pPinkα-LC vector (V013974)

Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V013974	pPinkα-LC	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:: pPinkα-LC

Antibiotic Resistance:: Ampicillin

Length:: 7973 bp

Type:: Protein expression

Replication origin:: ori

Host:: Yeast

Selection Marker:: ADE2

Promoter:: AOX1

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

pPinkα-LC vector Vector Map

View Map in NovoBuilder

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.

pPinkα-LC vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       62056_18515        7973 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7973)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             947..1201
                     /codon_start=1
                     /label=alpha-factor secretion signal
                     /note="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
     terminator      1254..1503
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        1523..1604
                     /label=PpADE2 LC promoter
                     /note="strong 82-bp promoter fragment that requires only 
                     low-copy integration for efficient expression"
     CDS             1605..3293
                     /codon_start=1
                     /label=PpADE2
                     /note="phosphoribosylaminoimidazole carboxylase from Pichia
                     pastoris"
                     /translation="MDSQVIGILGGGQLGRMIVEAASRLNIKTVILDDGFSPAKHINAA
                     QDHIDGSFKDEEAIAKLAAKCDVLTVEIEHVNTDALKRVQDRTGIKIYPLPETIELIKD
                     KYLQKEHLIKHNISVTKSQGIESNEKALLLFGEENGFPYLLKSRTMAYDGRGNFVVESK
                     EDISKALEFLKDRPLYAEKFAPFVKELAVMVVRSLEGEVFSYPTVETVHKDNICHIVYA
                     PARVNDTIQKKAQILAENTVKTFPGAGIFGVEMFLLSDGELLVNEIAPRPHNSGHYTID
                     ACVTSQFEAHVRAITGLPMPLDFTKLSTSNTNAIMLNVLGAEKSHGELEFCRRALETPG
                     ASVYLYGKTTRLARKMGHINIIGSSMLEAEQKLEYILEESTHLPSSTVSADTKPLVGVI
                     MGSDSDLPVISKGCDILKQFGVPFEVTIVSAHRTPQRMTRYAFEAASRGIKAIIAGAGG
                     AAHLPGMVAAMTPLPVIGVPVKGSTLDGVDSLHSIVQMPRGVPVATVAINNATNAALLA
                     IRILGTIDHKWQKEMSKYMNAMETEVLGKASNLESEGYESYLKNRL"
     promoter        3523..3672
                     /label=PpTRP2 promoter
                     /note="promoter for Pichia pastoris TRP2"
     CDS             3673..5286
                     /codon_start=1
                     /label=PpTRP2
                     /note="anthranilate synthase from Pichia pastoris"
                     /translation="MSVSDSHLATDYFGTQLTKQIHPSAESFLDIVKNDRQSHSPETVP
                     NLYPIYKFIQSDFLTPHLAYIKLTNSSETPSPSFLLESAKNGDTVDRYTFMGHSPRKII
                     KTGPLEGAEVDPLVLLEKELKGTRQAQLPGIPRLSGGAIGYISYDCIKYFEPKTERKLK
                     DVLQLPEAALMLFDTIVAFDNVYQRFQVIGNVSLSVDDSDEAILEKYYKTREEVEKISK
                     VVFDNKTVPYYEQKDIIQGQTFTSNIGQEGYENHVRKLKEHILKGDIFQAVPSQRVARP
                     TSLHPFNIYRHLRTVNPSPYMFYIDYLDFQVVGASPELLVKSDNNNKIITHPIAGTLPR
                     GKTIEEDDNYAKQLKSSLKDRAEHVMLVDLARNDINRVCEPTSTTVDRLLTVERFSHVM
                     HLVSEVSGTLRPNKTRFDAFRSIFPAGTVSGAPKVRAMQLIGELEGEKRGVYAGAVGHW
                     SYDGKSMDTCIALRTMVVKDGVAYLQAGGGIVYDSDPYDEYIETMNKMRSNNNTILEAE
                     KIWTDRLARDENQSESEENDQ"
     primer_bind     complement(5351..5367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5375..5391)
                     /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(5399..5429)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5444..5465)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5753..6341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6515..7372)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7373..7477)
                     /label=AmpR promoter
     primer_bind     7951..7967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"