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pTC223 vector (V016614)

Price Information

Cat No. Plasmid Name Availability Add to cart
V016614 pTC223 In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
pTC223
Antibiotic Resistance:
Kanamycin
Length:
17961 bp
Type:
Gene knockout
Replication origin:
ori
Host:
Plants
Selection Marker:
Neo/G418
Promoter:
AtU6-26
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pTC223 vector Map

Copy Sequence View Map
pTC22317961 bp8001600240032004000480056006400720080008800960010400112001200012800136001440015200160001680017600LB T-DNA repeatCaMV 35S promoterCas9HSP terminatorAtU6-26gRNA scaffoldNOS promoterNeoR/KanRCaMV poly(A) signalCaMV 35S promoterReplication-associated protein ARB T-DNA repeatpVS1 StaApVS1 RepApVS1 oriVbomoriKanR

Plasmid 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 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

pTC223 vector Sequence

LOCUS       V016614                17961 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V016614
VERSION     V016614
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 17961)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..17961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..25
                     /label="LB T-DNA repeat"
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        966..1311
                     /label="CaMV 35S promoter"
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             1345..5448
                     /label="Cas9"
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     terminator      5492..5740
                     /label="HSP terminator"
                     /note="efficient transcription terminator from the
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya
                     et al., 2010)"
     promoter        5785..6208
                     /label="AtU6-26"
                     /note="Arabidopsis U6-26 gene promoter"
     misc_RNA        6228..6303
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     promoter        7452..7635
                     /label="NOS promoter"
                     /note="nopaline synthase promoter"
     CDS             7699..8487
                     /label="NeoR/KanR"
                     /note="aminoglycoside phosphotransferase"
     polyA_signal    8516..8690
                     /label="CaMV poly(A) signal"
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        8901..9246
                     /label="CaMV 35S promoter"
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     CDS             complement(10435..11310)
                     /note="Replication-associated protein A from Bean yellow
                     dwarf virus. Accession#: O39521"
                     /label="Replication-associated protein A"
     misc_feature    11684..11708
                     /label="RB T-DNA repeat"
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             13008..13634
                     /label="pVS1 StaA"
                     /note="stability protein from the Pseudomonas plasmid pVS1
                     (Heeb et al., 2000)"
     CDS             14071..15135
                     /label="pVS1 RepA"
                     /note="replication protein from the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     rep_origin      15204..15398
                     /label="pVS1 oriV"
                     /note="origin of replication for the Pseudomonas plasmid
                     pVS1 (Heeb et al., 2000)"
     misc_feature    15742..15882
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(16068..16656)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(16746..17537)
                     /label="KanR"
                     /note="aminoglycoside phosphotransferase"