pK18mobsacB vector (V005269)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V005269	pK18mobsacB	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 pK18mobsacB vector contains the sacB element. The SacB gene, from Bacillus subtilis and related to sugar metabolism, converts sucrose to levan.
In Escherichia coli lacking genes to process levan, expressing SacB leads to the accumulation of levan and causes bacterial death. Thus, by simply adding 10% sucrose to the medium, E. coli can be screened.
In the CRISPR Cas9 system for gene knockout and targeted insertion in microorganisms, including E. coli, the sacB gene in the Cas9 plasmid helps eliminate the plasmid post-recombination. When sucrose is present, the protein encoded by SacB converts sucrose to fructose, and fructose accumulation is toxic to E. coli. Strains that can lose the plasmid survive under this pressure, while those unable to do so die.

Vector Name:: pK18mobsacB

Antibiotic Resistance:: Kanamycin

Length:: 5719 bp

Type:: Corynebacterium glutamicum knockout vectors

Replication origin:: ori

Source/Author:: Kvitko BH, Collmer A.

Promoter:: sacB

Cloning Method:: Enzyme digestion and ligation

Growth Strain(s):: JM108

Growth Temperature:: 37℃

pK18mobsacB 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

Fu WL, Duan PF, Wang Q, Liao YX, Wang YS, Xu MJ, Jiang HH, Zhang X, Rao ZM. Transcriptomics reveals the effect of ammonia nitrogen concentration on Pseudomonas stutzeri F2 assimilation and the analysis of amtB function. Synth Syst Biotechnol. 2023 Mar 6;8(2):262-272. doi: 10.1016/j.synbio.2023.03.002. PMID: 37033292; PMCID: PMC10074406.

pK18mobsacB vector Sequence

LOCUS       Exported                5719 bp DNA     circular SYN 29-AUG-2024
DEFINITION  Cloning vector pK18mobsacB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5719)
  AUTHORS   Kvitko BH, Collmer A.
  TITLE     Construction of Pseudomonas syringae pv. tomato DC3000 mutant and 
            polymutant strains
  JOURNAL   Methods Mol. Biol. 712, 109-128 (2011)
  PUBMED    21359804
REFERENCE   2  (bases 1 to 5719)
  AUTHORS   Kvitko BH, Collmer A.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-NOV-2008) Plant Pathology and Plant-Microbe Biology, 
            Cornell University, 334 Plant Science, Ithaca, NY 14853, USA
REFERENCE   3  (bases 1 to 5719)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5719)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 5719)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods
            Mol. Biol. 712, 109-128 (2011)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (04-NOV-2008) Plant Pathology and Plant-Microbe Biology, Cornell
            University, 334 Plant Science, Ithaca, NY 14853, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             346..1137
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        1186..1631
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             1632..3050
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     oriT            complement(3407..3516)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     promoter        complement(3751..3841)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     promoter        complement(4035..4125)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      4485..5073
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5361..5382
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5397..5427
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5435..5451
                     /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)."
     primer_bind     5459..5475
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    5484..5540
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5544..5560)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"

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