pGAPZα A vector (V010273)

Price Information

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

The pGAPZα A vector is designed for expression in Pichia pastoris under the control of the constitutive GAP promoter. This vector features a Zeocin resistance marker for easy selection of transformed cells. One of its key characteristics is the ability to produce high levels of recombinant proteins without the need for induction.
pGAPZα A offers several advantages, such as consistent expression, reduced production costs due to the lack of inducer requirements, and suitability for large-scale production. It is ideal for expressing proteins that need continuous production or when rapid expression is desired without the complexity of induction. Additionally, it can be easily manipulated for cloning and downstream processing.

pGAPZα A vector Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

• Sams L, Amara S, Chakroun A, Coudre S, Paume J, Giallo J, Carrière F. Constitutive expression of human gastric lipase in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues. Biochim Biophys Acta Mol Cell Biol Lipids. 2017 Oct;1862(10 Pt A):1025-1034. doi: 10.1016/j.bbalip.2017.07.002. Epub 2017 Jul 8. PMID: 28694218.
• Li Z, Xiong F, Lin Q, d'Anjou M, Daugulis AJ, Yang DS, Hew CL. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif. 2001 Apr;21(3):438-45. doi: 10.1006/prep.2001.1395. PMID: 11281719.
• Luo HY, Huang HQ, Bai YG, Wang YR, Yang PL, Meng K, Yuan TZ, Yao B. [Improving phytase expression by increasing the gene copy number of appA-m in Pichia pastoris]. Sheng Wu Gong Cheng Xue Bao. 2006 Jul;22(4):528-33. Chinese. doi: 10.1016/s1872-2075(06)60041-1. PMID: 16894882.

pGAPZα A vector Sequence

LOCUS       Exported                3149 bp DNA     circular SYN 02-SEP-2024
DEFINITION  Pichia pastoris vector for constitutive expression of a secreted 
            protein. For other reading frames, use pGAPZ-alpha B or pGAPZ-alpha 
            C.
ACCESSION   .
VERSION     .
KEYWORDS    pGAPZ-A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3149)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3149)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3149)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 3149)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     Linearize within the GAP promoter for integration.
FEATURES             Location/Qualifiers
     source          1..3149
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..483
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             493..759
                     /label=alpha-factor secretion signal
                     /note="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
     misc_feature    760..828
                     /label=MCS
                     /note="MCS"
                     /note="MCS;multiple cloning site"
     CDS             827..856
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             872..889
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      968..1214
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        1229..1642
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        1649..1696
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             1715..2086
                     /label=BleoR
                     /note="antibiotic-binding protein"
     terminator      2155..2402
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(2477..3065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"