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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pGADT7-AD
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pGADT7-AD酵母双杂交质粒
别称: pGADT7-AD
质粒属性
质粒简介
pGADT7 AD is a yeast expression vector that is designed to express a protein of interestfused to a GAL4 activation domain (AD; amino acids 768–881). Transcription of the GAL4 ADfusion is driven by the constitutively active ADH1 promoter (PADH1), and is terminated at theADH1 transcription termination signal (TADH1). The GAL4 AD fusion contains an N-terminalSV40 nuclear localization signal (SV40 NLS; 1) that targets the protein to the yeast nucleus,and a hemagglutinin epitope tag (HA Tag), located between the GAL4 AD and the proteinof interest, that allows the protein to be easily detected with HA-tag antibodies.
质粒图谱
质粒序列
LOCUS Exported 7987 bp ds-DNA circular SYN 09-SEP-2012
DEFINITION Yeast two-hybrid ""prey"" vector for expressing proteins fused to
the GAL4 activation domain.
KEYWORDS pGADT7 AD
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7987)
AUTHORS Clontech
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7987
/organism="synthetic DNA construct"
/lab_host="Saccharomyces cerevisiae"
/mol_type="other DNA"
promoter 771..1475
/gene="S. cerevisiae ADH1"
/note="ADH1 promoter"
/note="promoter for alcohol dehydrogenase 1"
CDS 1491..1493
/codon_start=1
/product="start codon for expression in yeast"
/note="ATG"
/translation="M"
CDS 1521..1541
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1557..1898
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="activation domain of the GAL4 transcriptional
activator"
/note="GAL4 activation domain"
/translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
EDTPPNPKKE"
promoter 1904..1922
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1935..1937
/codon_start=1
/product="start codon for in vitro
transcription/translation"
/note="ATG"
/translation="M"
CDS 1941..1967
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/note="HA"
/translation="YPYDVPDYA"
misc_feature 1968..2035
/label=MCS
/note="MCS"
/note="multiple cloning site"
terminator 2415..2602
/gene="S. cerevisiae ADH1"
/note="ADH1 terminator"
/note="transcription terminator for alcohol dehydrogenase
1"
CDS complement(2719..3813)
/codon_start=1
/gene="S. cerevisiae LEU2"
/product="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/note="LEU2"
/note="yeast auxotrophic marker"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIEATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(3814..4219)
/gene="S. cerevisiae LEU2"
/note="LEU2 promoter"
primer_bind complement(4261..4277)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 4285..4301
/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)."
promoter complement(4309..4339)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(4814..5402)
/direction=LEFT
/note="ori"
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5573..6433)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6434..6538)
/gene="bla"
/note="AmpR promoter"
rep_origin 6820..7984
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
ORIGIN
1 tgcatgcctg caggtcgaga tccgggatcg aagaaatgat ggtaaatgaa ataggaaatc
61 aaggagcatg aaggcaaaag acaaatataa gggtcgaacg aaaaataaag tgaaaagtgt
121 tgatatgatg tatttggctt tgcggcgccg aaaaaacgag tttacgcaat tgcacaatca
181 tgctgactct gtggcggacc cgcgctcttg ccggcccggc gataacgctg ggcgtgaggc
241 tgtgcccggc ggagtttttt gcgcctgcat tttccaaggt ttaccctgcg ctaaggggcg
301 agattggaga agcaataaga atgccggttg gggttgcgat gatgacgacc acgacaactg
361 gtgtcattat ttaagttgcc gaaagaacct gagtgcattt gcaacatgag tatactagaa
421 gaatgagcca agacttgcga gacgcgagtt tgccggtggt gcgaacaata gagcgaccat
481 gaccttgaag gtgagacgcg cataaccgct agagtacttt gaagaggaaa cagcaatagg
541 gttgctacca gtataaatag acaggtacat acaacactgg aaatggttgt ctgtttgagt
601 acgctttcaa ttcatttggg tgtgcacttt attatgttac aatatggaag ggaactttac
661 acttctccta tgcacatata ttaattaaag tccaatgcta gtagagaagg ggggtaacac
721 ccctccgcgc tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt
781 tgtttccggg tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga
841 ttcctataat accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat
901 agaacagata ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc
961 attgatggtg gtacataacg aactaatact gtagccctag acttgatagc catcatcata
1021 tcgaagtttc actacccttt ttccatttgc catctattga agtaataata ggcgcatgca
1081 acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat
1141 gacaaaaaaa tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag
1201 atgtcgttgt tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct
1261 tcattcacgc acactactct ctaatgagca acggtatacg gccttccttc cagttacttg
1321 aatttgaaat aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat
1381 taatcttttg tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca
1441 caatatttca agctatacca agcatacaat caactccaag ctttgcaaag atggataaag
1501 cggaattaat tcccgagcct ccaaaaaaga agagaaaggt cgaattgggt accgccgcca
1561 attttaatca aagtgggaat attgctgata gctcattgtc cttcactttc actaacagta
1621 gcaacggtcc gaacctcata acaactcaaa caaattctca agcgctttca caaccaattg
1681 cctcctctaa cgttcatgat aacttcatga ataatgaaat cacggctagt aaaattgatg
1741 atggtaataa ttcaaaacca ctgtcacctg gttggacgga ccaaactgcg tataacgcgt
1801 ttggaatcac tacagggatg tttaatacca ctacaatgga tgatgtatat aactatctat
1861 tcgatgatga agatacccca ccaaacccaa aaaaagagat ctttaatacg actcactata
1921 gggcgagcgc cgccatggag tacccatacg acgtaccaga ttacgctcat atggccatgg
1981 aggccagtga attccacccg ggtgggcatc gatacgggat ccatcgagct cgagctgcag
2041 atgaatcgta gatactgaaa aaccccgcaa gttcacttca actgtgcatc gtgcaccatc
2101 tcaatttctt tcatttatac atcgttttgc cttcttttat gtaactatac tcctctaagt
2161 ttcaatcttg gccatgtaac ctctgatcta tagaattttt taaatgacta gaattaatgc
2221 ccatcttttt tttggaccta aattcttcat gaaaatatat tacgagggct tattcagaag
2281 ctttggactt cttcgccaga ggtttggtca agtctccaat caaggttgtc ggcttgtcta
2341 ccttgccaga aatttacgaa aagatggaaa agggtcaaat cgttggtaga tacgttgttg
2401 acacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa taagttataa
2461 aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat
2521 tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagcat gaggtcgctc
2581 ttattgacca cacctctacc ggccggtcga aattccccta ccctatgaac atattccatt
2641 ttgtaatttc gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca
2701 taaaaaaaga gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc
2761 gacttcggtg gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac
2821 ctttttaact gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa
