Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V010258 | pKLAC2 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pKLAC2 is an expression vector capable both of replication in E. coli and stable integration into the genome of the yeast Kluyveromyces lactis.
- Vector Name:
- pKLAC2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9106 bp
- Type:
- Yeast Plasmids
- Replication origin:
- ori
- Source/Author:
- New England Biolabs
- Copy Number:
- High copy number
- Promoter:
- LAC4 (3')
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pKLAC2 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
- Brain-Isasi S, Álvarez-Lueje A, Higgins TJV. Heterologous expression of an α-amylase inhibitor from common bean (Phaseolus vulgaris) in Kluyveromyces lactis and Saccharomyces cerevisiae. Microb Cell Fact. 2017 Jun 15;16(1):110.
- De Silva C, Dhanapala P, King S, Doran T, Tang M, Suphioglu C. Immunological Comparison of Native and Recombinant Hen's Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis. Nutrients. 2018 Jun 12;10(6):757.
- Stressler T, Leisibach D, Lutz-Wahl S, Kuhn A, Fischer L. Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing. Appl Microbiol Biotechnol. 2016 Jun;100(12):5401-14.
pKLAC2 vector Sequence
LOCUS Exported 9106 bp DNA circular SYN 01-DEC-2025
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pKLAC2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9106)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..9106
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 989..1691
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 257..354
/label=Multiple Cloning Site
/note="A synthetic DNA sequence containing multiple
restriction enzyme recognition sites, used for inserting
foreign DNA fragments in molecular cloning experiments.;
id: 4352484"
terminator 371..988
/label=LAC4 terminator
/note="transcription terminator for Kluyveromyces lactis
LAC4"
promoter 989..1691
/label=ADH1 promoter
/note="promoter for alcohol dehydrogenase 1"
CDS 1712..3358
/codon_start=1
/gene="amdS"
/product="acetamidase from Aspergillus nidulans"
/label=amdS
/note="confers ability to process acetamide for selection
in fungal cells"
/translation="MPQSWEELAADKRARLAKTIPDEWKVQTLPAEDSVIDFPKKSGIL
SEAELKITEASAADLVSKLAAGELTSVEVTLAFCKRAAIAQQLTNCAHEFFPDAALAQA
RELDEYYAKHKRPVGPLHGLPISLKDQLRVKGYETSMGYISWLNKYDEGDSVLTTMLRK
AGAVFYVKTSVPQTLMVCETVNNIIGRTVNPRNKNWSCGGSSGGEGAIVGIRGGVIGVG
TDIGGSIRVPAAFNFLYGLRPSHGRLPYAKMANSMEGQETVHSVVGPITHSVEDLRLFT
KSVLGQEPWKYDSKVIPMPWRQSESDIIASKIKNGGLNIGYYNFDGNVLPHPPILRGVE
TTVAALAKAGHTVTPWTPYKHDFGHDLISHIYAADGSADVMRDISASGEPAIPNIKDLL
NPNIKAVNMNELWDTHLQKWNYQMEYLEKWREAEEKAGKELDAIIAPITPTAAVRHDQF
RYYGYASVINLLDFTSVVVPVTFADKNIDKKNESFKAVSELDALVQEEYDPEAYHGAPV
AVQVIGRRLSEERTLAIAEEVGKLLGNVVTP"
promoter 3994..4650
/label=LAC4 promoter (5')
/note="5' portion of the Kluyveromyces lactis LAC4
promoter"
promoter complement(4667..4685)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 4704..4826
/label=lac operon promoter
/note="Promoter region that regulates transcription of the
lactose operon genes, including lacZ, controlling bacterial
metabolism of lactose in response to environmental
conditions; id: 3799053"
rep_origin 4857..5859
/label=pUC19 replication origin
/note="Origin of replication derived from pUC19 plasmid,
containing the ColE1 origin sequence that enables
autonomous DNA replication in bacterial hosts.; id:
4370962"
CDS complement(5860..6720)
/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 6721..6851
/label=AmpR promoter
/note="Regulatory region that initiates transcription of
the ampicillin resistance gene in bacterial plasmids; id:
3672594"
rep_origin 6852..7307
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 7448..7464
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 7478..9006
/label=LAC4 promoter (3')
/note="3' portion of a modified Kluyveromyces lactis LAC4
promoter"
protein_bind 9012..9028
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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)."