pCold I
Catalog No. PVT10598
Packing 2ug
pCold I Information
Function E.coli expression plasmid
Promoter: CspA promoter
Replicator: Ori, F1 ori
Plasmid classification: large intestine plasmid; large intestine expression plasmid; pCold series plasmid.
Plasmid size: 4407bp
Plasmid label: N-His, N-Factor Xa
Prokaryotic resistance: ampicillin Amp (100 u g/ml)
Cloned strains of Escherichia coli, DH5 A and other Escherichia coli
Culture conditions: 37 centigrade, aerobic, LB
Expression of host: BL21 and other Escherichia coli
Culture conditions: 37 centigrade, aerobic, LB
Inducement: IPTG or lactose and its analogues
5'sequencing primers: pCold-F (ACGCCATATCGCCGAAAGG)
3'sequencing primers: pCold-R (GGCAGGGATCTTAGATTCTG)
Remarks: cold start expression plasmid
pCold I Description
When the temperature of the cultured Escherichia coli was reduced, most of the protein expression was reduced almost temporarily, while a group of proteins called "cold shock protein" was specifically induced. The cold shock expression vector, pCold I-IV, uses the cold shock gene CSPA promoter to design an efficient protein expression vector. The lac operon is inserted at the downstream of CSPA promoter to control expression. In addition, the 5'non translation area (5'UTR), the translation enhancement element (TEE), the His tag sequence, the Xa factor cutting site and the polyclonal site (MCS) are located downstream of the CSPA promoter. Most Escherichia coli strains can be used as the host of the product because of the Escherichia coli promoter. There are four types of pCold vectors, the location of their following parts is slightly different, TEE, His tag sequence and Factor XA enzyme site.
PColdI plasmid is designed by using cold shock gene CSPA promoter to design high efficiency protein expression vector. At the downstream of the promoter, lac operon was inserted to control expression. In addition, the 5 'untranslated region (5' UTR), the translation enhancement element (TEE), the His label sequence, the Xa factor cutting site and the polyclonal site (MCS) are located downstream of the CAPS promoter. When the culture temperature was switched to low temperature, the Escherichia coli stopped growing temporarily and most of the Escherichia coli protein expression decreased, but a class of proteins called cold shock proteins were specifically induced.
pCold I Sequence
LOCUS Exported 4407 bp ds-DNA circular SYN 13-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 3
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4407)
TITLE Direct Submission
JOURNAL Exported Tuesday, September 13, 2016
FEATURES Location/Qualifiers
source 1..4407
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 84..100
/bound_moiety="lac repressor encoded by lacI"
/note="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)."
CDS 271..288
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS 289..300
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
rep_origin complement(711..1166)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1266..1370
/gene="bla"
/note="AmpR promoter"
CDS 1371..2231
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2402..2990
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3162..4244)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(4245..4322)
/gene="lacI"
/note="lacI promoter"
ORIGIN
1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt
241 aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcatat cgaaggtagg
301 catatggagc tcggtaccct cgagggatcc gaattcaagc ttgtcgacct gcagtctaga
361 taggtaatct ctgcttaaaa gcacagaatc taagatccct gccatttggc ggggattttt
421 ttatttgttt tcaggaaata aataatcgat cgcgtaataa aatctattat tatttttgtg
481 aagaataaat ttgggtgcaa tgagaatgcg caggcccttt cgtctcgcgc gtttcggtga
541 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
601 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
661 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata aaattgtaaa
721 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
781 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag
841 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
901 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt
961 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
1021 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
1081 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
1141 gcttaatgcg ccgctacagg gcgcgtacta tggttgcttt gacgtatgcg gtgtgaaata
1201 ccgcacagat gcgtaaggag aaaataccgc atcaggcgtc aggtggcact tttcggggaa
1261 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
1321 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
1381 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
1441 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
1501 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
1561 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
1621 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
1681 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
1741 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
1801 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
1861 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
1921 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
1981 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
2041 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
2101 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
2161 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
2221 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
2281 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
2341 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
2401 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
2461 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
2521 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
2581 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
2641 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
2701 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
2761 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
2821 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
2881 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
2941 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
3001 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacata gtcatgcccc
3061 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
3121 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
3181 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
3241 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
3301 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
3361 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
3421 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
3481 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
3541 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
3601 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
3661 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
3721 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
3781 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
3841 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
3901 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
3961 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
4021 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
4081 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
4141 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
4201 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
4261 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
4321 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
4381 gttgaggccg ttgagcaccg ccgccgc
//