p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A vector (V010155)

MSDS

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 Vector Map

Plasmid Resuspension 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 ℃.

p2CT-His-MBP-Lbu_C2c2_R472A_H477A_R1048A_H1053A vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       p2CT-His-MBP-Lbu        9437 bp DNA     circular SYN 10-JAN-2018
DEFINITION  Bacterial expression of L. buccalis C2c2 CRISPR effector (double 
            HEPN nuclease 1 and 2 inactive mutant)..
ACCESSION   .
VERSION     .
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."
COMMENT     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=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"

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