Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006715 | pCRISPomyces-1 | 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:
- pCRISPomyces-1
- Antibiotic Resistance:
- Apramycin
- Length:
- 11473 bp
- Type:
- Bacterial Expression, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- rpsL
pCRISPomyces-1 vector 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.
pCRISPomyces-1 vector Sequence
LOCUS 40924_13415 11473 bp DNA circular SYN 13-MAY-2021
DEFINITION Streptomyces expression of codon-optimized Cas9, tracrRNA, and
custom crRNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11473)
AUTHORS Cobb RE, Wang Y, Zhao H
TITLE High-Efficiency Multiplex Genome Editing of Streptomyces Species
Using an Engineered CRISPR/Cas System.
JOURNAL ACS Synth Biol. 2014 Dec 8.
PUBMED 25458909
REFERENCE 2 (bases 1 to 11473)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 11473)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
Biol. 2014 Dec 8."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..11473
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 79..380
/label=rpsL promoter
/note="promoter for the Xylanimonas cellulosilytica
ribosomal protein S12 gene"
CDS 381..4484
/codon_start=1
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/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"
promoter 4687..5047
/label=rpsL promoter
/note="promoter for the Cellulomonas flavigena ribosomal
protein S12 gene"
misc_RNA 5048..5126
/label=tracrRNA
/note="trans-activating CRISPR RNA for the Streptococcus
pyogenes CRISPR/Cas9 system"
terminator 5150..5183
/label=lambda t0 terminator
/note="minimal transcription terminator from phage lambda
(Scholtissek and Grosse, 1987)"
promoter 5184..5468
/label=gapdh promoter
/note="glyceraldehyde-3-phosphate dehydrogenase promoter
from Eggerthella lenta"
primer_bind 5658..5680
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 5672..5689
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 5673..5689
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 5699..5717
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter complement(5765..5783)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(5804..5820)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5804..5820)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5817..5839)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5828..5844
/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)."
promoter complement(5852..5882)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(5897..5918)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
terminator 5991..6038
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin complement(6081..6669)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 6873..7673
/codon_start=1
/label=ApmR
/note="aminoglycoside 3-N-acetyltransferase type IV"
/translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
CDS complement(8615..10054)
/codon_start=1
/label=pSG5 Rep
/note="replication initiator protein from the Streptomyces
ghanaensis plasmid pSG5"
/translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
APVLQPWGQHPGDCLRVDTAVA"
oriT 11221..11330
/label=oriT
/note="incP origin of transfer"