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pIEx-4 vector (V011625)

Price Information

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V011625 pIEx-4 In stock, instant shipping

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

Basic Vector Information

pIEx vectors are designed for cloning and high-level expression of proteins in transiently transfected Spodoptera derived insect cells.

Vector Name:
pIEx-4
Antibiotic Resistance:
Ampicillin
Length:
3780 bp
Type:
Insect Cell Vectors
Replication origin:
ori
Source/Author:
Novagen (EMD Millipore)
Copy Number:
High copy number
Promoter:
IE1
Cloning Method:
Enzyme Cut

pIEx-4 vector Map

Copy Sequence View Map
pIEx-43780 bp60012001800240030003600hr5 enhancerIE1 promoterMCSS-Tag8xHisIE1 terminatorAmpR promoterAmpRoriCAP binding sitelac promoterlac operatorM13 rev

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.

pIEx-4 vector Sequence

LOCUS       pIEx-4.        3780 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Vector for high-level expression in insect cells of proteins with a 
            C-terminal S-Tag-8xHis cassette.
ACCESSION   .
VERSION     .
KEYWORDS    pIEx-4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3780)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3780)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..483
                     /label=hr5 enhancer
                     /note="baculovirus early transcription enhancer"
     promoter        487..1078
                     /label=IE1 promoter
                     /note="promoter of the ie1 gene from the baculovirus
                     Autographa californica"
     misc_feature    1082..1178
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             1084..1086
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             1189..1233
                     /label=S-Tag
                     /note="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
     CDS             1255..1278
                     /label=8xHis
                     /note="8xHis affinity tag"
     terminator      1297..1604
                     /label=IE1 terminator
                     /note="terminator of the ie1 gene from the baculovirus 
                     Autographa californica"
     promoter        1634..1738
                     /label=AmpR promoter
     CDS             1739..2596
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      2770..3358
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    3646..3667
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3682..3712
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3720..3736
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3744..3760
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"