Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010245 | pPIC9K | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pPIC9K vector is designed for expression in Pichia pastoris. It features a strong AOX1 promoter for high-level expression. This vector also has an alpha-factor secretion signal, which can direct the secretion of target proteins.
pPIC9K offers several advantages, such as efficient protein production, post-translational modifications similar to mammalian cells, and the ability to scale up production. It is ideal for expressing large amounts of recombinant proteins, especially those that require proper folding and modifications.
- Vector Name:
- pPIC9K
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9268 bp
- Type:
- Yeast Plasmids
- Replication origin:
- ori
- Source/Author:
- Invitrogen (Life Technologies)
- Copy Number:
- High copy number
- Promoter:
- AOX1
pPIC9K 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
- Ma L, Liang X, Yu S, Zhou J. Expression, characterization, and application potentiality evaluation of recombinant human-like collagen in Pichia pastoris. Bioresour Bioprocess. 2022 Nov 17;9(1):119. doi: 10.1186/s40643-022-00606-3. PMID: 38647896; PMCID: PMC10992492.
- Huang T, Qi J, Yang G, Ye X. [Expression, purification and bioactivity analysis of a recombinant fusion protein rHSA-hFGF21 in Pichia pastoris]. Sheng Wu Gong Cheng Xue Bao. 2022 Sep 25;38(9):3419-3432. Chinese. doi: 10.13345/j.cjb.220161. PMID: 36151810.
pPIC9K vector Sequence
LOCUS Exported 9268 bp DNA circular SYN 02-SEP-2024
DEFINITION Pichia pastoris multi-copy integrating vector for methanol-inducible
expression of a secreted protein.
ACCESSION .
VERSION .
KEYWORDS pPIC9K
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9268)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 9268)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9268)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9268
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 2..937
/label=AOX1 promoter
/note="inducible promoter, regulated by methanol"
CDS 949..1215
/label=alpha-factor secretion signal
/note="N-terminal secretion signal from S. cerevisiae
alpha-factor"
misc_feature 1216..1241
/label=MCS
/note="MCS"
/note="multiple cloning site"
terminator 1321..1567
/label=AOX1 terminator
/note="transcription terminator for AOX1"
CDS complement(1980..4514)
/codon_start=1
/gene="Pichia pastoris HIS4"
/product="multifunctional enzyme, required for histidine
biosynthesis"
/label=Pichia pastoris HIS4
/note="PpHIS4"
/note="auxotrophic marker for Pichia pastoris"
/translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
CDS complement(4930..5742)
/label=KanR
/note="aminoglycoside phosphotransferase"
misc_feature 6121..6869
/label=AOX1 3' fragment
/note="region downstream of Pichia pastoris AOX1 gene"
misc_feature 7013..7153
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(7339..7927)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8101..8958)
/label=AmpR
/note="beta-lactamase"
promoter complement(8959..9063)
/label=AmpR promoter