Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000010 | pC0061 PsmCas13 (B05) His6-TwinStrep-SUMO-BsaI | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pC0061 PsmCas13 (B05) His6-TwinStrep-SUMO-BsaI
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10050 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
pC0061 PsmCas13 (B05) His6-TwinStrep-SUMO-BsaI 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.
pC0061 PsmCas13 (B05) His6-TwinStrep-SUMO-BsaI vector Sequence
LOCUS 40924_7846 10050 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses PsmCas13b from a SUMO-tagged bacterial expression
construct.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10050)
AUTHORS Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang
F
TITLE Multiplexed and portable nucleic acid detection platform with Cas13,
Cas12a, and Csm6.
JOURNAL Science. 2018 Apr 27;360(6387):439-444. doi:
10.1126/science.aaq0179. Epub 2018 Feb 15.
PUBMED 29449508
REFERENCE 2 (bases 1 to 10050)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10050)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1126/science.aaq0179"; journalName: "Science"; date:
"2018-04-27- 27"; volume: "360"; issue: "6387"; pages: "439-444"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10050
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 1..25
/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)."
RBS 40..62
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 81..98
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 108..125
/codon_start=1
/label=thrombin site
/note="thrombin recognition and cleavage site"
/translation="LVPRGS"
CDS 138..221
/codon_start=1
/label=Twin-Strep-tag
/note="two Strep-Tag(R)II moieties connected by a linker
for enhanced binding to Strep-Tactin(R), an engineered form
of streptavidin (Schmidt et al., 2013)"
/translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
CDS 222..515
/codon_start=1
/label=SUMO
/note="cleavable ubiquitin-like protein tag"
/translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
terminator 4546..4593
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
promoter complement(4768..4796)
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
primer_bind complement(4823..4841)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4909..5013
/label=AmpR promoter
CDS 5014..5871
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 6045..6633
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6787..6804
/label=L4440
/note="L4440 vector, forward primer"
misc_feature complement(6819..6958)
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 7044..7066
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(7063..7251)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
protein_bind complement(8529..8550)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(8566..9645)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(9646..9723)
/label=lacI promoter
primer_bind 9929..9948
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 10032..10050
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"