pKK233-2 vector (V005224)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V005224	pKK233-2	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

The pKK233-2 plasmid features a robust hybrid trp/lac promoter, a lacZ ribosome-binding site (RBS), the multiple cloning site from pUC18, and the rrnB transcription terminators.

Vector Name:: pKK233-2

Antibiotic Resistance:: Ampicillin

Length:: 4593 bp

Type:: Cloning vector

Replication origin:: ori

Source/Author:: Amann E, Brosius J.

Promoter:: trc

Growth Strain(s):: DH5a

Growth Temperature:: 37℃

pKK233-2 vector Map

Copy Sequence View 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

Amann E, Ochs B, Abel KJ. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988;69(2):301-315. doi:10.1016/0378-1119(88)90440-4

pKK233-2 vector Sequence

LOCUS       Exported                4593 bp DNA     circular SYN 23-AUG-2024
DEFINITION  Cloning vector pKK233-2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4593)
  AUTHORS   Amann E, Brosius J.
  TITLE     'ATG vectors' for regulated high-level expression of cloned genes in
            Escherichia coli
  JOURNAL   Gene 40 (2-3), 183-190 (1985)
  PUBMED    3007288
REFERENCE   2  (bases 1 to 4593)
  AUTHORS   Kitts PA.
  TITLE     CLONTECH Vectors On Disc version 1.3
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 4593)
  AUTHORS   Kitts PA.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-OCT-1993) Paul A. Kitts, CLONTECH Laboratories, Inc., 
            1020 East Meadow Circle, Palo Alto, CA 94303, USA
REFERENCE   4  (bases 1 to 4593)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 4593)
  TITLE     Direct Submission
REFERENCE   6  (bases 1 to 4593)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene 40
            (2-3), 183-190 (1985)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted
            (07-OCT-1993) Paul A. Kitts, CLONTECH Laboratories, Inc., 1020 East
            Meadow Circle, Palo Alto, CA 94303, USA"
COMMENT     SGRef: number: 4; type: "Journal Article"
COMMENT     SGRef: number: 5; type: "Journal Article"
COMMENT     This vector can be obtained from CLONTECH Laboratories, Inc., 1020 
            East Meadow Circle, Palo Alto, CA 94303, USA. To place an order call
            (415) 424-8222 or (800) 662-2566, extension 1. International 
            customers, please contact your local distributor.  For technical 
            information, call (415) 424- 8222 or (800) 662-2566, extension 3. 
            This sequence was compiled by Jurgen Brosius. If you suspect there 
            is an error in this sequence, please contact CLONTECH's Technical 
            Service Department at (415) 424-8222 or (800) 662-2566, extension 3 
            or E-mail TECH@CLONTECH.COM.
FEATURES             Location/Qualifiers
     source          1..4593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        204..233
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    241..257
                     /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)."
     terminator      498..584
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      676..703
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        723..814
                     /label=AmpR promoter
     CDS             815..1672
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      1846..2434
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(2620..2760)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(2865..3053)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3692..4477)
                     /codon_start=1
                     /label=tetracycline efflux MFS transporter Tet(C)
                     /translation="MSACFGVGMVAGPVAGGLLGAISLHAPFLAAAVLNGLNLLLGCFL
                     MQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIMQLVGQVPAALWVIFGED
                     RFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAIIAGMAADALGYVLLAFAT
                     RGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSLAALTSLTSITGPLIVTA
                     IYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"