| 质粒名称: | pET21 MBPx-SpyCatcher |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 72327 |
| 存储实验室: | Mark Howarth |
|
载体骨架基本信息 |
|
| 载体名称: | pET21b |
| 载体出品公司: | Novagen |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5442 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: | pET21 MBPx-SpyCatcher,MBPx-SpyCatcher |
| 物种来源: | Synthetic |
| 插入大小: | 2661 bp |
| 基因突变: | MBP包含A312V、I317V突变和172、173、175和176个缺失 |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | N端子His6标签 |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | TAA TAC GAC TCA CTA TAG GG |
| 3' 测序引物 | GCT AGT TAT TGC TCA GCG G |
|
文献引用方法 |
|
| 材料和方法部分: | pET21 MBPx-SpyCatcher was a gift from Mark Howarth (Addgene plasmid # 72327 ; http://n2t.net/addgene:72327 ; RRID:Addgene_72327) |
| 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 pET21 MBPx-SpyCatcher 8043 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bacterial expression of tandem high affinity maltose binding protein
domains linked to SpyCatcher, for solid-phase polyprotein
synthesis..
ACCESSION .
VERSION .
KEYWORDS pET21 MBPx-SpyCatcher
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8043)
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 8043)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8043
/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(2783..2800)
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS complement(2810..2827)
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
RBS 2847..2869
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind 2884..2908
/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(2908..2927)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2909..2927)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3011..3030)
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
promoter 3236..3313
/gene="lacI"
/label=lacI promoter
CDS 3314..4396
/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(3333..3352)
/label=LacI-R
/note="LacI, reverse primer"
CDS 5205..5396
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
primer_bind complement(5390..5412)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
misc_feature 5498..5640
/label=bom
/note="basis of mobility region from pBR322"
primer_bind 5553..5572
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind complement(5655..5672)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5826..6414)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5906..5925)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6585..7445)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind 7208..7227
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7446..7550)
/gene="bla"
/label=AmpR promoter
rep_origin complement(7577..8032)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(7714..7735)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 7926..7945
/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 gcaagcttct
181 attaaatatg agcgtcacct ttagttgctt tgccatttac agtaacctga ccttgctcat
241 taactgtaaa ggtaatagca gttgctacct cataaccgtc tggtgctgcg gtttcgacaa
301 atgtatattt tcctggatac aggtagaaat ctttcacttg tccatctgaa atccatgtac
361 taatagtttt accagatgaa tcacgcaact ccatagttgc accagctaac tctttgccgt
421 cctcatcacg ttttgagaat ttaatatggg tagcactatc ttcttcaatt gtcatatcac
481 cggactgacc ttgctcactt gataaacctg ataaggtatc aaccatggca ccactaccgc
541 ccgaacccga gctcgaatta gtctgcgcgt ctttcagggc ttcatcgaca gtctgacgac
601 cgctggcggc gttgatcacc gcagtacgca cggcatacca gaaagcggac atctgcggga
661 tgttcggcat gatttcacct ttctgggcgt tttccatagt ggcggccaca cgtggatctt
721 tcaccaactc ttcctcgtaa gacttcagcg ctacggcacc cagcggtttg tctttattaa
781 ccgcttccag accttcatca gtcagcagat agttttcgag gaactctttt gccagctctt
841 tgttcggact ggcggcgtta atacctgcgc tcagcacgcc aacgaacggt ttggatggtt
901 gacccttgaa ggtcggcagt accgttacac cataattcac tttgctggtg tcgatgttgg
961 accatgccca cgggccgttg atggtcatcg ctgtttcgcc tttattaaag gcagcttctg
1021 cgatggagta atcggtgtct gcattcatgt gtttgttttt aatcaggtca accaggaagg
1081 tcagacccgc tttcgcgcca gcgttatcca cgcccacgtc tttaatgtcg ccatacttga
1141 acgcataacc cccgtcagca gcaatcagcg gccaggtgaa gtacggttct tgcaggttga
1201 acatcagcgc gctcttacct ttcgctttca gttctttatc cagcgccggg atctcttccc
1261 aggtttttgg cgggttcggc agcagatctt tgttataaat cagcgataac gcttcaacag
1321 cgatcgggta agcaatcagc ttgccgttgt aacgtacggc atcccaggta aacggataca
1381 gcttgtcctg gaacgctttg tccggggtga tttcagccaa caggccagat tgagcgtagc
1441 caccaaagcg gtcgtgtgcc cagaagataa tgtcagggcc atcgccagtt gccgcaacct
1501 gtgggaattt ctcttccagt ttatccggat gctcaacggt gactttaatt ccggtatctt
1561 tctcgaattt cttaccgact tcagcgagac cgttatagcc tttatcgccg ttaatccaga
1621 ttaccagttt accctcctct atctttccgg atccacctcc ggatccgccg gaactagaat
1681 tcgtctgcgc gtctttcagg gcttcatcga cagtctgacg accgctggcg gcgttgatca
1741 ccgcagtacg cacggcatac cagaaagcgg acatctgcgg gatgttcggc atgatttcac
1801 ctttctgggc gttttccata gtggcggcca cacgtggatc tttcaccaac tcttcctcgt
1861 aagacttcag cgctacggca cccagcggtt tgtctttatt aaccgcttcc agaccttcat
1921 cagtcagcag atagttttcg aggaactctt ttgccagctc tttgttcgga ctggcggcgt
1981 taatacctgc gctcagcacg ccaacgaacg gtttggatgg ttgacccttg aaggtcggca
2041 gtaccgttac accataattc actttgctgg tgtcgatgtt ggaccatgcc cacgggccgt
2101 tgatggtcat cgctgtttcg cctttattaa aggcagcttc tgcgatggag taatcggtgt
2161 ctgcattcat gtgtttgttt ttaatcaggt caaccaggaa ggtcagaccc gctttcgcgc
2221 cagcgttatc cacgcccacg tctttaatgt cgccatactt gaacgcataa cccccgtcag
2281 cagcaatcag cggccaggtg aagtacggtt cttgcaggtt gaacatcagc gcgctcttac
2341 ctttcgcttt cagttcttta tccagcgccg ggatctcttc ccaggttttt ggcgggttcg
2401 gcagcagatc tttgttataa atcagcgata acgcttcaac agcgatcggg taagcaatca
