Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006225 | pFLP2 | 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 temperature sensitive λ cI repressor protein has a cI857 mutation that results in denaturation of the repressor when the temperature is raised from 30 to 42°C, thereby allowing lambda promoter expression. The repressor normally negatively regulates the expression of genes from the bacteriophage lambda pL and pR promoters. This repressive action is strongest at 30°C. However, when the temperature is raised, typically to 42°C, the functionality of the protein is lost and the cI repressor is no longer able to bind to the operators on its promoter. Therefore, lambda promoter(pL/pR) expression increases.
- Vector Name:
- pFLP2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9297 bp
- Type:
- Site-specific excision vector
- Replication origin:
- ori
- Source/Author:
- Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
- Promoter:
- sacB
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 30℃
pFLP2 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
- Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene. 1998 May 28;212(1):77-86. doi: 10.1016/s0378-1119(98)00130-9. PMID: 9661666.
pFLP2 vector Sequence
LOCUS Exported 9297 bp DNA circular SYN 25-OCT-2024
DEFINITION Site-specific excision vector pFLP2, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9297)
AUTHORS Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
TITLE A broad-host-range Flp-FRT recombination system for site-specific
excision of chromosomally-located DNA sequences: application for
isolation of unmarked Pseudomonas aeruginosa mutants
JOURNAL Gene 212 (1), 77-86 (1998)
PUBMED 9661666
REFERENCE 2 (bases 1 to 9297)
AUTHORS Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP.
TITLE Direct Submission
JOURNAL Submitted (17-FEB-1998) Microbiology, Colorado State University,
Fort Collins, CO 80523, USA
REFERENCE 3 (bases 1 to 9297)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 9297)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 9297)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Gene";
date: "1998"; volume: "212"; issue: "1"; pages: "77-86"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(17-FEB-1998) Microbiology, Colorado State University, Fort Collins,
CO 80523, USA"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9297
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(232..583)
/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 597..1430
/codon_start=1
/product="replication protein for the broad-host-range
plasmid pRO1600 from Pseudomonas aeruginosa "
/label=pRO1600 Rep
/translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
primer_bind 1591..1607
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(2338..3759)
/codon_start=1
/gene="Bacillus subtilis sacB"
/product="secreted levansucrase that renders bacterial
growth sensitive to sucrose"
/label=SacB
/note="negative selection marker"
/translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
VKDSILEQGQLTVNK"
promoter complement(3760..4205)
/label=sacB promoter
/note="sacB promoter and control region"
CDS complement(4426..5697)
/codon_start=1
/product="site-specific recombinase"
/label=FLP
/note="FLP is a site-specific recombinase from
Saccharomyces cerevisiae. Recombination occurs at FRT
sequences."
/translation="MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
SYNKALKKNAPYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
YPAWNGIISQEVLDYLSSYINRRI"
RBS 5701..5709
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
promoter complement(5726..5755)
/label=lambda PR promoter
promoter 5762..5791
/label=pRM promoter
CDS 5798..6511
/codon_start=1
/gene="cIts"
/product="temperature-sensitive variant of the phage lambda
repressor"
/label=lambda repressor (ts)
/note="thermosensitivity is conferred by the A67T mutation"
/translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
VGKVIASQWPEETFG"
primer_bind complement(6800..6816)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6824..6840)
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(6848..6878)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(6893..6914)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
oriT 7298..7406
/label=oriT
/note="incP origin of transfer"
rep_origin complement(7478..8066)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8237..9097)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9098..9202)
/label=AmpR promoter