewaert(1) (2).
(1) Department for Molecular Biomedical Research, VIB, Ghent, Belgium
(2) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
Plasmid reference: -
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)
Cultivation temperature: 37°C
Biosafety level: L1
Cultivation remark: -
Other culture collection numbers: -

Refer in your Materials and Methods:

pAG25-AOX1-MFhIL6 (LMBP 5074) is available at BCCM/GeneCorner. This plasmid was deposited by Prof. Dr N. Callewaert .

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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