pACT vector (V006726)

Price Information

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

Basic Vector Information

The pACT Vector is a high-copy plasmid in which the human cytomegalovirus (CMV) immediate early promoter drives expression of the herpes virus VP16 activation domain (amino acids 411–456). A chimeric intron is located 5´ of the gene segment, and a multiple cloning region is located 3´ of the gene segment for insertion of cDNA clones of interest (Figures 2 and 3). The presence of this chimeric intron, in context with the CMV promoter and polyadenylation signal, can result in increased protein expression of cDNA genes linked to these elements (6). The stop codons and SV40 late polyadenylation region, demonstrated to be efficient for mRNA production (7), are at the 3´ end of the fusion gene. The fusion gene region is flanked by T7 and T3 RNA polymerase promoters for the synthesis of sense and antisense RNA products. The T7 promoter allows the construct to be translated using the TNT T7 Quick Coupled Transcription/Translation System (Cat.# L1170). Also located on this vector is the neomycin phosphotransferase gene (from Tn5) driven by the SV40 early promoter and followed by a synthetic polyadenylation cassette. This gene confers resistance to the neomycin analog, G418 (Geneticin; 8). The plasmid backbone contains an f1 origin of replication for the production of single-stranded DNA (ssDNA) and the β-lactamase (Ampr) gene for selection of the vector DNA in E. coli.

pACT 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.

pACT vector Sequence

LOCUS       40924_3891        5566 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 5566)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5566)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5566
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1149..1169
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     promoter        complement(1403..1420)
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    complement(1438..1559)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      1693..2148
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2163..2520
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2571..3362
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    3429..3477
                     /label=poly(A) signal
                     /note="synthetic polyadenylation signal"
     promoter        3769..3873
                     /label=AmpR promoter
     CDS             3874..4731
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4905..5493
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"