pET28a-MH6-EsCas13d vector (V010138)

Basic Vector Information

      • Vector Name:
      • pET28a-MH6-EsCas13d
      • Antibiotic Resistance:
      • Kanamycin
      • Length:
      • 8248 bp
      • Type:
      • Bacterial Expression, CRISPR
      • Copy Number:
      • Low Copy
      • Promoter:
      • lac
      • Cloning Method:
      • Restriction Enzyme
      • 5' Primer:
      • atgggcaaaaaaatccatgcac

pET28a-MH6-EsCas13d vector Vector Map

pET28a-MH6-EsCas13d8248 bp400800120016002000240028003200360040004400480052005600600064006800720076008000T7 promoterlac operatorlac promoterRBS6xHisJ23119 promoterT7 terminatorf1 oriKanRoriL4440bompGEX 3'CAP binding sitelacIlacI promoterpBRrevBam

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 ℃.

pET28a-MH6-EsCas13d vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       40924_18406        8248 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Expresses E. coli codon optimized EsCas13d in the pET28a backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8248)
  AUTHORS   Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA
  TITLE     Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively
            Modulated by a WYL-Domain-Containing Accessory Protein.
  JOURNAL   Mol Cell. 2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028.
  PUBMED    29551514
REFERENCE   2  (bases 1 to 8248)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8248)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Cell. 
            2018 Mar 9. pii: S1097-2765(18)30173-4. doi: 
            10.1016/j.molcel.2018.02.028."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=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        53..82
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             102..124
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             156..173
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     promoter        3054..3088
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological
                     Parts BBa_J23119)"
     terminator      3197..3244
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3281..3736
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3831..4643)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      4765..5353
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5507..5524
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(5539..5678)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5764..5786
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5783..5971)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6746..6767)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6783..7862)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7863..7940)
                     /label=lacI promoter
     primer_bind     8146..8165
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"

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