Basic Vector Information
pET28a-MH6-EsCas13d 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 ℃.
pET28a-MH6-EsCas13d vector Sequence
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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