| 质粒名称: | MSP573 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 98796 |
| 存储实验室: | David Liu |
|
载体骨架基本信息 |
|
| 载体名称: | pBR322 |
| 载体出品公司: | ATCC |
| 完整质粒大小: | 7091 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | Kanamycin |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | pET28 MBP-ENLYFQS-TEV WT |
| 物种来源: | Synthetic |
| 基因突变: | 无 |
| 克隆方法: | 未知 |
|
文献引用方法 |
|
| 材料和方法部分: |
MSP573 was a gift from David Liu (Addgene plasmid # 98796 ; http://n2t.net/addgene:98796 ; RRID:Addgene_98796)
|
| 参考文献部分: | Phage-assisted continuous evolution of proteases with altered substrate specificity. Packer MS, Rees HA, Liu DR. Nat Commun. 2017 Oct 16;8(1):956. doi: 10.1038/s41467-017-01055-9. 10.1038/s41467-017-01055-9 [pii] PubMed 29038472 |
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 MSP573 7091 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression Vector.
ACCESSION .
VERSION .
KEYWORDS MSP573
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7091)
AUTHORS Packer MS, Rees HA, Liu DR
TITLE Phage-assisted continuous evolution of proteases with altered
substrate specificity.
JOURNAL Nat Commun. 2017 Oct 16;8(1):956. doi: 10.1038/s41467-017-01055-9.
PUBMED 29038472
REFERENCE 2 (bases 1 to 7091)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7091
/organism="synthetic DNA construct"
/mol_type="other DNA"
terminator 26..73
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind 69..87
/label=T7 Term
/note="T7 terminator, reverse primer"
CDS complement(140..157)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS complement(161..868)
/codon_start=1
/product="tobacco etch virus protease (Kapust et al.,
2001)"
/label=TEV protease
/translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
EPFQPVKEATQLMN"
CDS complement(869..889)
/codon_start=1
/product="6xHis affinity tag"
/label=7xHis
/translation="HHHHHHH"
CDS complement(890..910)
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQS"
CDS complement(926..2026)
/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="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
primer_bind complement(929..952)
/label=MBP-F
/note="Maltose binding protein, forward primer"
regulatory 1060..1069
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
RBS 2032..2054
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 2069..2093
/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 complement(2093..2112)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2094..2112)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2196..2215)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 2421..2498
/gene="lacI"
/label=lacI promoter
CDS 2499..3581
/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(2518..2537)
/label=LacI-R
/note="LacI, reverse primer"
CDS 4390..4581
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind complement(4575..4597)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 4683..4822
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 4738..4757
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(4837..4854)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5008..5596)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5088..5107)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind complement(5707..5726)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 5718..6533
/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 complement(5791..5810)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin complement(6625..7080)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6762..6783)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 6974..6993
/label=F1ori-R
/note="F1 origin, reverse primer"
ORIGIN
1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
121 tgttagcagc cggatctcag tggtggtggt ggtggtgtta attcatgagt tgagtcgctt
181 ccttaactgg ctgaaaaggc tcttcaggtt tcaccatgaa aactttatgg cccccccaca
241 atactgagtc agcatttaat cgccaaccac taacccactg ctgcgcctcc tgatttgtca
301 acaattccat gaagtttttc ggcacgcttg tgaaataatt gtttgtgttg gtgaaattcg
361 atgctgagtg tataccaaca atgaacccat ctctagttga tactaatgga ctgccacact
421 gcccatcctt ggtttgaatc caatgcttcc agaatatgcc atcagatgaa gggaatgtgc
481 aactagtgtc tgacaccatg ctagacatgc tcttagtttg gaagttggtt gtcacaagac
541 atatgcgctc ttccctttgt ggctctctaa atttcagctt ttgaggaaat ggtgggaaat
601 ccttaggcat gcgaataatt atcatgtccc tcccatcaat gaggtgttgt tgcaaagtcg
661 tggtgttctt gaccttgaat acaccatgta gtgattggac caacagtgtt ccattatttc
721 ttctaaacaa gtgcttgttt gtaatgatga agggaccaaa tccaatacca tacaacgatg
781 ttgtgtgccc atcagattca ttcgtcaaat gacaaatggt gctcgatatc gggttgtaat
841 cacgcggccc cttaaacaag ctttctccat gatgatgatg atgatgatga ctttgaaaat
901 aaagattttc tcccgagctc gaattagtct gcgcgtcttt cagggcttca tcgacagtct
961 gacgaccgct ggcggcgttg atcaccgcag tacgcacggc ataccagaaa gcggacatct
1021 gcgggatgtt cggcatgatt tcacctttct gggcgttttc catggtggcg gcaatacgtg
1081 gatctttcgc caactcttcc tcgtaagact tcagcgctac ggcacccagc ggtttgtctt
1141 tattaaccgc ttccagacct tcatcagtca gcagatagtt ttcgaggaac tcttttgcca
1201 gctctttgtt cggactggcg gcgttaatac ctgcgctcag cacgccaacg aacggtttgg
1261 atggttgacc cttgaaggtc ggcagtaccg ttacaccata attcactttg ctggtgtcga
1321 tgttggacca tgcccacggg ccgttgatgg tcatcgctgt ttcgccttta ttaaaggcag
1381 cttctgcgat ggagtaatcg gtgtctgcat tcatgtgttt gtttttaatc aggtcaacca
1441 ggaaggtcag acccgctttc gcgccagcgt tatccacgcc cacgtcttta atgtcgtact
1501 tgccgttttc atacttgaac gcataacccc cgtcagcagc aatcagcggc caggtgaagt
1561 acggttcttg caggttgaac atcagcgcgc tcttaccttt cgctttcagt tctttatcca
1621 gcgccgggat ctcttcccag gtttttggcg ggttcggcag cagatctttg ttataaatca
1681 gcgataacgc ttcaacagcg atcgggtaag caatcagctt gccgttgtaa cgtacggcat
