Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V007060 | pREDKI | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
Kanamycin low (25 ug/ml)
- Vector Name:
- pREDKI
- Antibiotic Resistance:
- Kanamycin
- Length:
- 9028 bp
- Type:
- Bacterial Expression ; Red recombinase and I-SceI
- Replication origin:
- pSC101 ori
- Copy Number:
- Low Copy
- Promoter:
- araBAD
- Cloning Method:
- Restriction Enzyme
pREDKI 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.
pREDKI vector Sequence
LOCUS Exported 9028 bp DNA circular SYN 06-AUG-2024
DEFINITION E coli Genetic Modification.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9028)
AUTHORS Yang J, Sun B, Huang H, Jiang Y, Diao L, Chen B, Xu C, Wang X, Liu
J, Jiang W, Yang S
TITLE High-Efficiency Scarless Genetic Modification in Escherichia coli by
Using Lambda Red Recombination and I-SceI Cleavage.
JOURNAL Appl Environ Microbiol. 2014 Jul 1;80(13):3826-3834. Epub 2014 Apr
18.
PUBMED 24747889
REFERENCE 2 (bases 1 to 9028)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9028)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 9028)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Appl
Environ Microbiol."; date: "2014-07-1"; volume: "80(13)"; pages:
"3826-3834. Epub 2014 Apr 18"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9028
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(22..897)
/codon_start=1
/label=araC
/note="L-arabinose regulatory protein"
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 924..1208
/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)"
RBS 1235..1243
/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)"
CDS 1252..1665
/codon_start=1
/label=Gam
/note="inhibitor of the host RecBCD nuclease in the lambda
Red system"
/translation="MDINTETEIKQKHSLTPFPVFLISPAFRGRYFHSYFRSSAMNAYY
IQDRLEAQSWARHYQQLAREEKEAELADDMEKGLPQHLFESLCIDHLQRHGASKKSITR
AFDDDVEFQERMAEHIRYMVETIAHHQVDIDSEV"
CDS 1674..2456
/codon_start=1
/label=Beta
/note="single-stranded DNA binding recombinase in the
lambda Red system"
/translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI
ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC
TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA
FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP
LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA"
CDS 2456..3133
/codon_start=1
/label=Exo
/note="5' to 3' double-stranded DNA exonuclease in the
lambda
Red system"
/translation="MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPR
SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP
IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW
VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG
EQWR"
terminator 3137..3381
/label=lambda tL3 terminator
/note="transcription terminator tL3 from phage lambda"
CDS complement(3417..4262)
/codon_start=1
/label=rhaR
/note="transcriptional activator of rhaR and rhaS"
/translation="VAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
D"
CDS complement(4339..5172)
/codon_start=1
/label=rhaS
/note="positive regulator of the rhaB promoter"
/translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
promoter 5318..5436
/label=rhaB promoter
/note="promoter of the E. coli rhaBAD operon, conferring
tight induction with L-rhamnose and repression with
D-glucose in the presence of RhaR and RhaS (Giacalone et
al., 2006)"
CDS 5459..6094
/codon_start=1
/product="Intron-encoded endonuclease"
/label=I-SceI
/translation="MHQKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGDAYI
RSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSFKHQAFNKLANLFIVNNKKTIPNNL
VENYLTPMSLAYWFMDDGGKWDYNKNSTNKSIVLNTQSFTFEEVEYLVKGLRNKFQLNC
YVKINKNKPIIYIDSMSYLIFYNLIKPYLIPQMMYKLPNTISSETFLK"
CDS complement(6208..7155)
/codon_start=1
/label=Rep101
/note="RepA protein needed for replication with the pSC101
origin"
/translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
rep_origin complement(7203..7425)
/direction=LEFT
/label=pSC101 ori
/note="low-copy replication origin that requires the Rep101
protein"
CDS 8191..9003
/codon_start=1
/label=KanR
/note="aminoglycoside phosphotransferase"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"