Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012664 | pLCA.66/2272 | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pLCA.66/2272
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6678 bp
- Type:
- Cre/Lox ; BAC recombineering
- Replication origin:
- ori
- Selection Marker:
- Neomycin (select with G418) ; ganciclovir
- Copy Number:
- High Copy
- Promoter:
- mPGK
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T3
- 3' Primer:
- T7
pLCA.66/2272 vector 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.
pLCA.66/2272 vector Sequence
LOCUS 40924_27598 6678 bp DNA circular SYN 13-MAY-2021
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6678)
AUTHORS Chen SX, Osipovich AB, Ustione A, Potter LA, Hipkens S, Gangula R,
Yuan W, Piston DW, Magnuson MA
TITLE Quantification of factors influencing fluorescent protein expression
using RMCE to generate an allelic series in the ROSA26 locus in
mice.
JOURNAL Dis Model Mech. 2011 Feb 14. ():.
PUBMED 21324933
REFERENCE 2 (bases 1 to 6678)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6678)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Dis Model
Mech. 2011 Feb 14. ():."
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6678
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..500
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
regulatory 514..523
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 526..1119
/codon_start=1
/label=PuroR
/note="puromycin N-acetyltransferase"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 1126..2118
/codon_start=1
/label=Delta-TK
/note="truncated herpes simplex virus thymidine kinase
(Salomon et al., 1995)"
/translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
polyA_signal 2149..2356
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 2419..2466
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 2485..3285
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase from Tn5"
/translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3326..3550
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 3616..3649
/label=lox2272
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are
compatible with each other, but incompatible with loxP or
loxN sites (Lee and Saito, 1988)."
promoter complement(3726..3744)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3751..3767)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3909..4364
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4390..4494
/label=AmpR promoter
CDS 4495..5352
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5526..6114
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6268..6285
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 6402..6423
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6438..6468
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6476..6492
/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)."
primer_bind 6500..6516
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 6537..6555
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
protein_bind 6613..6646
/label=lox66
/note="Right element (RE) mutant of loxP (Araki et al.,
2010). Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
promoter 6672..6678
/label=mPGK
/note="Mouse phosphoglycerate kinase 1 promoter"