pCold-SUMO质粒 货号：kl-zl-0734 规格：20ul
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启动子： |
CspA promoter |
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复制子： |
ColE1 ori，f1 ori |
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质粒分类： |
大肠系列质粒；大肠表达质粒；pCold系列质粒 |
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质粒大小： |
4740bp |
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质粒标签： |
N-His, N-SUMO |
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原核抗性： |
氨苄青霉素Amp（100μg/ml） |
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克隆菌株： |
DH5α等大肠杆菌 |
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培养条件： |
37℃，有氧，LB |
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表达宿主： |
BL21等大肠杆菌 |
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诱导方式： |
IPTG或乳糖及其类似物 |
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5'测序引物： |
pCold-F（ACGCCATATCGCCGAAAGG） |
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3'测序引物： |
pCold-R（GGCAGGGATCTTAGATTCTG） |
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备注： |
冷休克表达载体 |
质粒简介：
原核表达载体(pCold-SUMO)是在 pCold 载体基础上改造而成，该载体启动子(CS Promoter)来源于南极嗜冷细菌，在低温下(15 度)才能启动蛋白的表达。低温下细菌生长缓慢，使得蛋白合成速度减慢，从而最大限度的提高了蛋白正确折叠的几率，提高了蛋白可溶性表达，增强了活性蛋白的表达比率。该表达系统所包含的 SUMO tag可以极大地提高小分子量蛋白的表达量，而且进一步提高了蛋白的可溶表达几率。同时，试剂盒配备的SUMO Protease(含6×His标签)可特异性去除 SUMO tag，从而得到不含任何标签的重组蛋白。TEE信号肽可增强冷启动子调控下目的蛋白的高表达。BL21(DE3)ChaproneE.coli 细菌中含有分子伴侣蛋白质粒，其表达产物可协助重组蛋白正确折叠形成可溶活性蛋白。分子伴侣蛋白质粒为氯霉素抗性（chloramphenicol，Cmr）的表达受控于四环素（tetR）操纵子。
pCold-SUMO质粒是利用冷休克基因CSPA启动子设计高效率蛋白质表达载体。在E.COLI启动子下游处，插入了lac操纵子，以便严格控制表达。此外，5’非翻译区（5’UTR），翻译增强元件（TEE）,His标签序列，Xa因子切割位点和多克隆位点（MCS）位于CAPS启动子的下游。当培养温度切换到低温时，大肠杆菌暂时停止生长，大部分的大肠杆菌蛋白质表达减少，但是一类称为冷休克蛋白的蛋白质被特异性诱导表达。
质粒图谱：
序列：
LOCUS Exported 4740 bp ds-DNA circular SYN 22-JUN-2017
DEFINITION synthetic circular DNA
KEYWORDS pCold-SUMO
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
source 1..4740
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 15..81
/note="cspA promoter"
misc_feature 82..121
/note="lac operator"
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)."
misc_feature 122..255
/note="cspA 5'UTR"
misc_feature 243..246
/note="SD"
misc_feature 256..270
/note="TEE"
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"
CDS 340..633
/codon_start=1
/gene="S. cerevisiae SMT3 (truncated)"
/product="cleavable ubiquitin-like protein tag"
/note="SUMO"
/translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
misc_feature 634..693
/note="mcs"
misc_feature 701..845
/note="cspA 3'UTR"
misc_feature 729..746
/note="transcription terminator"
rep_origin complement(1044..1499)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1599..1703
/gene="bla"
/note="AmpR promoter"
CDS 1704..2564
/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 2735..3323
/direction=RIGHT
/note="ColE1 ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3495..4577)
/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(4578..4655)
/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 catatgggcg gtggcggtag cggcggtggc ggtagcggta tgtcggactc agaagtcaat
361 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
421 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
481 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
541 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
601 attattgagg ctcacagaga acagattggt ggtactagtg agctcggtac cctcgaggga
661 tccgaattca agcttgtcga cctgcagtct agataggtaa tctctgctta aaagcacaga
721 atctaagatc cctgccattt ggcggggatt tttttatttg ttttcaggaa ataaataatc
781 gatcgcgtaa taaaatctat tattattttt gtgaagaata aatttgggtg caatgagaat
841 gcgcaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
901 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
961 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
1021 gattgtactg agagtgcacc ataaaattgt aaacgttaat attttgttaa aattcgcgtt
1081 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
1141 taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
1201 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
1261 cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact
1321 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
1381 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
1441 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta
1501 ctatggttgc tttgacgtat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
1561 cgcatcaggc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
1621 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
1681 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
1741 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
1801 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
1861 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
1921 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
1981 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
2041 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
2101 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
2161 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
2221 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
2281 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
2341 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
2401 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
2461 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
2521 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
2581 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
2641 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
2701 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
2761 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
2821 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
2881 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
2941 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
3001 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
3061 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
3121 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
3181 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
3241 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
3301 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
3361 tttgctggcc ttttgctcac atagtcatgc cccgcgccca ccggaaggag ctgactgggt
3421 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
3481 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
3541 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
3601 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
3661 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
3721 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
3781 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
3841 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
3901 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
3961 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
4021 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
4081 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
4141 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
4201 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
4261 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
4321 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
4381 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
4441 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
4501 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
4561 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
4621 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
4681 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
