pTRKH3-ldhGFP vector (V000751)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V000751	pTRKH3-ldhGFP	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

The promoters of L. acidophilus lactate dehydrogenase gene (ldhL) fused to the coding sequence of GFP and inserted into the backbone of the pTRKH3 shuttle vector.

Vector Name:: pTRKH3-ldhGFP

Antibiotic Resistance:: Erythromycin

Length:: 8509 bp

Type:: Lactobacillus expression vectors

Replication origin:: p15A ori

Selection Marker:: mgfp5

Growth Strain(s):: Top10

Growth Temperature:: 37℃

pTRKH3-ldhGFP vector Map

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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

Lizier M, Sarra PG, Cauda R, Lucchini F. Comparison of expression vectors in Lactobacillus reuteri strains. FEMS Microbiol Lett. 2010 Jul 1;308(1):8-15.

pTRKH3-ldhGFP vector Sequence

LOCUS       V000751                 8509 bp    DNA     circular SYN 11-MAR-2024
DEFINITION  Exported.
ACCESSION   V000751
VERSION     V000751
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8509)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8509)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1322..2056
                     /gene="ermBP"
                     /label="rRNA adenine N-6-methyltransferase"
                     /note="rRNA adenine N-6-methyltransferase from Enterococcus
                     faecalis. Accession#: P0A4D5"
     CDS             3643..5130
                     /codon_start=1
                     /label="repS"
                     /note="pLannotate"
                     /note="/database=swissprot"
                     /note="/fragment=False"
                     /note="/identity=97.8"
                     /note="/match_length=100.0"
                     /note="/other=CDS"
                     /translation="MNIPFVVETVLHDGLLKYKFKNSKIRSITTKPGKSKGAIFAYRSK
                     SSMIGGRGVVLTSEEAIQENQDTFTHWTPNVYRYGTYADENRSYTKGHSENNLRQINTF
                     FIDFDIHTAKETISASDILTTAIDLGFMPTMIIKSDKGYQAYFVLETPVYVTSKSEFKS
                     VKAAKIISQNIREYFGKSLPVDLTCNHFGIARIPRTDNVEFFDPNYRYSFKEWQDWSFK
                     QTDNKGFTRSSLTVLSGTEGKKQVDEPWFNLLLHETKFSGEKGLIGRNNVMFTLSLAYF
                     SSGYSIETCEYNMFEFNNRLDQPLEEKEVIKIVRSAYSENYQGANREYITILCKAWVSS
                     DLTSKDLFVRQGWFKFKKKRSERQRVHLSEWKEDLMAYISEKSDVYKPYLVTTKKEIRE
                     VLGIPERTLDKLLKVLKANQEIFFKIKPGRNGGIQLASVKSLLLSIIKVKKEEKESYIK
                     ALTNSFDLEHTFIQETLNKLAERPKTDTQLDLFSYDTG"
     promoter        complement(5843..5945)
                     /label="cat promoter"
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(6471..7016)
                     /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."
     promoter        7128..7156
                     /label="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             complement(7502..8215)
                     /label="mgfp5"
                     /note="GFP with folding enhancement mutations"
     promoter        complement(8223..8507)
                     /label="ldhL promoter"
                     /note="L. acidophilus lactate dehydrogenase gene (ldhL)
                     promoter"