2881 catcattgca gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg
2941 agcagaaccg tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc
3001 caaggacgca gatggcaaca aacccaagga acctgggata acggaggctt catcggagat
3061 gatatcacca aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac
3121 taggatcatg gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt
3181 cttgatggtt tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt
3241 atccaaggac caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct
3301 tgtgattctt tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc
3361 atcgtcttcc tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa
3421 gtcagtacct ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc
3481 aaagttacat ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc
3541 ttgttcaggt ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac
3601 ggcatcagcc ttcttggagg cttccagcgc ctcatctgga agtgggacac ctgtagcttc
3661 gatagcagca ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc
3721 agaaatagct ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc
3781 tggcaaaacg acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata
3841 tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac
3901 ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa
3961 gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc gctctcacct
4021 ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat
4081 taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc
4141 gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata
4201 cctgagtatt cccacagttg ggggatctcg actctagcta gaggatcaat tcgtaatcat
4261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
4321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctga taacttcgta
4441 taatgtatgc tatacgaagt tattaggtct gaagaggagt ttacgtccag ccaagctagc
4501 ttggctgcag gtcgagcggc cgcgatccgg aacccttaat ataacttcgt ataatgtatg
4561 ctatacgaag ttatcagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
4621 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
4681 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
4741 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
4801 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
4861 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
4921 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
4981 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
5041 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
5101 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
5161 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
5221 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
5281 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
5341 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
5401 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
5461 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
5521 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
5581 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
5641 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
5701 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
5761 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
5821 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
5881 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
5941 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
6001 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
6061 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
6121 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
6181 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
6241 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
6301 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
6361 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
6421 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
6481 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
6541 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
6601 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
6661 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
6721 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
6781 atgcggcatc agagcagatt gtactgagag tgcaccataa cgcatttaag cataaacacg
6841 cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata taggtgcgac
6901 gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg
6961 aaacgctttg aagttcctat tccgaagttc ctattctcta gctagaaagt ataggaactt
7021 cagagcgctt ttgaaaacca aaagcgctct gaagacgcac tttcaaaaaa ccaaaaacgc
7081 accggactgt aacgagctac taaaatattg cgaataccgc ttccacaaac attgctcaaa
7141 agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc atccaccttt
7201 cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat ctctagtatt
7261 actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga aaacaatacg
7321 aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa ccgttcataa
7381 ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag tatgcgcaat
7441 ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc acccgcagct
7501 tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga agagaaaata
7561 gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag ttatcaagag
7621 actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt gttagaaaaa
7681 tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct ttgttggtaa
7741 aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga
7801 aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag attcttcgtt
7861 ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg
7921 tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc acagatgctt
7981 cgttgct
//
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：pGADT7-AD酵母双杂交质粒
别称: pGADT7-AD
| 启动子: | ADH1 |
|---|---|
| 复制子: | 2U |
| 质粒分类: | 酵母系列，酵母双杂交载体 |
| 质粒大小: | 7987bp |
| 原核抗性: | Amp |
| 筛选标记: | LEU2 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 5'测序引物: | T7:TAATACGACTCACTATAGGG |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 载体宿主： | 酵母菌 |
|---|---|
| 载体用途： | 杂交 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | LEU2 |
| 荧光蛋白： |
质粒简介
pGADT7 AD is a yeast expression vector that is designed to express a protein of interestfused to a GAL4 activation domain (AD; amino acids 768–881). Transcription of the GAL4 ADfusion is driven by the constitutively active ADH1 promoter (PADH1), and is terminated at theADH1 transcription termination signal (TADH1). The GAL4 AD fusion contains an N-terminalSV40 nuclear localization signal (SV40 NLS; 1) that targets the protein to the yeast nucleus,and a hemagglutinin epitope tag (HA Tag), located between the GAL4 AD and the proteinof interest, that allows the protein to be easily detected with HA-tag antibodies.
