Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V003400 | pSD1 | 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 pSD1 is a E. coli-S. aureus shuttle plasmid for constitutive expression of the sgRNA and conditional expression of dCas9 under the control of an anhydrotetracycline (ATc)-inducible promoter, making this system both inducible and reversible. It reuires both the dCas9 and sgRNA to construct an efficient system for gene knockdown in S. aureus. SapI-digested plasmid pSD1 was used to insert sgRNA.
- Vector Name:
- pSD1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10904 bp
- Type:
- Staphylococcus aureus knockdown vector
- Replication origin:
- ori
- Source/Author:
- Zhao C, Sun B.
- Selection Marker:
- Tetracycline
- Growth Strain(s):
- DH5alpha
- Growth Temperature:
- 37℃
pSD1 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
- Zhao C, Shu X, Sun B. Construction of a Gene Knockdown System Based on Catalytically Inactive ("Dead") Cas9 (dCas9) in Staphylococcus aureus. Appl Environ Microbiol. 2017;83(12):e00291-17. Published 2017 May 31. doi:10.1128/AEM.00291-17
pSD1 vector Sequence
LOCUS pSD1 10904 bp DNA circular SYN 02-OCT-2024
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pSD1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10904)
AUTHORS 111111111
TITLE Direct Submission
REFERENCE 2 (bases 1 to 10904)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 10904)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10904
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 379..395
/label=M13 fwd
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
misc_RNA complement(408..483)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
regulatory complement(508..692)
/note="sgRNA; constitutive expression"
/regulatory_class="promoter"
terminator complement(705..732)
/label=T7Te terminator
/note="phage T7 early transcription terminator"
terminator complement(748..819)
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
CDS complement(835..4941)
/codon_start=1
/product="catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes Type II
CRISPR/Cas system"
/label=dCas9
/note="dCas9"
/note="RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10 mutation in the
RuvC-like domain and the H840A mutation in the HNH domain"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
protein_bind complement(4993..5011)
/label=tet operator
/note="bacterial operator O1 for the tetR and tetA genes"
CDS 5121..5744
/codon_start=1
/gene="tetR from transposon Tn10"
/product="tetracycline repressor TetR"
/label=tetR from transposon Tn10
/note="TetR"
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
CDS complement(5985..5999)
/codon_start=1
/product="enterokinase recognition and cleavage site"
/label=enterokinase recognition and cleavage site
/note="enterokinase site"
/translation="DDDDK"
CDS 7490..8137
/gene="cat"
/label=Chloramphenicol acetyltransferase
/note="Chloramphenicol acetyltransferase from
Staphylococcus aureus. Accession#: P00485"
primer_bind complement(8683..8699)
/label=M13 rev
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 8707..8723
/label=lac repressor encoded by lacI binding site
/bound_moiety="lac repressor encoded by lacI"
/note="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 8731..8761
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 8776..8797
/label=E. coli catabolite activator protein binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(9085..9673)
/direction=LEFT
/label=ori
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(9844..10704)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=bla
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(10705..10809)
/gene="bla"
/label=bla promoter
/note="AmpR promoter"