pCBC-DT3T4 vector (V017494)

Price Information

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

Basic Vector Information

Bacterial Resistance(s)Chloramphenicol, 25 μg/mL. Used as CRISPR/Cas based plant genome editing and gene regulation; used as template for making expression cassette with multiple gRNA target sites.

Vector Name:
pCBC-DT3T4
Antibiotic Resistance:
Chloramphenicol
Length:
3656 bp
Type:
Plant Expression, CRISPR
Source/Author:
Qi-Jun Chen
Copy Number:
High copy number
Promoter:
U6
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

pCBC-DT3T4 vector Map

pCBC-DT3T43656 bp60012001800240030003600CAT-RccdBIn lacZ geneT7gRNA scaffoldSP6 promoterIn lacZ geneEM7 promoterpENTR-FrrnB T2 terminatorrrnB T1 terminatorori

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.

pCBC-DT3T4 vector Sequence

LOCUS       Exported                3656 bp DNA     circular SYN 29-JUN-2024
DEFINITION  CRISPR/Cas based plant genome editing and gene regulation; used as 
            template for making expression cassette with multiple gRNA target 
            sites.
ACCESSION   .
VERSION     .
KEYWORDS    pCBC-DT3T4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3656)
  AUTHORS   Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ
  TITLE     A CRISPR/Cas9 toolkit for multiplex genome editing in plants.
  JOURNAL   BMC Plant Biol. 2014 Nov 29;14(1):327.
  PUBMED    25432517
REFERENCE   2  (bases 1 to 3656)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC Plant 
            Biol."; date: "2014-11-29"; volume: "14(1)"; pages: "327"
FEATURES             Location/Qualifiers
     source          1..3656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     235..254
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             complement(669..971)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(771..790)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     1094..1116
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1108..1125
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     1109..1125
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1132..1151
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1132..1150
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        complement(1808..1883)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1954..1972)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1955..1972)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1990..2006)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1990..2006)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2003..2025)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        complement(2045..2092)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     primer_bind     complement(2103..2126)
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     terminator      2134..2161
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      2253..2339
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     rep_origin      complement(2504..3092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2584..2603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3317..3656,1..320))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKSKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"