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pKK223-3 vector (V001042)

Price Information

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V001042 pKK223-3 In stock, instant shipping

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

Basic Vector Information

The pKK223-3 is an E.coli expression vector

Vector Name:
pKK223-3
Antibiotic Resistance:
Ampicillin
Length:
4584 bp
Type:
E. coli Expression Vectors
Replication origin:
ori
Promoter:
tac
Cloning Method:
Enzyme digestion and ligation
5' Primer:
P0084-F：gtgaaattgttatccgctcac
3' Primer:
P0084-R：ttctgataaagcgggccatg
Growth Strain(s):
DH5a
Growth Temperature:
37℃

pKK223-3 vector Map

Copy Sequence View Map
pKK223-34584 bp600120018002400300036004200rrnB T1 terminatorrrnB T2 terminatorAmpR promoterAmpRoribomropTetA(C)tac promoterlac operator

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

  • Xu D, Zhang L. Metabolic engineering of Escherichia coli for agmatine production. Eng Life Sci. 2018;19(1):13-20. Published 2018 Oct 4. doi:10.1002/elsc.201800104

pKK223-3 vector Sequence

LOCUS       Exported                4584 bp DNA     circular SYN 26-AUG-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4584)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4584)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4584)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      236..322
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      414..441
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        460..551
                     /label=AmpR promoter
     CDS             552..1097
                     /codon_start=1
                     /label=AmpR
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTML"
     rep_origin      1582..2170
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2356..2496
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2601..2789)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3428..4213)
                     /codon_start=1
                     /product="Tetracycline resistance protein, class C"
                     /label=TetA(C)
                     /translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCFL
                     MQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVIFGED
                     RFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVLLAFAT
                     RGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITGPLIVTA
                     IYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     promoter        4513..4541
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    4549..4565
                     /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)."