Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017498 | pRECas1 | 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 pRECas1 system is a versatile CRISPR-Cas9 gene editing platform designed for widespread use in Clostridia and other anaerobic Gram-positive species. It includes five vectors, collectively referred to as pRECas1. A component of this system, RiboCas, features the Cas9 enzyme regulated by a precisely controlled, theophylline-responsive riboswitch. This innovative approach addresses many challenges associated with Cas9 toxicity in both the shuttle host and target organisms.
- Vector Name:
- pRECas1
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 8842 bp
- Type:
- CRISPR Plasmids
- Source/Author:
- Cañadas IC, Groothuis D, Zygouropoulou M, Rodrigues R, Minton NP.
- Promoter:
- Pfdx
- Growth Strain(s):
- DH10b
- Growth Temperature:
- 37℃
pRECas1 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
- Zhou P, G C B, Stolte F, Wu C. Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum. Appl Environ Microbiol. 2024 Feb 21;90(2):e0166523. doi: 10.1128/aem.01665-23. Epub 2024 Jan 8. PMID: 38185820; PMCID: PMC10880640.
- Poulalier-Delavelle M, Baker JP, Millard J, Winzer K, Minton NP. Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum. Front Bioeng Biotechnol. 2023 Jun 23;11:1213236. doi: 10.3389/fbioe.2023.1213236. PMID: 37425362; PMCID: PMC10328091.
- Dykstra JC, van Oort J, Yazdi AT, Vossen E, Patinios C, van der Oost J, Sousa DZ, Kengen SWM. Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO. Microb Cell Fact. 2022 Nov 23;21(1):243. doi: 10.1186/s12934-022-01964-5. PMID: 36419165; PMCID: PMC9686113.
pRECas1 vector Sequence
LOCUS Exported 8842 bp DNA circular SYN 04-SEP-2024
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pRECas1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8842)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8842
/label=source
/organism="synthetic DNA construct"
terminator 140..193
/label=terminator
primer_bind 194..210
/label=M13 rev
CDS complement(249..4355)
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
misc_feature 4356..4608
/label=Pfdx-E
misc_feature 4615..4883
/label=p1339 (Cac araE)
misc_feature 4890..4892
/label=sgRNA insertion site
misc_feature 4899..4901
/label=HA insertion site
misc_feature 4904..6528
/label=pCB102
gene 6617..7240
/label=catP
rep_origin 7417..8005
/label=ori
oriT 8326..8435
/label=incP origin of transfer
CDS 8468..8839
/label=traJ