Basic Vector Information
ptCC9 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 ℃.
ptCC9 vector Sequence
LOCUS 40924_42714 8857 bp DNA circular SYN 18-DEC-2018 DEFINITION Shuttle vector ptCC9, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8857) AUTHORS Stukenberg D, Zauner S, Dell'Aquila G, Maier UG. TITLE Optimizing CRISPR/Cas9 for the Diatom Phaeodactylum tricornutum JOURNAL Front Plant Sci 9, 740 (2018) PUBMED 29928285 REFERENCE 2 (bases 1 to 8857) AUTHORS Stuckenberg D, Zauner S. TITLE Direct Submission JOURNAL Submitted (29-MAR-2018) Philipps-University Marburg, Cellbiology, Karl-von-Frisch Str. 8, Marburg 35043, Deutschland REFERENCE 3 (bases 1 to 8857) TITLE Direct Submission REFERENCE 4 (bases 1 to 8857) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Front Plant Sci 9, 740 (2018)" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (29-MAR-2018) Philipps-University Marburg, Cellbiology, Karl-von-Frisch Str. 8, Marburg 35043, Deutschland" COMMENT SGRef: number: 3; type: "Journal Article" COMMENT ##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..8857 /mol_type="other DNA" /organism="synthetic DNA construct" regulatory 1..415 /note="nitrate reductase; derived from Phaeodactylum tricornutum" /regulatory_class="promoter" CDS 431..496 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 503..523 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 548..4648 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 4649..4696 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" regulatory 4700..4972 /note="nitrate reductase; derived from Phaeodactylum tricornutum" /regulatory_class="terminator" regulatory 5010..5288 /note="U6-RNA promoter; derived from Phaeodactylum tricornutum" /regulatory_class="promoter" misc_RNA 5310..5385 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" regulatory 5386..5723 /note="U6-RNA terminator; derived from Phaeodactylum tricornutum" /regulatory_class="terminator" regulatory 5724..5916 /note="fcpB promoter; derived from Phaeodactylum tricornutum" /regulatory_class="promoter" CDS 5917..6288 /label=BleoR /note="antibiotic-binding protein" regulatory 6292..6532 /note="fcpB teminator; derived from Phaeodactylum tricornutum" /regulatory_class="terminator" promoter complement(6543..6561) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" rep_origin complement(6819..7407) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7581..8438) /label=AmpR /note="beta-lactamase" promoter complement(8439..8543) /label=AmpR promoter
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