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- 详细信息
- 文献和实验
- 技术资料
- 库存：
100
- 英文名：
Plasmid#102962;pMalTAviHisBirA
- 保质期：
一年
- 供应商：
上海烜雅生物科技有限公司
- 保存条件：
-20
- 规格：
0.5ug
基本信息
名称：pMal-T-Avi-His/BirA大肠表达质粒
别称: Plasmid#102962;pMalTAviHisBirA
质粒属性
质粒简介
pMal-T-Avi-His/BirA大肠表达质粒
质粒图谱
质粒序列
LOCUS Exported 7292 bp ds-DNA circular SYN 10-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pMal-T-Avi-His-BirA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7292)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7292
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(101..292)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
promoter 788..865
/gene="lacI (mutant)"
/note="lacIq promoter"
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 866..1948
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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"
promoter 2191..2219
/note="tac promoter"
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 2227..2243
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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)."
CDS 2313..3413
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/note="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
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
CDS 3462..3473
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
CDS 3551..3571
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/note="TEV site"
/translation="EDLYFQS"
misc_feature 3590..3631
/note="AviTag"
CDS 3638..3655
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS 3664..3687
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
terminator 3699..3785
/gene="Escherichia coli rrnB"
/note="T1"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 3877..3904
/note="T2"
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 3932..4022
/gene="bla"
/note="Amp promoter"
CDS 4023..4883
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="Amp"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4971..5559
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 5847..5871
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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 5886..5908
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5915..6877
/codon_start=1
/gene="birA"
/product="E. coli biotin protein ligase"
/note="BirA"
/translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
terminator 7000..7047
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
ORIGIN
1 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
61 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
121 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac
181 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
241 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
301 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
361 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
421 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
481 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
541 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
601 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
661 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
721 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
781 aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
841 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
901 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
961 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
1021 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
1081 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
1141 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
1201 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
1261 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
1321 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
1381 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
1441 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
1501 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
1561 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
1621 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatattt cggtagtggg
1681 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
1741 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
1801 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
1861 caatacacaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
1921 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
1981 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
2041 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
2101 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
2161 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
2221 gtggaattgt gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt
2281 cacgagcaat tgaccaacaa ggaccataga ttatgaaaat cgaagaaggt aaactggtaa
2341 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
2401 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
2461 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
2521 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
2581 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
2641 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
2701 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
2761 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
2821 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
2881 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
2941 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
3001 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
3061 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaatgccg
3121 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
3181 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
3241 aagagttggt gaaagatccg cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
3301 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
3361 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
3421 gttctaacaa caacaacaat aacaataaca acaacctcgg gatcgaggga aggatttcac
3481 atatgtccat gggcggccgc gatatcgtcg acaagaggtg atatcgatcg gagctctcga
3541 gccaaccact gaggatctgt actttcagag cgataacgat ggatccctag gactgaacga
3601 catcttcgaa gctcagaaaa tcgaatggca cggatcccat catcaccatc accactagaa
3661 ttcgattaca aggatgacga cgataagtaa ataagcttca aataaaacga aaggctcagt
3721 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
3781 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
3841 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
3901 tttttgcgtt tctacaaact ctttcggtcc gttgtttatt tttctaaata cattcaaata
3961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
4021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
4081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
4141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
4201 ccgaagaacg tttcccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
4261 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
4321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
4381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
4441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
4501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
4561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
