Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000868 | pC013 - Twinstrep-SUMO-huLwCas13a | 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:
- pC013 - Twinstrep-SUMO-huLwCas13a
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9542 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Copy Number:
- Low Copy
pC013 - Twinstrep-SUMO-huLwCas13a 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.
pC013 - Twinstrep-SUMO-huLwCas13a vector Sequence
LOCUS 40924_7856 9542 bp DNA circular SYN 13-MAY-2021
DEFINITION Twinstrep-SUMO-huLwCas13a for recombinant protein bacterial
expression. Insert is human codon optimized but expresses well in
bacteria..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9542)
AUTHORS Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung
J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C,
Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC,
Collins JJ, Zhang F
TITLE Nucleic acid detection with CRISPR-Cas13a/C2c2.
JOURNAL Science. 2017 Apr 13. pii: eaam9321. doi: 10.1126/science.aam9321.
PUBMED 28408723
REFERENCE 2 (bases 1 to 9542)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9542)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Science.
2017 Apr 13. pii: eaam9321. doi: 10.1126/science.aam9321."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9542
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 278..417
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(603..1191)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1365..2222)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(2223..2327)
/label=AmpR promoter
promoter 2440..2468
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
terminator complement(2643..2690)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(2754..6209)
/codon_start=1
/label=LwCas13a
/note="Leptotrichia wadei CRISPR-Cas effector that acts as
an RNA-guided RNAse (Abudayyeh et al., 2017)"
/translation="MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIY
IKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEY
DLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINEN
NVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEK
YKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLD
KEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLL
NKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETE
NENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNE
IEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLK
IFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFW
SVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQ
KFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIE
SNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKY
TENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDF
ELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEK
IADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAI
GNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEE
IFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNY
IAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKK
IEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE"
CDS complement(6213..6506)
/codon_start=1
/label=SUMO
/note="cleavable ubiquitin-like protein tag"
/translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
CDS complement(6507..6590)
/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 complement(6603..6620)
/codon_start=1
/label=thrombin site
/note="thrombin recognition and cleavage site"
/translation="LVPRGS"
CDS complement(6630..6647)
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
RBS complement(6666..6688)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(6703..6727)
/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(6728..6746)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 7055..7132
/label=lacI promoter
CDS 7133..8212
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
protein_bind 8228..8249
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS join(9527..9542,1..173)
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"