p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A vector (V010155)

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Cat No.	Plasmid Name	Availability	Add to cart
V010155	p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A	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

Gene/Insert name Lbu_C2c2_R472A_H477A_R1048A_H1053A Species Leptotrichia buccalis Insert Size (bp) 3477 Mutation R472A, H477A, R1048A, H1053A GenBank IDGI_506250229 Tag / Fusion Protein His6-MBP (N terminal on insert)

Vector Name:: p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A

Antibiotic Resistance:: Ampicillin

Length:: 9437 bp

Type:: CRISPR Plasmids

Replication origin:: ori

Source/Author:: Jennifer Doudna

Copy Number:: High copy number

Promoter:: tet

Cloning Method:: Enzyme Cut

5' Primer:: T7 promoter

3' Primer:: T7 terminator

Fusion Tag:: MBP

Expression Method:: IPTG induced

p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A vector Map

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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.

p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A vector Sequence

LOCUS       V010155                 9437 bp    DNA     circular SYN 10-JAN-2018
DEFINITION  Exported.
ACCESSION   V010155
VERSION     V010155
KEYWORDS    p2CT-His-MBP-Lbu-C2c2-R472A-H477A-R1048A-H1053A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9437)
  AUTHORS   East-Seletsky A, O'Connell MR, Knight SC, Burstein D, Cate JH, Tjian
            R, Doudna JA
  TITLE     Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA
            processing and RNA detection.
  JOURNAL   Nature. 2016 Sep 26. doi: 10.1038/nature19802.
   PUBMED   27669025
REFERENCE   2  (bases 1 to 9437)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9437)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature.
            2016 Sep 26. doi: 10.1038/nature19802."
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        104..208
                     /label="AmpR promoter"
     CDS             209..1066
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      1240..1828
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    complement(2014..2156)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(2261..2449)
                     /label="rop"
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     promoter        4010..4028
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4117..4139
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4159..4176
                     /label="6xHis"
                     /note="6xHis affinity tag"
     CDS             4186..5286
                     /label="MBP"
                     /note="maltose binding protein from E. coli"
     CDS             5341..5361
                     /label="TEV site"
                     /note="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
     CDS             5368..8844
                     /gene="cas13a"
                     /label="CRISPR-associated endoribonuclease Cas13a"
                     /note="CRISPR-associated endoribonuclease Cas13a from
                     Leptotrichia buccalis (strain ATCC 14201 / DSM 1135 / JCM
                     12969 / NCTC 10249 / C-1013-b). Accession#: C7NBY4"
     terminator      8987..9034
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     promoter        complement(9400..9428)
                     /label="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"