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Cat No. Plasmid Name Availability Buy one, get one free! (?)
V008316 pCold I In stock, instant shipping

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

Basic Vector Information

The pCold I plasmid is an E. coli cold-shock expression vector. It uses the ​cspA promoter​ to induce high-yield recombinant protein expression at low temperatures (e.g., 15°C), which enhances solubility and reduces host protein interference. It features an N-terminal ​6xHis-tag​ and a ​Factor Xa cleavage site​ for purification and tag removal.

Vector Name:
pCold I
Antibiotic Resistance:
Ampicillin
Length:
4407 bp
Type:
Expression vector
Replication origin:
ori
Source/Author:
Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M.
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pCold I vector Map

Copy Sequence View Map
pCold I4407 bp600120018002400300036004200cspA promoterlac operator5'UTRTEE6xHisFactor Xa siteMCS = Multiple cloning sites contain the follow restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI, HindIII, SalI, PstI, XbaI3'UTRf1 oriAmpR promoterAmpRoriCAP binding sitelacIlacI 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

  • Liu Z, Yang Y, Qi R, Gu H, Chen M, Feng K, Jiang Q, Jia H, Kang H, Liu J. A colloidal gold immunochromatographic test strip based on mAbs anti-N protein to detect feline coronavirus. Microbiol Spectr. 2025 Jul;13(7):e0183024. doi: 10.1128/spectrum.01830-24. Epub 2025 Jun 2. PMID: 40454842; PMCID: PMC12210966.

  • Zhang R, Yu Y, Huang L, Chen S, Hu R, Wang X, Huang D, Song C, Lu J, Bao Q, Hu Y, Jiang P, Pan W. Characterization of FosA13, a novel fosfomycin glutathione transferase identified in a Morganella morganii isolate from poultry. Front Cell Infect Microbiol. 2025 Mar 11;15:1534084. doi: 10.3389/fcimb.2025.1534084. PMID: 40134785; PMCID: PMC11933065.

pCold I vector Sequence

LOCUS       40924_12650        4407 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pColdI DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4407)
  AUTHORS   Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, 
            Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M.
  TITLE     Cold-shock induced high-yield protein production in Escherichia coli
  JOURNAL   Nat. Biotechnol. 22 (7), 877-882 (2004)
  PUBMED    15195104
REFERENCE   2  (bases 1 to 4407)
  AUTHORS   Takayama MM, Inouye M.
  TITLE     Direct Submission
  JOURNAL   Submitted (04-AUG-2004) Masanori Mitta Takayama, UMDNJ-Robert Wood 
            Johnson Medical School, Department of Biochemistry; 675 Hoes Lane, 
            Piscataway, NJ 08854-5635, USA (E-mail:mitsutam@takara-bio.co.jp, 
            Tel:81-77-543-7200, Fax:81-77-543-2494)
REFERENCE   3  (bases 1 to 4407)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4407)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol."; date: "2004"; volume: "22"; issue: "7"; pages: 
            "877-882"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (04-AUG-2004) Masanori Mitta Takayama, UMDNJ-Robert Wood Johnson 
            Medical School, Department of Biochemistry"; volume: " 675 Hoes 
            Lane, Piscataway, NJ 08854-5635, USA 
            (E-mail:mitsutam@takara-bio.co.jp, Tel:81-77-543-7200, Fax"; pages: 
            "81-77-543-2494"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..81
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    84..100
                     /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)."
     5'UTR           122..255
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     CDS             256..270
                     /label=TEE
                     /note="translation enhancing element for E. coli (Qing et
                     al., 2004)"
     CDS             271..288
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             289..300
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
     misc_feature    301..360
                     /gene="cspA"
                     /note="MCS = Multiple cloning sites contain the follow 
                     restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI, 
                     HindIII, SalI, PstI, XbaI"
     3'UTR           368..512
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     rep_origin      complement(711..1166)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1266..1370
                     /label=AmpR promoter
     CDS             1371..2228
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      2402..2990
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    complement(3128..3149)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3165..4244)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(4245..4322)
                     /label=lacI promoter