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pG-Tf2 vector (V012822)

Price Information

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V012822 pG-Tf2 In stock, instant shipping

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

Basic Vector Information

Plasmid pG-Tf2 could express TF and GroEL-GroES together under the tetracycline-inducible promoter (Pzt-1)

Vector Name:
pG-Tf2
Antibiotic Resistance:
Chloramphenicol
Length:
8739 bp
Type:
E.coli expression plasmid
Replication origin:
p15A ori
Source/Author:
Takashi Yura
Copy Number:
Medium copy number
Promoter:
Pzt-1
5' Primer:
GGTAGCTCAGAGAACCTTCG
3' Primer:
gccatctcttcgatcaggc
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

pG-Tf2 vector Map

Copy Sequence View Map
pG-Tf28739 bp4008001200160020002400280032003600400044004800520056006000640068007200760080008400RBSS loopChaperonin GroELTrigger FactorrrnB T1 terminatortetR/tetA promotersTetRCmRcat promoterp15A ori

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

  • Yasamut U, Thongheang K, Weechan A, Sornsuwan K, Juntit OA, Tayapiwatana C. Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli. J Biosci Bioeng. 2024;138(3):232-238. doi:10.1016/j.jbiosc.2024.06.005

pG-Tf2 vector Sequence

LOCUS       Exported                8739 bp DNA     circular SYN 10-OCT-2024
DEFINITION  Exported.
ACCESSION   V012822
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8739)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8739)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8739)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8739
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          3573..3958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          4763..4822
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             98..109
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             206..259
                     /label=S loop
                     /note="GroES chaperone mobile loop that interacts with
                     GroEL"
     CDS             498..2141
                     /gene="groEL"
                     /label=Chaperonin GroEL
                     /note="Chaperonin GroEL from Escherichia coli O8 (strain
                     IAI1). Accession#: B7M8Q4"
     CDS             2202..3497
                     /label=Trigger Factor
                     /note="ribosome-associated chaperone from E. coli (Hoffmann
                     et al., 2010)"
     terminator      4833..4919
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     promoter        complement(4979..5034)
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     CDS             5050..5673
                     /label=TetR
                     /note="tetracycline repressor TetR"
     CDS             complement(6696..7352)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(7353..7455)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(7981..8526)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."