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pEE12.4 vector (V012681)

Price Information

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V012681 pEE12.4 In stock, instant shipping

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

Basic Vector Information

Vector Name:
pEE12.4
Antibiotic Resistance:
Ampicillin
Length:
7569 bp
Type:
Mammalian Expression Vectors
Replication origin:
ori
Source/Author:
Lonza biologics
Selection Marker:
GS(Glutamine Synthetase)
Copy Number:
High copy number
Promoter:
SV40
Cloning Method:
Enzyme Cut
5' Primer:
CMV fwd
Expression Method:
Constiutive, Stable

pEE12.4 vector Map

Copy Sequence View Map
pEE12.47569 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600690072007500SV40 poly(A) signaloriAmpRAmpR promoterT7Te terminatorSV40 promoterGlutamine synthetasesmall t intronSV40 NLSSV40 poly(A) signalCMV enhancerCMV promoterCMV intron A

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

  • Lu Z, Zhang TY, Han MM, Bai FL, Wu W, Tian GY, Ren GP, Li DS. [Antitumor activity of the recombinant rClone30-CD/5-FC system]. Yao Xue Xue Bao. 2013 Feb;48(2):261-8. Chinese. PMID: 23672024.

  • Sun T, Niu ZS, Liu XY, Tian GY, Bai Y, Bai FL, Yin JC, Yu D, Wu YZ, Li DS, Yu QZ, Li SM, Ren GP. [The synergism and mechanism of action of rClone30-hDR5 in combination with TRAIL on HCC]. Yao Xue Xue Bao. 2014 Jul;49(7):985-92. Chinese. PMID: 25233628.

pEE12.4 vector Sequence

LOCUS       V012681                 7569 bp    DNA     circular SYN 23-SEP-2020
DEFINITION  Exported.
ACCESSION   V012681
VERSION     V012681
KEYWORDS    pEE12.4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7569)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7569)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7569
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    116..237
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(999..1587)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(1761..2618)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(2619..2723)
                     /label="AmpR promoter"
     terminator      2892..2919
                     /label="T7Te terminator"
                     /note="phage T7 early transcription terminator"
     promoter        3002..3331
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     CDS             3370..4488
                     /gene="GLUL"
                     /label="Glutamine synthetase"
                     /note="Glutamine synthetase from Acomys cahirinus.
                     Accession#: Q9QY94"
     intron          4623..4688
                     /label="small t intron"
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4818..4838
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     polyA_signal    5263..5397
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     enhancer        5966..6345
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6346..6549
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          6685..7511
                     /label="CMV intron A"
                     /note="human cytomegalovirus intron A (Chapman et al.,
                     1991)"