4621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
4681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
4741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
4801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
4861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
4921 atttccttag gactgagcgt caaccccgta gaaaagatca aaggatcttc ttgagatcct
4981 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
5041 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
5101 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
5161 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
5221 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
5281 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
5341 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
5401 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
5461 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
5521 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
5581 ttacggttcc tggccttttg ctggcctttt gctcacatgt tccggtaccg cgcccaacag
5641 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
5701 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
5761 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
5821 gcgaaattaa tacgactcac tagaggggaa ttgtgagcgg ataacaattc ccctctagaa
5881 ataattttgt ttaactttaa gaaggagata taccatgaag gataacaccg tgccactgaa
5941 attgattgcc ctgttagcga acggtgaatt tcactctggc gagcagttgg gtgaaacgct
6001 gggaatgagc cgggcggcta ttaataaaca cattcagaca ctgcgtgact ggggcgttga
6061 tgtctttacc gttccgggta aaggatacag cctgcctgag cctatccagt tacttaatgc
6121 taaacagata ttgggtcagc tggatggcgg tagtgtagcc gtgctgccag tgattgactc
6181 cacgaatcag taccttcttg atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc
6241 agaataccag caggctggcc gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc
6301 aaacttatat ttgtcgatgt tctggcgtct ggaacaaggc ccggcggcgg cgattggttt
6361 aagtctggtt atcggtatcg tgatggcgga agtattacgc aagctgggtg cagataaagt
6421 tcgtgttaaa tggcctaatg acctctatct gcaggatcgc aagctggcag gcattctggt
6481 ggagctgact ggcaaaactg gcgatgcggc gcaaatagtc attggagccg ggatcaacat
6541 ggcaatgcgc cgtgttgaag agagtgtcgt taatcagggg tggatcacgc tgcaggaagc
6601 ggggatcaat ctcgatcgta atacgttggc ggccatgcta atacgtgaat tacgtgctgc
6661 gttggaactc ttcgaacaag aaggattggc accttatctg tcgcgctggg aaaagctgga
6721 taattttatt aatcgcccag tgaaacttat cattggtgat aaagaaatat ttggcatttc
6781 acgcggaata gacaaacagg gggctttatt acttgagcag gatggaataa taaaaccctg
6841 gatgggcggt gaaatatccc tgcgtagtgc agaaaaaggt aagctttgat gaggactagt
6901 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
6961 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
7021 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atgctagcac
7081 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
7141 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
7201 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
7261 taaggtgcac tgtgactggg tcatggctgc gc
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pMal-T-Avi-His/BirA大肠表达质粒
别称: Plasmid#102962;pMalTAviHisBirA
| 启动子: | Tac |
|---|---|
| 复制子: | pBR322 |
| 原核抗性: | Amp |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 大肠杆菌 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 真核抗性： | |
| 荧光蛋白： |
质粒简介
pMal-T-Avi-His/BirA大肠表达质粒
质粒图谱
质粒序列
LOCUS Exported 7292 bp ds-DNA circular SYN 10-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pMal-T-Avi-His-BirA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7292)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7292
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(101..292)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
promoter 788..865
/gene="lacI (mutant)"
/note="lacIq promoter"
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 866..1948
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="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"
promoter 2191..2219
/note="tac promoter"
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 2227..2243
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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)."
CDS 2313..3413
/codon_start=1
/gene="malE (mutated)"
/product="maltose binding protein from E. coli"
/note="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
ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
WYAVRTAVINAASGRQTVDEALKDAQT"
CDS 3462..3473
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
CDS 3551..3571
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/note="TEV site"
/translation="EDLYFQS"
misc_feature 3590..3631
/note="AviTag"
CDS 3638..3655
/codon_start=1
/product="6xHis affinity tag"
/note="6xHis"
/translation="HHHHHH"
CDS 3664..3687
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
terminator 3699..3785
/gene="Escherichia coli rrnB"
/note="T1"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 3877..3904
/note="T2"
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 3932..4022
/gene="bla"
/note="Amp promoter"
CDS 4023..4883
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="Amp"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 4971..5559
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 5847..5871
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/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 5886..5908
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5915..6877
/codon_start=1
/gene="birA"
/product="E. coli biotin protein ligase"
/note="BirA"
/translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
terminator 7000..7047
/note="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
ORIGIN
1 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
61 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
121 caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac
181 agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct
241 ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct
301 ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc
361 tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac
421 aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct
481 tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc
541 ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac
601 cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg
661 ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc
721 gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ccaggaccca acgctgcccg
781 aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga
841 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
901 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
961 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
1021 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
1081 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
1141 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
1201 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
1261 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
1321 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
1381 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
1441 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
1501 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
1561 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
