Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V008105 | pCW-LIC | 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 sequencing results indicat that SacB contains a W88C mutation, but it does not affect its function. The bacteria containing mutant W88C SacB is still sensitive to sucrose.
- Vector Name:
- pCW-LIC
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6980 bp
- Type:
- Expression vector
- Replication origin:
- ori
- Source/Author:
- Loppnau P, Kozieradzki I, Arrowsmith C.
- Promoter:
- sacB
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pCW-LIC 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.
pCW-LIC vector Sequence
LOCUS Exported 6980 bp DNA circular SYN 12-NOV-2024
DEFINITION Expression vector pCW-LIC, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6980)
AUTHORS Loppnau P, Kozieradzki I, Arrowsmith C.
TITLE Expression vectors for high-throughput In-Fusion cloning
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6980)
AUTHORS Loppnau P, Kozieradzki I, Arrowsmith C.
TITLE Direct Submission
JOURNAL Submitted (27-FEB-2007) Structural Genomics Consortium, University
of Toronto, 100 College Street, Toronto, ON M5G1L5, Canada
REFERENCE 3 (bases 1 to 6980)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6980)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 6980)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName:
"Unpublished"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(27-FEB-2007) Structural Genomics Consortium, University of Toronto,
100 College Street, Toronto, ON M5G1L5, Canada"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6980
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 12..465
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 492..596
/label=AmpR promoter
CDS 597..1454
/label=AmpR
/note="beta-lactamase"
rep_origin 1628..2216
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(2402..2544)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(2953..3126)
/label=lacZ-alpha
/note="LacZ-alpha fragment of beta-galactosidase"
protein_bind complement(3146..3162)
/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(3170..3200)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3215..3236)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3252..4331)
/label=lacI
/note="lac repressor"
promoter complement(4332..4409)
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
protein_bind 4461..4482
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4497..4527
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
protein_bind 4535..4551
/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 4606..4634
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 4640..4660
/label=lac repressor protein binding site
/bound_moiety="lac repressor protein"
protein_bind 4642..4658
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
regulatory 4701..4729
/label=Ptac
/note="Ptac"
/regulatory_class="promoter"
promoter 4701..4729
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
regulatory 4701..4706
/label=derived from trp promoter
/note="derived from trp promoter"
/regulatory_class="minus_35_signal"
regulatory 4723..4729
/label=derived from lacUV5 promoter
/note="derived from lacUV5 promoter"
/regulatory_class="minus_10_signal"
protein_bind 4735..4755
/label=lac repressor protein binding site
/bound_moiety="lac repressor protein"
protein_bind 4737..4753
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."
misc_feature 4784..4798
/note="5'-cloning sequence; add to upstream PCR primer"
protein_bind complement(4826..4859)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
promoter 4874..5319
/label=sacB promoter
/note="sacB promoter and control region"
CDS 5320..6738
/codon_start=1
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSCPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
misc_feature 6814..6828
/note="3'-cloning sequence; add complement to downstream
PCR primer"
regulatory 6862..6889
/label=trp operon transcription termination sequence
/note="trp operon transcription termination sequence"
/regulatory_class="terminator"