2461 gcttgccgtt gtaacgtacg gcatcccagg taaacggata cagcttgtcc tggaacgctt
2521 tgtccggggt gatttcagcc aacaggccag attgagcgta gccaccaaag cggtcgtgtg
2581 cccagaagat aatgtcaggg ccatcgccag ttgccgcaac ctgtgggaat ttctcttcca
2641 gtttatccgg atgctcaacg gtgactttaa ttccggtatc tttctcgaat ttcttaccga
2701 cttcagcgag accgttatag cctttatcgc cgttaatcca gattaccagt ttaccttctt
2761 cgattttcat gctagccata tggctgccgc gcggcaccag gccgctgctg tgatgatgat
2821 gatgatggct gctgcccata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
2881 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
2941 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
3001 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
3061 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
3121 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
3181 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
3241 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
3301 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
3361 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
3421 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
3481 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
3541 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
3601 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
3661 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
3721 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
3781 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
3841 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
3901 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
3961 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
4021 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
4081 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
4141 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
4201 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
4261 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
4321 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
4381 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
4441 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
4501 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
4561 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
4621 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
4681 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
4741 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
4801 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
4861 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
4921 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
4981 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
5041 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
5101 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
5161 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
5221 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
5281 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
5341 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
5401 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
5461 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
5521 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
5581 gcaccatata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
5641 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
5701 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
5761 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
5821 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
5881 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
5941 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
6001 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
6061 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
6121 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
6181 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
6241 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
6301 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
6361 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
6421 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
6481 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
6541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
6601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
6661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
6721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
6781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
6841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
6901 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
6961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
7021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
7081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
7141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
7201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
7261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
7321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
7381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
7441 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
7501 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
7561 cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt
7621 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
7681 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
7741 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
7801 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
7861 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
7921 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
7981 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
8041 cca
//