1741 cccaggtaaa cggatacagc ttgtcctgga acgctttgtc cggggtgatt tcagccaaca
1801 ggccagattg agcgtagcca ccaaagcggt cgtgtgccca gaagataatg tcagggccat
1861 cgccagttgc cgcaacctgt gggaatttct cttccagttt atccggatgc tcaacggtga
1921 ctttaattcc ggtatctttc tcgaatttct taccgacttc agcgagaccg ttatagcctt
1981 tatcgccgtt aatccagatt accagtttac cttcttcgat tttcatgtat atctccttct
2041 taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat tcccctatag
2101 tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac gcatcgtggc
2161 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
2221 ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg gtatggtggc
2281 aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat tccttgcggc
2341 ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg agtcgcataa
2401 gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc ggtatggcat
2461 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
2521 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
2581 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
2641 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
2701 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
2761 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
2821 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
2881 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
2941 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
3001 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
3061 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
3121 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
3181 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
3241 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
3301 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
3361 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
3421 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
3481 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
3541 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
3601 agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc cttcaaccca
3661 gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc
3721 tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac
3781 cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac
3841 gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga gaagcaggcc
3901 attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc gttgaggacc
3961 cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga
4021 acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc
4081 ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc
4141 cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg
4201 aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt
4261 tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg
4321 agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg
4381 gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc
4441 cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc
4501 tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt
4561 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
4621 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
4681 ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
4741 catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt
4801 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
4861 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
4921 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
4981 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
5041 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
5101 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
5161 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
5221 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
5281 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
5341 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
5401 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
5461 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
5521 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
5581 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
5641 cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca
5701 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat
5761 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
5821 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
5881 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
5941 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
6001 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
6061 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
6121 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
6181 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
6241 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
6301 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
6361 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
6421 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
6481 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
6541 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
6601 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
6661 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
6721 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
6781 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
6841 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
6901 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
6961 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
7021 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
7081 cccattcgcc a
//
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