pCold- GST

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  • Model: PVT0608
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pCold-GST

PVT0608     2ug

 

pCold-GST Information

Promoter: CSPA

Replicon: ColE1 ori

Plasmid classification: Escherichia coli vector; pCold series expression plasmid

Plasmid size: 5097bp

Plasmid labeling: N-GST, N-HRV 3C protease cutting site, N-His

Prokaryotic resistance: ampicillin Amp

Cloned strain: Escherichia coli DH5 alpha

Culture conditions: 37 centigrade, aerobic, LB

Expression host: Escherichia coli

Culture conditions: 37 centigrade, aerobic, LB

Inducement: IPTG or lactose and its analogues

5'sequencing primers: pCold Forward: 5-ACGCCATATCGCCGAAAGG-3

3'sequencing primers: pCold Reverse 5-GGCAGGGATCTTAGATTCTG-3

Note: the pCold-GST vector expresses GST fusion protein, and the GST label can improve the soluble protein of the target protein and improve the purity of the target protein.

 

pCold-GST Description

Based on the cold shock vector, pCold-GST DNA integrated glutathione S- transferase (glutathione S-transferases, GST) soluble label from Schistosoma japonicum. The fusion expression of GST at the end of the target protein N can improve the stability and solubility of the fusion protein. This product inserts 5 'non coding region (5' -UTR), translation enhancer element (TEE), His tag, GST tag and polyclonal site (MCS) at the downstream of cspA promoter. In addition, the lac operator, which can strictly regulate the expression of the target gene, is also inserted at the lower reaches of the cspA promoter. GST tag fusion protein can be purified for high affinity. The recognition sequence of high specific HRV 3C Protease was inserted between the GST label and the polyclonal site (MCS), which could remove the label from the fusion protein. The optimum temperature of HRV 3C Protease is 4~5 C, and the label removal reaction of the target protein can be carried out under mild conditions. The promoter of the pCold series is derived from Escherichia coli, so most Escherichia coli can be used as an expression host.

pCold-GST-polyclonal-site

 

pCold-GST Sequence

LOCUS       Exported                5097 bp ds-DNA     circular SYN 06-JUL-2017

DEFINITION  synthetic circular DNA

FEATURES             Location/Qualifiers

     source          1..5097

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     promoter        86..163

                     /gene="lacI"

                     /label=lacI promoter

     CDS             164..1246

                     /codon_start=1

                     /gene="lacI"

                     /product="lac repressor"

                     /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)."

                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL

                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV

                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH

                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA

                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC

                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR

                     ALADSLMQLARQVSRLESGQ"

     rep_origin      complement(1418..2006)

                     /direction=LEFT

                     /label=ori

                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 

                     replication"

     CDS             complement(2177..3037)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(3038..3142)

                     /gene="bla"

                     /label=AmpR promoter

     rep_origin      3242..3697

                     /direction=RIGHT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     CDS             complement(4111..4134)

                     /codon_start=1

                     /product="recognition and cleavage site for human 

                     rhinovirus 3C and PreScission proteases"

                     /label=HRV 3C site

                     /translation="LEVLFQGP"

     CDS             complement(4141..4794)

                     /codon_start=1

                     /product="glutathione S-transferase from Schistosoma 

                     japonicum"

                     /label=GST

                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK

                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY

                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL

                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"

     CDS             complement(4798..4809)

                     /codon_start=1

                     /product="Factor Xa recognition and cleavage site"

                     /label=Factor Xa site

                     /translation="IEGR"

     CDS             complement(4810..4827)

                     /codon_start=1

                     /product="6xHis affinity tag"

                     /label=6xHis

                     /translation="HHHHHH"

     protein_bind    4998..5014

                     /label=lac operator

                     /bound_moiety="lac repressor encoded by lacI"

                     /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)."

ORIGIN

        1 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg

       61 cataagggag agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat

      121 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt

      181 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca

      241 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa

      301 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt

      361 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg

      421 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc

      481 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta

      541 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt

      601 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg

      661 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc

      721 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac

      781 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt

      841 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa

      901 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc

      961 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt

     1021 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca

     1081 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag

     1141 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt

     1201 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta

     1261 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca

     1321 acccagtcag ctccttccgg tgggcgcggg gcatgactat gtgagcaaaa ggccagcaaa

     1381 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg

     1441 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa

     1501 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc

     1561 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac

     1621 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac

     1681 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg

     1741 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt

     1801 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa

     1861 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct

     1921 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga

     1981 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg

     2041 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct

     2101 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt

     2161 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc

     2221 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg

     2281 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag

     2341 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt

     2401 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag

     2461 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt

     2521 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca

     2581 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg

     2641 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat

     2701 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta

     2761 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca

     2821 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct

     2881 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat

     2941 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa

     3001 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt

     3061 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa

     3121 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gcctgatgcg

     3181 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag

     3241 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc

     3301 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc

     3361 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt

     3421 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg

     3481 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt

     3541 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta

     3601 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt

     3661 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc

     3721 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc

     3781 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc

     3841 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctgcgc

     3901 attctcattg cacccaaatt tattcttcac aaaaataata atagatttta ttacgcgatc

     3961 gattatttat ttcctgaaaa caaataaaaa aatccccgcc aaatggcagg gatcttagat

     4021 tctgtgcttt taagcagaga ttacctatct agactgcagg tcgacaagct tgaattcgga

     4081 tccctcgagg gtaccgagct ccatatgtcc cgggccctgg aacagaactt ccagatccga

     4141 ttttggagga tggtcgccac caccaaacgt ggcttgccag ccctgcaaag gccatgctat

     4201 atacttgctg gatttcaagt acttatcaat ttgtgggata gcttcaatac gttttttaaa

     4261 acaaactaat tttgggaacg catccaggca cattgggtcc atgtataaaa caacatcaag

     4321 agcgtcatac aacatgaagt caggatgggt tacatgatca ccatttaaat atgttttatg

     4381 acataaacga tcttcgaaca ttttcagcat ttcaggtagc ttgctaagaa aatcaacttt

     4441 gagagtttca aagtctttac tatatgcaat tctcgaaaca ccgtatctaa tatccaaaac

     4501 cgctccttca agcattgaaa tctctgcacg ctcttttgga caaccaccca acatgttgtg

     4561 cttgtcagct atataacgta tgatggccat agactgtgtt aatttaacat caccatcaat

     4621 ataataagga agattgggaa actccaaacc caattcaaac tttttgtttc gccatttatc

     4681 accttcatcg cgctcataca aatgctcttc atatttttct tcaagatatt ccaaaagaag

     4741 tcgagtgggt tgcacaaggc ccttaatttt ccaataacct agtatagggg acatgtgcct

     4801 accttcgata tgatgatgat gatgatgcac tttgtgattc atggtgtatt acctcttaat

     4861 aattaagtgt gcctttcggc gatatggcgt gctttacaga ttttgaagcg ttaaaggaat

     4921 gtgcactacg aggggtatca acgataactc ttgaagggac ttgccttact acactggata

     4981 tgcgctagca catcaaattg ttatccgctc acaatttgat gtgcattaag ccacgcattg

     5041 gcgggtgatg caacaattat ttttcatatt tatgattaat cggccacacc attcctt

//

 

Caution:
Product is for research use only!

 

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pCold-GST,Plasmid pCold-GST,pCold-GST vector

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