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pTK-Hyg vector (V000776)

Price Information

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V000776 pTK-Hyg In stock, instant shipping

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

pTK-Hyg is a selection vector that confers hygromycin resistance in mammalian cells for the selection of stably transformed cells. pTK-Hyg is especially useful for selection of double-stable cell lines using the Tet-On or Tet-Off Gene Expression Systems because the absence of an enhancer element on pTK-Hyg prevents the unwanted activation of pTRE- and pBI-derived plasmids upon cointegration into the host cell's genome.pTK-Hyg can be cotransfected into the host cell line with an expression plasmid that contains a gene of interest using any standard transfection technique. Most mammalian cell lines require 200 μg/ml of Hygromycin to select for stably transformed cells.

Vector Name:
pTK-Hyg
Antibiotic Resistance:
Ampicillin
Length:
5066 bp
Type:
Tetracycline Regulatory System
Replication origin:
ori
Selection Marker:
Hyg
Promoter:
HSV TK
Cloning Method:
Enzyme digestion and ligation
Growth Strain(s):
Top10
Growth Temperature:
37℃

pTK-Hyg vector Map

Copy Sequence View Map
pTK-Hyg5066 bp6001200180024003000360042004800SV40 poly(A) signalHSV TK poly(A) signalHygRHSV TK promoteroriAmpRAmpR promoter

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

  • Ren ST, Yu LH, Zhang J, Han SP, Xu CF. Developing a system for regulating expression of human hepatocyte growth factor using tetracycline in NRK52E cells. Urol Int. 2010;85(2):228-236. doi:10.1159/000314956

pTK-Hyg vector Sequence

LOCUS       40924_43503        5066 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5066)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5066)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    complement(29..163)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    complement(1450..1498)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(1543..2577)
                     /codon_start=1
                     /label=HygR
                     /note="hygromycin B phosphotransferase"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRGDRE
                     MGEAN"
     promoter        complement(2612..2757)
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     rep_origin      complement(3214..3802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3976..4833)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4834..4938)
                     /label=AmpR promoter