1621 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatattt cggtagtggg
1681 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
1741 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
1801 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
1861 caatacacaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
1921 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
1981 attaggcaca attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc
2041 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
2101 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
2161 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
2221 gtggaattgt gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt
2281 cacgagcaat tgaccaacaa ggaccataga ttatgaaaat cgaagaaggt aaactggtaa
2341 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
2401 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
2461 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
2521 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
2581 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
2641 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
2701 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
2761 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
2821 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcga
2881 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
2941 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
3001 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
3061 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaatgccg
3121 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
3181 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
3241 aagagttggt gaaagatccg cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
3301 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
3361 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
3421 gttctaacaa caacaacaat aacaataaca acaacctcgg gatcgaggga aggatttcac
3481 atatgtccat gggcggccgc gatatcgtcg acaagaggtg atatcgatcg gagctctcga
3541 gccaaccact gaggatctgt actttcagag cgataacgat ggatccctag gactgaacga
3601 catcttcgaa gctcagaaaa tcgaatggca cggatcccat catcaccatc accactagaa
3661 ttcgattaca aggatgacga cgataagtaa ataagcttca aataaaacga aaggctcagt
3721 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
3781 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
3841 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
3901 tttttgcgtt tctacaaact ctttcggtcc gttgtttatt tttctaaata cattcaaata
3961 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
4021 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
4081 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
4141 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
4201 ccgaagaacg tttcccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
4261 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
4321 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
4381 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
4441 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
4501 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
4561 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
4621 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
4681 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
4741 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
4801 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
4861 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
4921 atttccttag gactgagcgt caaccccgta gaaaagatca aaggatcttc ttgagatcct
4981 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
5041 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
5101 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
5161 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
5221 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
5281 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
5341 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
5401 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
5461 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
5521 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
5581 ttacggttcc tggccttttg ctggcctttt gctcacatgt tccggtaccg cgcccaacag
5641 tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa
5701 gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc
5761 tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc
5821 gcgaaattaa tacgactcac tagaggggaa ttgtgagcgg ataacaattc ccctctagaa
5881 ataattttgt ttaactttaa gaaggagata taccatgaag gataacaccg tgccactgaa
5941 attgattgcc ctgttagcga acggtgaatt tcactctggc gagcagttgg gtgaaacgct
6001 gggaatgagc cgggcggcta ttaataaaca cattcagaca ctgcgtgact ggggcgttga
6061 tgtctttacc gttccgggta aaggatacag cctgcctgag cctatccagt tacttaatgc
6121 taaacagata ttgggtcagc tggatggcgg tagtgtagcc gtgctgccag tgattgactc
6181 cacgaatcag taccttcttg atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc
6241 agaataccag caggctggcc gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc
6301 aaacttatat ttgtcgatgt tctggcgtct ggaacaaggc ccggcggcgg cgattggttt
6361 aagtctggtt atcggtatcg tgatggcgga agtattacgc aagctgggtg cagataaagt
6421 tcgtgttaaa tggcctaatg acctctatct gcaggatcgc aagctggcag gcattctggt
6481 ggagctgact ggcaaaactg gcgatgcggc gcaaatagtc attggagccg ggatcaacat
6541 ggcaatgcgc cgtgttgaag agagtgtcgt taatcagggg tggatcacgc tgcaggaagc
6601 ggggatcaat ctcgatcgta atacgttggc ggccatgcta atacgtgaat tacgtgctgc
6661 gttggaactc ttcgaacaag aaggattggc accttatctg tcgcgctggg aaaagctgga
6721 taattttatt aatcgcccag tgaaacttat cattggtgat aaagaaatat ttggcatttc
6781 acgcggaata gacaaacagg gggctttatt acttgagcag gatggaataa taaaaccctg
6841 gatgggcggt gaaatatccc tgcgtagtgc agaaaaaggt aagctttgat gaggactagt
6901 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
6961 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
7021 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg atgctagcac
7081 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
7141 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
7201 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
7261 taaggtgcac tgtgactggg tcatggctgc gc
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P3871/pCMV-SPORT6-NCAPD3人源基因质粒 |
| P3872/pCMV-SPORT6-ORC1人源基因质粒 |
| P3873/pCMV-SPORT6-PTPN4人源基因质粒 |
| P3874/pCMV-SPORT6-MAP3K11（1同义突变）人源基因质粒 |
| P3875/pCMV-SPORT6-MED24（2同义突变）人源基因质粒 |
| P3876/pCMV-SPORT6-GPT人源基因质粒 |
| P3877/pCMV-SPORT6-PHYHIPL(1点突变）人源基因质粒 |
| P3878/pCMV-SPORT6-DVL2人源基因质粒 |
| P3879/pCMV-SPORT6-GAS2L1（1同义突变）人源基因质粒 |
| P3880/pCMV-SPORT6-LYAR人源基因质粒 |
| P3881/pCMV-SPORT6-GPRASP2人源基因质粒 |
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文献和实验相关实验
α基因,目的基因的插入导致其失活,通过X-gal平板可进行蓝白斑筛选.pMAL载体都含有一段编码蛋白酶识别位点的序列,融合蛋白纯化后,通过Factor Xa(X),Enterokinase (E)或 Genenase™ I (G)可将目的蛋白与MBP切割分离.如果不想在切割后留下任何外源氨基酸可以在设计引物时引入XmnⅠ或KpnⅠ或SnaBⅠ位点. MBP融合表达更能提高E.coli表达蛋白的可溶性,可是它在大肠杆菌的表达含量不如T7等系列高.形成包涵体的主要原因之一是其表达速度
或KpnⅠ或SnaBⅠ位点。MBP融合表达更能提高E.coli表达蛋白的可溶性，可是它在大肠杆菌的表达含量不如T7等系列高。形成包涵体的主要原因之一是其表达速度过快，如果大肠杆菌自身蛋白表达过快也会以包涵体形式出现。这个问题在以后的原核表达终结篇里再做详细讨论。pMAL™系统采用麦芽糖温和洗脱，无去污剂或变性剂对蛋白活性的影响。其中的pMAL-p2X载体带有信号肽，可使外源蛋白在细胞质或周质中表达。周质表达可提高二硫键的形成，促进蛋白折叠的形成。此系统还有配套的，用于纯化的多糖树脂，它是一亲和基质
pMBP-P 大肠杆菌质粒 Amp 分泌表达，His和MBP标签，带凝血酶位点 pMal-C2x 大肠杆菌质粒 Amp 表达MBP标签蛋白，原核表达载体 pMal-P2x 大肠杆菌质粒 Amp pMal-P
pMal-T-Avi-His/BirA大肠表达质粒
￥580 - 1280
