| 质粒名称: | pET28-MBP-TEV |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 69929 |
| 存储实验室: | Zita Balklava, Thomas Wassmer |
|
载体骨架基本信息 |
|
| 载体名称: | pET28 |
| 载体出品公司: | Novagen |
| 完整质粒大小: | 7034bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | Kanamycin, 50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 启动子: | T7 |
| 标签/融合蛋白: | MBP-TEV (N terminal on backbone) |
| 克隆方法: | Restriction Enzyme |
| 5’测序引物 | T7 |
| 3’测序引物 | T7 terminal |
|
文献引用方法 |
|
| 材料和方法部分: |
pET28-MBP-TEV was a gift from Zita Balklava & Thomas Wassmer (Addgene plasmid # 69929 ; http://n2t.net/addgene:69929 ; RRID:Addgene_69929)
|
| 参考文献部分: | APP controls the formation of PI(3,5)P vesicles through its binding of the PIKfyve complex. Currinn H, Guscott B, Balklava Z, Rothnie A, Wassmer T. Cell Mol Life Sci. 2015 Jul 28. 10.1007/s00018-015-1993-0 PubMed 26216398 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pET28-MBP-TEV 7034 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION bacterial expression of MBP-TEV n-terminal fusions.
ACCESSION .
VERSION .
KEYWORDS pET28-MBP-TEV
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7034)
AUTHORS Currinn H, Guscott B, Balklava Z, Rothnie A, Wassmer T
TITLE APP controls the formation of PI(3,5)P vesicles through its binding
of the PIKfyve complex.
JOURNAL Cell Mol Life Sci. 2015 Jul 28.
PUBMED 26216398
REFERENCE 2 (bases 1 to 7034)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7034
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 106..125
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind 209..228
/label=T7
/note="T7 promoter, forward primer"
promoter 209..227
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 228..252
/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)."
RBS 267..289
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 308..325
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 335..352
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 368..1465
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/label=MBP
/note="This version of the gene does not encode a signal
sequence, so MBP will remain in the cytosol."
/translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
YAVRTAVINAASGRQTVDEALKDAQT"
regulatory 1322..1331
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind 1439..1462
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 1481..1501
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQG"
CDS 2107..2124
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(2177..2195)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 2191..2238
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2275..2730
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2362..2381)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2572..2593
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(2822..3637)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 3545..3564
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 3629..3648
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 3759..4347
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4248..4267
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4501..4518
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 4533..4672
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(4598..4617)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4758..4780
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4774..4965)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(5774..6856)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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"
primer_bind 6818..6837
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(6857..6934)
/gene="lacI"
/label=lacI promoter
ORIGIN
1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
301 cagcagccat catcatcatc atcacagcag cggcctggtg ccgcgcggca gccatatggc
361 tagcggtaaa atcgaagaag gtaaactggt aatctggatt aacggcgata aaggctataa
421 cggtctcgct gaagtcggta agaaattcga gaaagatacc ggaattaaag tcaccgttga
481 gcatccggat aaactggaag agaaattccc acaggttgcg gcaactggcg atggccctga
541 cattatcttc tgggcacacg accgctttgg tggctacgct caatctggcc tgttggctga
601 aatcaccccg gacaaagcgt tccaggacaa gctgtatccg tttacctggg atgccgtacg
661 ttacaacggc aagctgattg cttacccgat cgctgttgaa gcgttatcgc tgatttataa
721 caaagatctg ctgccgaacc cgccaaaaac ctgggaagag atcccggcgc tggataaaga
781 actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg caagaaccgt acttcacctg
841 gccgctgatt gctgctgacg ggggttatgc gttcaagtat gaaaacggca agtacgacat
901 taaagacgtg ggcgtggata acgctggcgc gaaagcgggt ctgaccttcc tggttgacct
961 gattaaaaac aaacacatga atgcagacac cgattactcc atcgcagaag ctgcctttaa
1021 taaaggcgaa acagcgatga ccatcaacgg cccgtgggca tggtccaaca tcgacaccag
1081 caaagtgaat tatggtgtaa cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt
1141 cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg aacaaagagc tggcgaaaga
1201 gttcctcgaa aactatctgc tgactgatga aggtctggaa gcggttaata aagacaaacc
1261 gctgggtgcc gtagcgctga agtcttacga ggaagagttg gcgaaagatc cacgtattgc
1321 cgccaccatg gaaaacgccc agaaaggtga aatcatgccg aacatcccgc agatgtccgc
1381 tttctggtat gccgtgcgta ctgcggtgat caacgccgcc agcggtcgtc agactgtcga
1441 tgaagccctg aaagacgcgc agactcgtat caccaagggt gaaaatctat acttccaagg
1501 atccctcagt acaggctgct actgacagat tacttaaaga accttctaga agactctgta
1561 gatcacagag acacccaaga tgccctggct gttgtcatag aggtagctaa ccatgccaat
1621 gacaccatga agcaagggga caactttcag aagctcatgc agattcagta cagcttaagt
1681 ggacaccatg aaattgtgca acctgggcgg gtttttctta aagaaggcac cctgatgaag
1741 ctgtctcgga aagtcatgca gccccggatg tttttcctgt ttaacgatgc tcttctctac
1801 accacaccca tgcagtctgg gatgtacaaa ctgaacaaca tgctctcgct ggctggaatg
1861 aaggtcagaa aacctaccca ggaggcatat cagaacgagt taaagattga aagtgtagag
1921 cgttctttca tcctctcagc tagttctgcc gcagaaagag acgactggct ggaggccata
1981 tccagctcca tagaagagta tgccaagaaa agaatcacat tctgccccag caggagtctt
2041 gatgaggact cagaaaggaa agaagaagtt agccccctcg gggcaaaggc tccctaaccg
2101 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
2161 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
2221 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
2281 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
2341 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
2401 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
2461 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
2521 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
2581 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
2641 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
2701 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
2761 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
2821 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
2881 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
2941 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
3001 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
3061 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
3121 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
3181 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
3241 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
3301 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
3361 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
3421 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
3481 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
3541 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
3601 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
3661 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
3721 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
3781 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
3841 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
3901 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
3961 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
4021 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
4081 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
4141 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
4201 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
4261 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
4321 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
4381 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
4441 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
4501 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
4561 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
4621 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
4681 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
4741 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
4801 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
4861 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
4921 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
4981 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
5041 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
5101 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
5161 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
5221 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
5281 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
5341 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
5401 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
5461 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
5521 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
5581 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
5641 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
5701 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
5761 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
5821 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
5881 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
5941 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
6001 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
6061 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
6121 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
6181 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
6241 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
6301 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
6361 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
6421 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
6481 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
6541 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
6601 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
6661 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
6721 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
6781 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
6841 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
6901 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
6961 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
7021 aggaatggtg catg
//
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