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pPIC9K-His vector (V000728)

Price Information

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V000728 pPIC9K-His In stock, instant shipping

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

Basic Vector Information

It is a Pichia Vector for Multicopy Integration and Secreted Expression, 6*his-tag included in the backbone.

Vector Name:
pPIC9K-His
Antibiotic Resistance:
Ampicillin
Length:
9294 bp
Type:
Pichia pastoris expression vectors
Replication origin:
ori
Selection Marker:
Neomycin (select with G418), HIS4
Copy Number:
High copy number
Promoter:
AOX1
Fusion Tag:
C-HIS
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

pPIC9K-His vector Map

Copy Sequence View Map
pPIC9K-His9294 bp400800120016002000240028003200360040004400480052005600600064006800720076008000840088009200AOX1 promoteralpha-factor secretion signal6xHisAOX1 terminatorPpHIS4KanRAOX1 3' fragmentbomoriAmpRAmpR promoter

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

  • Wang L, Wang YL, Lv ZL, Zhang EP, Guo AZ. Design of bovine lactoferricin-derived peptide and its expression and activity in Pichia pastoris. Biochem Biophys Res Commun. 2021 Jan 1;534:822-829.

pPIC9K-His vector Sequence

LOCUS       40924_34550        9294 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9294)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9294)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9294
                     /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"
     CDS             1246..1263
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      1339..1585
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(1998..4532)
                     /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(4949..5761)
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    6140..6896
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    7039..7179
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7365..7953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8127..8984)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(8985..9089)
                     /label=AmpR promoter