Plasmid_Backbone
pPICZα

Part:BBa_K805020

Designed by: Tang Yong   Group: iGEM12_HUST-China   (2012-09-20)

pPICZα

pPICZα A is a 3.6 kb vector purchased from Invitrogen. It is used to express and secrete recombinant proteins in Pichia pastoris. We import digesting site of EcoRⅠ and PstⅠinto polyclone sites to make it a standardized carrier.

Recombinant protein are expressed as fusions to an N-terminal peptide encoding the Saccharomyces cerevisiae α-factor secretion signal.The vector allows high-level, methanol inducible expression of the gene of interest in Pichia, and can be used in any Pichia strain including X-33, SMD1168H,and KM71H. pPICZα contains the following elements:

• 5′ fragment containing the AOX1 promoter for tightly regulated, methanolinduced expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a)

• α-factor secretion signal for directing secreted expression of the recombinant protein

• Zeocin™ resistance gene for selection in both E. coli and Pichia (Baron et al., 1992; Drocourt et al., 1990)

• C-terminal peptide containing the c-myc epitope and a polyhistidine (6×His) tag for detection and purification of a recombinant fusion protein (if desired) Three reading frames to facilitate in-frame cloning with the C-terminal peptide.


Usage and Biology

The series of products of Pichia pastoris expression systems developed by Invitrogen are mostwidely used ones and contains many advantages as follows:having a strong promoter which can be regulated by alcohol oxidase, being able to fermentate at high density and the recombinant protein having a high expression quantity. The exogenous genes integrated in the yeast's genome can be stable.Meanwhile, after the expression,translaion and modification, the exogenous genes will be secreted to extracellular by the efficient secretory expression plasmid,which can get the avtivity of the exprssion protein improved and be benefit for the purification of the product.


Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1208
    Illegal PstI site found at 2447
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1208
    Illegal PstI site found at 2447
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1208
    Illegal BglII site found at 1
    Illegal BamHI site found at 2087
    Illegal BamHI site found at 2861
    Illegal XhoI site found at 1184
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1208
    Illegal PstI site found at 2447
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 1208
    Illegal PstI site found at 2447
    Illegal NgoMIV site found at 1520
    Illegal NgoMIV site found at 1976
    Illegal NgoMIV site found at 3619
    Illegal NgoMIV site found at 3680
    Illegal AgeI site found at 2240
    Illegal AgeI site found at 2580
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2237
    Illegal BsaI.rc site found at 2844
    Illegal BsaI.rc site found at 3043
    Illegal BsaI.rc site found at 4041
    Illegal EcoRI site found at 1208
    Illegal PstI site found at 2447


[edit]
Categories
Parameters
resistanceZeocin