pPIC6α A vector (V010249) Gene synthesis in pPIC6α A backbone

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Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V010249	pPIC6α A	In stock, instant shipping

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

Basic Vector Information

pPIC6α A serves as a secretory expression vector for Pichia pastoris, enabling efficient expression of recombinant proteins. Its α-factor signal peptide mediates secretion of the target protein into the extracellular space, achieving high-yield expression and purification.

Vector Name:: pPIC6α A

Antibiotic Resistance:: Blasticidin

Length:: 3646 bp

Type:: Yeast Plasmids

Replication origin:: ori

Source/Author:: Invitrogen (Life Technologies)

Copy Number:: High copy number

Promoter:: AOX1

Growth Strain(s):: Top10

pPIC6α A vector Map

Copy Sequence View 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

Lai L, Zeff RA, Goldschneider I. A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells. Blood. 2006 Mar 1;107(5):1776-84. doi: 10.1182/blood-2005-08-3470. Epub 2005 Nov 22. PMID: 16304052; PMCID: PMC1895697.

pPIC6α A vector Sequence

LOCUS       pPIC6-A.        3646 bp DNA     circular SYN 13-SEP-2021
DEFINITION  Pichia pastoris vector for methanol-inducible expression of a 
            secreted protein. For other reading frames, use pPIC6-alpha B or 
            pPIC6-alpha C.
ACCESSION   .
VERSION     .
KEYWORDS    pPIC6-A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3646)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3646)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3646)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     Linearize with SacI, PmeI, or BstXI for integration at the AOX1 
            locus.
FEATURES             Location/Qualifiers
     source          1..3646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             941..1207
                     /label=alpha-factor secretion signal
                     /note="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
     misc_feature    1208..1276
                     /label=MCS
                     /note="MCS"
                     /note="MCS;multiple cloning site"
     CDS             1275..1304
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
     CDS             1320..1337
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      1417..1663
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        1678..2091
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2098..2145
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             2164..2559
                     /label=BSD
                     /note="blasticidin S deaminase"
     terminator      2656..2903
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(2978..3566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"