pZE21-MCS vector (Cat. No.: V039151)
- Name:
- pZE21-MCS
- Antibiotic Resistance:
- Kanamycin
- Length:
- 2265 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- E. coli
- Selection Marker:
- Neo/G418
- Copy Number:
- High
- Promoter:
- PLtetO-1
- 3' Primer:
- rrnBT1-R
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
- Expression Method:
- Transient
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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.
pZE21-MCS vector (Cat. No.: V039151) Sequence
LOCUS pZE21-MCS 2265 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION V039151
VERSION V039151
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 6..79
/note="modified phage lambda PL promoter with tet operator
sites (Lutz and Bujard, 1997)"
/label="PLtetO-1 promoter"
RBS 89..100
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
/label="RBS"
terminator 207..293
/gene="Escherichia coli rrnB"
/note="transcription terminator T1 from the E. coli rrnB
gene"
/label="rrnB T1 terminator"
rep_origin complement(458..1046)
/direction=LEFT
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/label="ori"
terminator complement(1134..1228)
/gene=""
/note="transcription terminator from phage lambda"
/label="lambda t0 terminator"
CDS complement(1259..2053)
/codon_start=1
/gene="aph(3')-II (or nptII)"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin®)"
/product="aminoglycoside phosphotransferase from Tn5"
/transl_table=1
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
/label="NeoR/KanR"