Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005261 | pK19mobsacB | 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 pK19mobsacB is a Corynebacterium glutamicum gene Knockout vector.
- Vector Name:
- pK19mobsacB
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5719 bp
- Type:
- Function E.coli Editing plasmids
- Replication origin:
- ori
- Source/Author:
- Okamoto S, Niki H.
- Promoter:
- sacB
- Growth Strain(s):
- JM109
- Growth Temperature:
- 37℃
pK19mobsacB 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.
References
- Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene. 1994 Jul 22;145(1):69-73.
pK19mobsacB vector Sequence
LOCUS 40924_26496 5719 bp DNA circular SYN 18-DEC-2018
DEFINITION Cloning vector pK19mobsacB DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5719)
AUTHORS Okamoto S, Niki H.
TITLE NBRP cloning vector collection sequence project
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 5719)
AUTHORS Okamoto S, Niki H.
TITLE Direct Submission
JOURNAL Submitted (07-APR-2017) Contact:Sho Okamoto National Institute of
Genetics, Microbial Genetics Laboratory; 1111 Yata, Mishima,
Shizuoka 411-8540, Japan URL
:https://www.nig.ac.jp/labs/MicroGen/index.html
REFERENCE 3 (bases 1 to 5719)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5719)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(07-APR-2017) Contact:Sho Okamoto National Institute of Genetics,
Microbial Genetics Laboratory; 1111 Yata, Mishima, Shizuoka
411-8540, Japan URL :https://www.nig.ac.jp/labs/MicroGen/index.html"
COMMENT SGRef: number: 3; 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 complement(5487..5543)
/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"