| 质粒名称: | pET28a SnoopTag-MBP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 72323 |
| 存储实验室: | Mark Howarth |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Novagen |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5369 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | pET28a SnoopTag-MBP,SnoopTag-MBP |
| 物种来源: | Synthetic |
| 插入大小: | 1233 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | N端子His6标签。MBP在SnoopTag的C端融合 |
| 克隆方法: | Ligation Independent克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | - |
| 5' 测序引物 | TAATACGACTCACTATAGGG |
| 3' 测序引物 | GCTAGTTATTGCTCAGCGG |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a SnoopTag-MBP was a gift from Mark Howarth (Addgene plasmid # 72323 ; http://n2t.net/addgene:72323 ; RRID:Addgene_72323) |
| 参考文献部分: | Programmable polyproteams built using twin peptide superglues. Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M. Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214. 10.1073/pnas.1519214113 PubMed 26787909 |
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 pET28a SnoopTag-MBP 6481 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bacterial expression of Maltose Binding Protein linked to SnoopTag,
a peptide tag forming a spontaneous covalent bond with its protein
partner Snoopcatcher..
ACCESSION .
VERSION .
KEYWORDS pET28a SnoopTag-MBP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6481)
AUTHORS Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV,
Howarth M
TITLE Programmable polyproteams built using twin peptide superglues.
JOURNAL Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214.
PUBMED 26787909
REFERENCE 2 (bases 1 to 6481)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6481
/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(194..1291)
/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"
primer_bind complement(197..220)
/label=MBP-F
/note="Maltose binding protein, forward primer"
CDS complement(1355..1372)
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS complement(1382..1399)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
RBS 1418..1440
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 1455..1479
/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(1479..1498)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(1480..1498)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(1582..1601)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 1807..1884
/gene="lacI"
/label=lacI promoter
CDS 1885..2967
/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(1904..1923)
/label=LacI-R
/note="LacI, reverse primer"
CDS 3776..3967
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind complement(3961..3983)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 4069..4211
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 4124..4143
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(4226..4243)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4397..4985)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4477..4496)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind complement(5096..5115)
/label=pENTR-R
/note="pENTR vectors, reverse primer"
CDS 5107..5922
/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(5180..5199)
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
rep_origin complement(6015..6470)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(6152..6173)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 6364..6383
/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 ggtggtgctc gagtgcggcc gcaagctttt
181 acgagctcga attagtctgc gcgtctttca gggcttcatc gacagtctga cgaccgctgg
241 cggcgttgat caccgcagta cgcacggcat accagaaagc ggacatctgc gggatgttcg
301 gcatgatttc acctttctgg gcgttttcca tagtggcggc aatacgtgga tctttcgcca
361 actcttcctc gtaagacttc agcgctacgg cacccagcgg tttgtcttta ttaaccgctt
421 ccagaccttc atcagtcagc agatagtttt cgaggaactc ttttgccagc tctttgttcg
481 gactggcggc gttaatacct gcgctcagca cgccaacgaa cggtttggat ggttgaccct
541 tgaaggtcgg cagtaccgtt acaccataat tcactttgct ggtgtcgatg ttggaccatg
601 cccacgggcc gttgatggtc atcgctgttt cgcctttatt aaaggcagct tctgcgatgg
661 agtaatcggt gtctgcattc atgtgtttgt ttttaatcag gtcaaccagg aaggtcagac
721 ccgctttcgc gccagcgtta tccacgccca cgtctttaat gtcgtacttg ccgttttcat
781 acttgaacgc ataacccccg tcagcagcaa tcagcggcca ggtgaagtac ggttcttgca
841 ggttgaacat cagcgcgctc ttacctttcg ctttcagttc tttatccagc gccgggatct
901 cttcccaggt ttttggcggg ttcggcagca gatctttgtt ataaatcagc gataacgctt
961 caacagcgat cgggtaagca atcagcttgc cgttgtaacg tacggcatcc caggtaaacg
1021 gatacagctt gtcctggaac gctttgtccg gggtgatttc agccaacagg ccagattgag
1081 cgtagccacc aaagcggtcg tgtgcccaga agataatgtc agggccatcg ccagttgccg
1141 caacctgtgg gaatttctct tccagtttat ccggatgctc aacggtgact ttaattccgg
1201 tatctttctc gaatttctta ccgacttcag cgagaccgtt atagccttta tcgccgttaa
1261 tccagattac cagtttacct tcttcgattt taccactttc accactacct ttgttcactt
1321 taataaattc aatatcgccc agttttccca tatggctgcc gcgcggcacc aggccgctgc
1381 tgtgatgatg atgatgatgg ctgctgccca tggtatatct ccttcttaaa gttaaacaaa
1441 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
1501 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
1561 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
1621 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
1681 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
1741 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
1801 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
1861 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
1921 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
1981 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
2041 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
2101 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
2161 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
2221 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
2281 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
2341 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
2401 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
2461 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
2521 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
2581 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
2641 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
2701 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
2761 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
2821 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
2881 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
2941 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
3001 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
3061 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
3121 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
3181 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
3241 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
3301 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
3361 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
3421 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
3481 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
3541 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
3601 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
3661 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
3721 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
3781 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
3841 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
3901 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
3961 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
4021 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
4081 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
4141 gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
4201 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
4261 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
4321 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
4381 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
4441 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
4501 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
4561 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
4621 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
4681 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
4741 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
4801 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
4861 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
4921 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
4981 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
5041 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
5101 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
5161 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
5221 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
5281 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
5341 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
5401 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
5461 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
5521 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
5581 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
5641 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
5701 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
5761 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
5821 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
5881 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
5941 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
6001 aaaagtgcca cctgaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg
6061 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
6121 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
6181 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
6241 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
6301 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
6361 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
6421 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
6481 a
//
