| 质粒名称: | pDZ2087 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 92414 |
| 存储实验室: | David Waugh |
|
载体骨架基本信息 |
|
| 载体名称: | pMalC2 |
| 载体出品公司: | New England Biolabs |
| 空载体大小: | 7393 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素100 μg/mL |
| 生长温度: | 30 ℃ |
| 宿主菌: | BL21-RIL |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | TEV protease |
| 物种来源: | tobacco etch virus |
| 插入大小: | -- |
| 基因突变: | L56V/S135G/S219V |
| GenBank编号 | -- |
| 启动子: | tac |
| 标签/融合蛋白: | MBP (self cleaving)（插入件上的N端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | SacI(未破坏) |
| 3’克隆位点: | BamHI(未破坏) |
| 5’测序引物 | MBP |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pDZ2087 was a gift from David Waugh (Addgene plasmid # 92414 ; http://n2t.net/addgene:92414 ; RRID:Addgene_92414) |
| 参考文献部分: | Removal of Affinity Tags with TEV Protease. Raran-Kurussi S, Cherry S, Zhang D, Waugh DS. Methods Mol Biol. 2017;1586:221-230. doi: 10.1007/978-1-4939-6887-9_14. 10.1007/978-1-4939-6887-9_14 PubMed 28470608 |
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 pDZ2087 7390 bp ds-DNA circular SYN 16-JUN-2021
DEFINITION Expresses His-tagged mutant (L56V/S135G/S219V) TEV protease in E.
coli.
ACCESSION .
VERSION .
KEYWORDS pDZ2087
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7390)
AUTHORS Raran-Kurussi S, Cherry S, Zhang D, Waugh DS
TITLE Removal of Affinity Tags with TEV Protease.
JOURNAL Methods Mol Biol. 2017;1586:221-230. doi:
10.1007/978-1-4939-6887-9_14.
PUBMED 28470608
REFERENCE 2 (bases 1 to 7390)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 16, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7390
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 13..30
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 45..185
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(111..130)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 271..293
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(287..478)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
promoter 974..1051
/gene="lacI (mutant)"
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 1052..2134
/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 complement(1071..1090)
/label=LacI-R
/note="LacI, reverse primer"
promoter 2377..2405
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 2413..2429
/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)."
primer_bind 2418..2440
/label=M13/pUC Reverse
/note="In lacZ gene"
CDS 2499..3599
/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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
regulatory 3456..3465
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind 3573..3596
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS 3648..3659
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
CDS 3663..3683
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQG"
CDS 3684..3704
/codon_start=1
/product="6xHis affinity tag"
/label=7xHis
/translation="HHHHHHH"
CDS 3705..4412
/codon_start=1
/product="tobacco etch virus protease (Kapust et al.,
2001)"
/label=TEV protease
/translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
HLFRRNNGTLVVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
REERICLVTTNFQTKSMSSMVSDTSCTFPSGDGIFWKHWIQTKDGQCGSPLVSTRDGFI
VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
EPFQPVKEATQLMN"
primer_bind complement(4454..4471)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4454..4470)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4463..4485)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(4649..4666)
/label=pBAD Reverse
/note="For vectors with E. coli araBAD promoter, reverse
primer"
terminator 4819..4905
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 4997..5024
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 5043..5134
/gene="bla"
/label=AmpR promoter
CDS 5135..5995
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5353..5372)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin complement(6037..6550)
/direction=LEFT
/label=M13 ori
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6232..6253)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 6444..6463
/label=F1ori-R
/note="F1 origin, reverse primer"
rep_origin 6661..7249
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7150..7169
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
61 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
121 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
181 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
241 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
301 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgcagcg
361 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
421 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
481 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
541 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
601 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
661 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
721 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
781 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
841 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
901 ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc
961 tgcccgaaat tccgacacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg
1021 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
1081 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
1141 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
1201 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
1261 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
1321 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
1381 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
1441 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
1501 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
1561 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
1621 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
1681 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
1741 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
1801 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt
1861 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
1921 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
1981 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
2041 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
2101 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
2161 tcactcatta ggcacaattc tcatgtttga cagcttatca tcgactgcac ggtgcaccaa
2221 tgcttctggc gtcaggcagc catcggaagc tgtggtatgg ctgtgcaggt cgtaaatcac
2281 tgcataattc gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca
2341 tcataacggt tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta
2401 taatgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagccag tccgtttagg
2461 tgttttcacg agcacttcac caacaaggac catagattat gaaaactgaa gaaggtaaac
2521 tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat
2581 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
2641 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
2701 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
2761 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
2821 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
2881 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
2941 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
3001 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
3061 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
3121 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
3181 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
3241 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
3301 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
3361 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
3421 acgaggaaga gttggcgaaa gatccacgta ttgccgccac catggaaaac gcccagaaag
3481 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
3541 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta
3601 attcgagctc gaacaacaac aacaataaca ataacaacaa cctcgggatc gagggaaggg
3661 gagaaaatct ttattttcaa ggtcatcatc atcatcatca tcatggagaa agcttgttta
3721 aggggccgcg tgattacaac ccgatatcga gcaccatttg tcatttgacg aatgaatctg
3781 atgggcacac aacatcgttg tatggtattg gatttggtcc cttcatcatt acaaacaagc
3841 acttgtttag aagaaataat ggaacactgg tggtccaatc actacatggt gtattcaagg
3901 tcaagaacac cacgactttg caacaacacc tcattgatgg gagggacatg ataattattc
3961 gcatgcctaa ggatttccca ccatttcctc aaaagctgaa atttagagag ccacaaaggg
4021 aagagcgcat atgtcttgtg acaaccaact tccaaactaa gagcatgtct agcatggtgt
4081 cagacactag ttgcacattc ccttcaggtg atggcatatt ctggaagcat tggattcaaa
4141 ccaaggatgg gcagtgtggc agtccattag tatcaactag agatgggttc attgttggta
4201 tacactcagc atcgaatttc accaacacaa acaattattt cacaagcgtg ccgaaaaact
4261 tcatggaatt gttgacaaat caggaggcgc agcagtgggt tagtggttgg cgattaaatg
4321 ctgactcagt attgtggggg ggccataaag ttttcatggt gaaacctgaa gagccttttc
4381 agccagttaa ggaagcgact caactcatga attaataagg atcctctaga gtcgacctgc
4441 aggcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
4501 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
4561 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cagcttggct
4621 gttttggcgg atgagataag attttcagcc tgatacagat taaatcagaa cgcagaagcg
4681 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
4741 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag
4801 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
4861 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
4921 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
4981 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
5041 cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
5101 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
5161 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
5221 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
5281 caacagcggt aagatccttg agagttttcg ccccgaagaa cgtttcccaa tgatgagcac
5341 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact
5401 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
5461 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
5521 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
5581 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
5641 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
5701 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
5761 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
5821 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
5881 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
5941 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
6001 agaccaagtt tactcatata tactttagat tgatttaccc cggttgataa tcagaaaagc
6061 cccaaaaaca ggaagattgt ataagcaaat atttaaattg taaacgttaa tattttgtta
6121 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
6181 aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg
6241 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
6301 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc
6361 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
6421 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
6481 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
6541 cagggcgcgt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
6601 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
6661 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
6721 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
6781 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
6841 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
6901 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
6961 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
7021 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
7081 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
7141 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
7201 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
7261 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
7321 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
7381 ccgcagccga
//
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