Basic Vector Information
This plasmid separately encodes a human codon-optimized SpCas9, a tracrRNA and customizable crRNA. Also known as pX260-U6-DR-BB-DR-Cbh-NLS-hSpCas9-NLS-H1-shorttracr-PGK-puro
pX260 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 ℃.
pX260 vector Sequence
LOCUS pX260. 10203 bp DNA circular SYN 11-SEP-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS pX260.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10203)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 10203)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..10203
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 157..442
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard CMV
enhancer"
repeat_region 493..528
/label=DR
/note="DR"
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
repeat_region 545..580
/label=DR
/note="direct repeat for the Streptococcus pyogenes
CRISPR/Cas system"
promoter complement(591..831)
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
rep_origin complement(893..1481)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1655..2512)
/label=AmpR
/note="beta-lactamase"
promoter complement(2513..2617)
/label=AmpR promoter
rep_origin complement(2899..3354)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(3630..4226)
/label=PuroR
/note="puromycin N-acetyltransferase"
promoter complement(4266..4765)
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
protein_bind complement(4826..4859)
/label=loxP
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_RNA complement(5030..5108)
/label=tracrRNA
/note="trans-activating CRISPR RNA for the Streptococcus
pyogenes CRISPR/Cas9 system"
promoter complement(5119..5333)
/label=H1 promoter
/note="human H1 RNA promoter"
polyA_signal complement(5347..5554)
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
CDS complement(5588..5635)
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=bipartite nuclear localization signal from
nucl
/note="nucleoplasmin NLS"
/translation="KRPAATKKAGQAKKKK"
CDS complement(5636..9736)
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS complement(9761..9781)
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/label=nuclear localization signal of SV40 large T
ant
/note="SV40 NLS"
/translation="PKKKRKV"
CDS complement(9788..9853)
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=three tandem FLAG
/note="3xFLAG"
/translation="DYKDHDGDYKDHDIDYKDDDDK"
intron complement(9874..10101)
/label=hybrid intron
/note="hybrid between chicken beta-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)"
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