质粒图谱
质粒序列
LOCUS Exported 7987 bp ds-DNA circular SYN 09-SEP-2012
DEFINITION Yeast two-hybrid ""prey"" vector for expressing proteins fused to
the GAL4 activation domain.
KEYWORDS pGADT7 AD
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7987)
AUTHORS Clontech
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..7987
/organism="synthetic DNA construct"
/lab_host="Saccharomyces cerevisiae"
/mol_type="other DNA"
promoter 771..1475
/gene="S. cerevisiae ADH1"
/note="ADH1 promoter"
/note="promoter for alcohol dehydrogenase 1"
CDS 1491..1493
/codon_start=1
/product="start codon for expression in yeast"
/note="ATG"
/translation="M"
CDS 1521..1541
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS 1557..1898
/codon_start=1
/gene="Saccharomyces cerevisiae GAL4 (truncated)"
/product="activation domain of the GAL4 transcriptional
activator"
/note="GAL4 activation domain"
/translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
EDTPPNPKKE"
promoter 1904..1922
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1935..1937
/codon_start=1
/product="start codon for in vitro
transcription/translation"
/note="ATG"
/translation="M"
CDS 1941..1967
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/note="HA"
/translation="YPYDVPDYA"
misc_feature 1968..2035
/label=MCS
/note="MCS"
/note="multiple cloning site"
terminator 2415..2602
/gene="S. cerevisiae ADH1"
/note="ADH1 terminator"
/note="transcription terminator for alcohol dehydrogenase
1"
CDS complement(2719..3813)
/codon_start=1
/gene="S. cerevisiae LEU2"
/product="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/note="LEU2"
/note="yeast auxotrophic marker"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIEATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(3814..4219)
/gene="S. cerevisiae LEU2"
/note="LEU2 promoter"
primer_bind complement(4261..4277)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 4285..4301
/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)."
promoter complement(4309..4339)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(4814..5402)
/direction=LEFT
/note="ori"
/note="high-copy-number colE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5573..6433)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6434..6538)
/gene="bla"
/note="AmpR promoter"
rep_origin 6820..7984
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
ORIGIN
1 tgcatgcctg caggtcgaga tccgggatcg aagaaatgat ggtaaatgaa ataggaaatc
61 aaggagcatg aaggcaaaag acaaatataa gggtcgaacg aaaaataaag tgaaaagtgt
121 tgatatgatg tatttggctt tgcggcgccg aaaaaacgag tttacgcaat tgcacaatca
181 tgctgactct gtggcggacc cgcgctcttg ccggcccggc gataacgctg ggcgtgaggc
241 tgtgcccggc ggagtttttt gcgcctgcat tttccaaggt ttaccctgcg ctaaggggcg
301 agattggaga agcaataaga atgccggttg gggttgcgat gatgacgacc acgacaactg
361 gtgtcattat ttaagttgcc gaaagaacct gagtgcattt gcaacatgag tatactagaa
421 gaatgagcca agacttgcga gacgcgagtt tgccggtggt gcgaacaata gagcgaccat
481 gaccttgaag gtgagacgcg cataaccgct agagtacttt gaagaggaaa cagcaatagg
541 gttgctacca gtataaatag acaggtacat acaacactgg aaatggttgt ctgtttgagt
601 acgctttcaa ttcatttggg tgtgcacttt attatgttac aatatggaag ggaactttac
661 acttctccta tgcacatata ttaattaaag tccaatgcta gtagagaagg ggggtaacac
721 ccctccgcgc tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt
781 tgtttccggg tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga
841 ttcctataat accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat
901 agaacagata ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc
961 attgatggtg gtacataacg aactaatact gtagccctag acttgatagc catcatcata
1021 tcgaagtttc actacccttt ttccatttgc catctattga agtaataata ggcgcatgca
1081 acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat
1141 gacaaaaaaa tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag
1201 atgtcgttgt tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct
1261 tcattcacgc acactactct ctaatgagca acggtatacg gccttccttc cagttacttg
1321 aatttgaaat aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat
1381 taatcttttg tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca
1441 caatatttca agctatacca agcatacaat caactccaag ctttgcaaag atggataaag
1501 cggaattaat tcccgagcct ccaaaaaaga agagaaaggt cgaattgggt accgccgcca
1561 attttaatca aagtgggaat attgctgata gctcattgtc cttcactttc actaacagta
1621 gcaacggtcc gaacctcata acaactcaaa caaattctca agcgctttca caaccaattg
1681 cctcctctaa cgttcatgat aacttcatga ataatgaaat cacggctagt aaaattgatg
1741 atggtaataa ttcaaaacca ctgtcacctg gttggacgga ccaaactgcg tataacgcgt
1801 ttggaatcac tacagggatg tttaatacca ctacaatgga tgatgtatat aactatctat
1861 tcgatgatga agatacccca ccaaacccaa aaaaagagat ctttaatacg actcactata
1921 gggcgagcgc cgccatggag tacccatacg acgtaccaga ttacgctcat atggccatgg
1981 aggccagtga attccacccg ggtgggcatc gatacgggat ccatcgagct cgagctgcag
2041 atgaatcgta gatactgaaa aaccccgcaa gttcacttca actgtgcatc gtgcaccatc
2101 tcaatttctt tcatttatac atcgttttgc cttcttttat gtaactatac tcctctaagt
2161 ttcaatcttg gccatgtaac ctctgatcta tagaattttt taaatgacta gaattaatgc
2221 ccatcttttt tttggaccta aattcttcat gaaaatatat tacgagggct tattcagaag
2281 ctttggactt cttcgccaga ggtttggtca agtctccaat caaggttgtc ggcttgtcta
