pCRISPomyces-2 vector (V011616)

Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V011616	pCRISPomyces-2	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-2

Antibiotic Resistance:: Apramycin

Length:: 10994 bp

Type:: Bacterial Expression, CRISPR

Replication origin:: ori

Copy Number:: High Copy

Promoter:: rpsL

pCRISPomyces-2 vector Vector Map

View Map in NovoBuilder

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-2 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       40924_13420       10994 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Streptomyces expression of codon-optimized Cas9 and custom gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10994)
  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 10994)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 10994)
  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..10994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(156..1595)
                     /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            2762..2871
                     /label=oriT
                     /note="incP origin of transfer"
     promoter        3093..3394
                     /label=rpsL promoter
                     /note="promoter for the Xylanimonas cellulosilytica
                     ribosomal protein S12 gene"
     CDS             3395..7498
                     /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        7701..7985
                     /label=gapdh promoter
                     /note="glyceraldehyde-3-phosphate dehydrogenase promoter
                     from Eggerthella lenta"
     primer_bind     8140..8162
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8154..8171
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     8155..8171
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        8181..8199
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(8247..8265)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(8286..8302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8286..8302)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8299..8321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8310..8326
                     /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(8334..8364)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(8379..8400)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_RNA        8443..8518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8540..8574
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     rep_origin      complement(8616..9204)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             9408..10208
                     /codon_start=1
                     /label=ApmR
                     /note="aminoglycoside 3-N-acetyltransferase type IV"
                     /translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
                     LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
                     KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
                     LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
                     AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"