Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005565 | pHERD30T | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pHERD30T is a bacterial broad-host vector, especially suitable for Pseudomonas aeruginosa.
- Vector Name:
- pHERD30T
- Antibiotic Resistance:
- Gentamicin
- Length:
- 5216 bp
- Type:
- Escherichia-Pseudomonas shuttle vector
- Replication origin:
- ori
- Source/Author:
- Qiu D, Damron FH, Mima T, Schweizer HP, Yu HD.
- Promoter:
- araBAD
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pHERD30T vector Map
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.
References
- Pinilla-Redondo R, Shehreen S, Marino ND, Fagerlund RD, Brown CM, Sørensen SJ, Fineran PC, Bondy-Denomy J. Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements. Nat Commun. 2020 Nov 6;11(1):5652.
pHERD30T vector Sequence
LOCUS 40924_24488 5216 bp DNA circular SYN 18-DEC-2018
DEFINITION Escherichia-Pseudomonas shuttle vector pHERD30T, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5216)
AUTHORS Qiu D, Damron FH, Mima T, Schweizer HP, Yu HD.
TITLE PBAD-based shuttle vectors for functional analysis of toxic and
highly regulated genes in Pseudomonas and Burkholderia spp. and
other bacteria
JOURNAL Appl. Environ. Microbiol. 74 (23), 7422-7426 (2008)
PUBMED 18849445
REFERENCE 2 (bases 1 to 5216)
AUTHORS Qiu D, Yu HD.
TITLE Direct Submission
JOURNAL Submitted (31-MAR-2008) Biochemistry and Microbiology, Marshall
University Joan C. Edwards School of Medicine, 1 John Marshall
Drive, Huntington, WV 25755, USA
REFERENCE 3 (bases 1 to 5216)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 5216)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
Environ. Microbiol."; date: "2008"; volume: "74"; issue: "23";
pages: "7422-7426"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(31-MAR-2008) Biochemistry and Microbiology, Marshall University
Joan C. Edwards School of Medicine, 1 John Marshall Drive,
Huntington, WV 25755, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5216
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(733..1263)
/codon_start=1
/label=GmR
/note="gentamycin acetyltransferase"
/translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
EEVMHFDIDPSTAT"
promoter complement(1452..1480)
/label=Pc promoter
/note="class 1 integron promoter"
rep_origin complement(1521..1872)
/direction=LEFT
/label=pRO1600 oriV
/note="broad-host-range origin of replication from
Pseudomonas aeruginosa plasmid pRO1600; requires the
pRO1600 Rep protein for replication (West et al., 1994)"
CDS 1886..2716
/codon_start=1
/label=pRO1600 Rep
/note="replication protein for the broad-host-range plasmid
pRO1600 from Pseudomonas aeruginosa"
/translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
primer_bind 2880..2896
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
misc_feature complement(2900..2956)
/label=MCS
/note="pUC18/19 multiple cloning site"
RBS complement(2982..3004)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
promoter complement(3031..3315)
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"
CDS 3342..4217
/codon_start=1
/label=araC
/note="L-arabinose regulatory protein"
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
oriT 4377..4485
/label=oriT
/note="incP origin of transfer"
rep_origin complement(4557..5145)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"