| 质粒名称: | pET-28a-hPKM1 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 44241 |
| 存储实验室: | Lewis Cantley, Matthew Vander Heiden |
|
载体骨架基本信息 |
|
| 载体名称: | pET-28a |
| 载体出品公司: | Novagen |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5400 bp |
| 完整质粒大小: | 7100 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Pyruvate Kinase M1,PKM1 |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 1700 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | T7 |
| 标签/融合蛋白: | 6xHis（主干网上的N个终端） |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | EcoRI(未破坏) |
| 3' 克隆位点: | EcoRI (未破坏) |
| 5' 测序引物 | T7 Promoter Primer |
| 3' 测序引物 | T7 Terminator Primer |
|
文献引用方法 |
|
| 材料和方法部分: | pET-28a-hPKM1 was a gift from Lewis Cantley & Matthew Vander Heiden (Addgene plasmid # 44241 ; http://n2t.net/addgene:44241 ; RRID:Addgene_44241) |
| 参考文献部分: | Pyruvate kinase M2 is a phosphotyrosine-binding protein. Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC. Nature. 2008 Mar 13;452(7184):181-6. doi: 10.1038/nature06667. 10.1038/nature06667 PubMed 18337815 |
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 pET-28a-hPKM1 6967 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pET-28a-hPKM1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6967)
AUTHORS Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC
TITLE Pyruvate kinase M2 is a phosphotyrosine-binding protein.
JOURNAL Nature. 2008 Mar 13;452(7184):181-6. doi: 10.1038/nature06667.
PUBMED 18337815
REFERENCE 2 (bases 1 to 6967)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6967
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 55..74
/label=T7
/note="T7 promoter, forward primer"
promoter 55..73
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 74..98
/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 113..135
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 154..171
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 181..198
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 202..234
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10"
/label=T7 tag (gene 10 leader)
/note="promotes efficient translation in E. coli"
/translation="MASMTGGQQMG"
CDS 1886..1903
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(1956..1974)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 1970..2017
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2054..2509
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2141..2160)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2351..2372
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(2601..3416)
/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 3324..3343
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 3408..3427
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 3538..4126
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4027..4046
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4280..4297
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 4312..4451
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(4377..4396)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4537..4559
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4553..4744)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(5553..6635)
/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 6597..6616
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(6636..6713)
/gene="lacI"
/label=lacI promoter
primer_bind 6919..6938
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
61 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
121 actttaagaa ggagatatac catgggcagc agccatcatc atcatcatca cagcagcggc
181 ctggtgccgc gcggcagcca tatggctagc atgactggtg gacagcaaat gggtcgcgga
241 tccgaattca tgtcgaagcc ccatagtgaa gccgggactg ccttcattca gacccagcag
301 ctgcacgcag ccatggctga cacattcctg gagcacatgt gccgcctgga cattgattca
361 ccacccatca cagcccggaa cactggcatc atctgtacca ttggcccagc ttcccgatca
421 gtggagacgt tgaaggagat gattaagtct ggaatgaatg tggctcgtct gaacttctct
481 catggaactc atgagtacca tgcggagacc atcaagaatg tgcgcacagc cacggaaagc
541 tttgcttctg accccatcct ctaccggccc gttgctgtgg ctctagacac taaaggacct
601 gagatccgaa ctgggctcat caagggcagc ggcactgcag aggtggagct gaagaaggga
661 gccactctca aaatcacgct ggataacgcc tacatggaaa agtgtgacga gaacatcctg
721 tggctggact acaagaacat ctgcaaggtg gtggaagtgg gcagcaagat ctacgtggat
781 gatgggctta tttctctcca ggtgaagcag aaaggtgccg acttcctggt gacggaggtg
841 gaaaatggtg gctccttggg cagcaagaag ggtgtgaacc ttcctggggc tgctgtggac
901 ttgcctgctg tgtcggagaa ggacatccag gatctgaagt ttggggtcga gcaggatgtt
961 gatatggtgt ttgcgtcatt catccgcaag gcatctgatg tccatgaagt taggaaggtc
1021 ctgggagaga agggaaagaa catcaagatt atcagcaaaa tcgagaatca tgagggggtt
1081 cggaggtttg atgaaatcct ggaggccagt gatgggatca tggtggctcg tggtgatcta
1141 ggcattgaga ttcctgcaga gaaggtcttc cttgctcaga agatgatgat tggacggtgc
1201 aaccgagctg ggaagcctgt catctgtgct actcagatgc tggagagcat gatcaagaag
1261 ccccgcccca ctcgggctga aggcagtgat gtggccaatg cagtcctgga tggagccgac
1321 tgcatcatgc tgtctggaga aacagccaaa ggggactatc ctctggaggc tgtgcgcatg
1381 cagcacctga tagctcgtga ggctgaggca gccatgttcc accgcaagct gtttgaagaa
1441 cttgtgcgag cctcaagtca ctccacagac ctcatggaag ccatggccat gggcagcgtg
1501 gaggcttctt ataagtgttt agcagcagct ttgatagttc tgacggagtc tggcaggtct
1561 gctcaccagg tggccagata ccgcccacgt gcccccatca ttgctgtgac ccggaatccc
1621 cagacagctc gtcaggccca cctgtaccgt ggcatcttcc ctgtgctgtg caaggaccca
1681 gtccaggagg cctgggctga ggacgtggac ctccgggtga actttgccat gaatgttggc
1741 aaggcccgag gcttcttcaa gaagggagat gtggtcattg tgctgaccgg atggcgccct
1801 ggctccggct tcaccaacac catgcgtgtt gttcctgtgc cgtgagaatt cgagctccgt
1861 cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa
1921 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
1981 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
2041 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
2101 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
2161 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
2221 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
2281 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
2341 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
2401 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
2461 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
2521 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
2581 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
2641 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
2701 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
2761 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
2821 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
2881 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
2941 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
3001 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
3061 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
3121 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
3181 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
3241 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
3301 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
3361 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
3421 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta
3481 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
3541 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
3601 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
3661 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa
3721 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
3781 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
3841 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
3901 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
3961 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
4021 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
4081 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
4141 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
4201 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
4261 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
4321 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca
4381 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
4441 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
4501 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
4561 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
4621 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
4681 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
4741 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
4801 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
4861 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
4921 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
4981 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
5041 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
5101 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
5161 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
5221 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
5281 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
5341 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
5401 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
5461 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
5521 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
5581 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
5641 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
5701 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
5761 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
5821 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
5881 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
5941 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
6001 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
6061 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
6121 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
6181 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
6241 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
6301 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
6361 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
6421 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
6481 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
6541 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
6601 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
6661 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
6721 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
6781 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
6841 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
6901 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
6961 gccggtg
//
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