pEX18Tc vector (V006436)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V006436	pEX18Tc	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

Suicide Plasmid

Vector Name:: pEX18Tc

Antibiotic Resistance:: Tetracycline

Length:: 6369 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.

Promoter:: sacB

Cloning Method:: restriction endonuclease,

5' Primer:: M13F (-47) CGCCAGGGTTTTCCCAGTCACGAC

3' Primer:: M13R (-48) AGCGGATAACAATTTCACACAGGA

pEX18Tc vector Map

Copy Sequence View 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.

References

Huang W, Wilks A. A rapid seamless method for gene knockout in Pseudomonas aeruginosa. BMC Microbiol. 2017 Sep 19;17(1):199. doi: 10.1186/s12866-017-1112-5. PMID: 28927382; PMCID: PMC5606073.

Wang Y, Zhang C, Gong T, Zuo Z, Zhao F, Fan X, Yang C, Song C. An upp-based markerless gene replacement method for genome reduction and metabolic pathway engineering in Pseudomonas mendocina NK-01 and Pseudomonas putida KT2440. J Microbiol Methods. 2015 Jun;113:27-33. doi: 10.1016/j.mimet.2015.03.022. Epub 2015 Mar 28. PMID: 25828098.

pEX18Tc vector Sequence

LOCUS       Exported                6369 bp DNA     circular SYN 02-SEP-2024
DEFINITION  Cloning vector pEX18Tc, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6369)
  AUTHORS   Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
  TITLE     A broad-host-range Flp-FRT recombination system for site-specific 
            excision of chromosomally-located DNA sequences: application for 
            isolation of unmarked Pseudomonas aeruginosa mutants
  JOURNAL   Gene 212 (1), 77-86 (1998)
  PUBMED    9661666
REFERENCE   2  (bases 1 to 6369)
  AUTHORS   Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-FEB-1998) Microbiology, Colorado State University, 
            Fort Collins, CO 80523, USA
REFERENCE   3  (bases 1 to 6369)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6369)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6369)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene"; 
            date: "1998"; volume: "212"; issue: "1"; pages: "77-86"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (10-FEB-1998) Microbiology, Colorado State University, Fort Collins,
            CO 80523, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2899..2921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(212..1630)
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     promoter        complement(1631..2076)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     oriT            complement(2450..2559)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(3002..3029)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(3121..3207)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     rRNA            complement(3208..3327)
                     /product="Eschericia coli 5S ribosomal RNA"
     primer_bind     3557..3573
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(3577..3633)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3643..3658)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3666..3682)
                     /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)."
     promoter        complement(3690..3720)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(3735..3756)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4044..4632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4908..6095)
                     /codon_start=1
                     /label=TcR
                     /note="tetracycline efflux protein"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     promoter        complement(6170..6274)
                     /label=AmpR promoter

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