2341 ccttgccaga aatttacgaa aagatggaaa agggtcaaat cgttggtaga tacgttgttg
2401 acacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa taagttataa
2461 aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat
2521 tcttgagtaa ctctttcctg taggtcaggt tgctttctca ggtatagcat gaggtcgctc
2581 ttattgacca cacctctacc ggccggtcga aattccccta ccctatgaac atattccatt
2641 ttgtaatttc gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca
2701 taaaaaaaga gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc
2761 gacttcggtg gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac
2821 ctttttaact gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa
2881 catcattgca gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg
2941 agcagaaccg tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc
3001 caaggacgca gatggcaaca aacccaagga acctgggata acggaggctt catcggagat
3061 gatatcacca aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac
3121 taggatcatg gcggcagaat caatcaattg atgttgaacc ttcaatgtag gaaattcgtt
3181 cttgatggtt tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt
3241 atccaaggac caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct
3301 tgtgattctt tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc
3361 atcgtcttcc tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa
3421 gtcagtacct ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc
3481 aaagttacat ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc
3541 ttgttcaggt ctaacactac ctgtacccca tttaggacca cccacagcac ctaacaaaac
3601 ggcatcagcc ttcttggagg cttccagcgc ctcatctgga agtgggacac ctgtagcttc
3661 gatagcagca ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc
3721 agaaatagct ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc
3781 tggcaaaacg acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata
3841 tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac
3901 ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa
3961 gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc gctctcacct
4021 ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat
4081 taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc
4141 gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata
4201 cctgagtatt cccacagttg ggggatctcg actctagcta gaggatcaat tcgtaatcat
4261 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
4321 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctga taacttcgta
4441 taatgtatgc tatacgaagt tattaggtct gaagaggagt ttacgtccag ccaagctagc
4501 ttggctgcag gtcgagcggc cgcgatccgg aacccttaat ataacttcgt ataatgtatg
4561 ctatacgaag ttatcagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
4621 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
4681 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
4741 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
4801 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
4861 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
4921 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
4981 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
5041 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
5101 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
5161 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
5221 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
5281 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
5341 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
5401 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
5461 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
5521 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
5581 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
5641 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
5701 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
5761 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
5821 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
5881 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
5941 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
6001 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
6061 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
6121 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
6181 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
6241 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
6301 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
6361 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
6421 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
6481 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
6541 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
6601 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
6661 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
6721 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
6781 atgcggcatc agagcagatt gtactgagag tgcaccataa cgcatttaag cataaacacg
6841 cactatgccg ttcttctcat gtatatatat atacaggcaa cacgcagata taggtgcgac
6901 gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc tcgttttcgg
6961 aaacgctttg aagttcctat tccgaagttc ctattctcta gctagaaagt ataggaactt
7021 cagagcgctt ttgaaaacca aaagcgctct gaagacgcac tttcaaaaaa ccaaaaacgc
7081 accggactgt aacgagctac taaaatattg cgaataccgc ttccacaaac attgctcaaa
7141 agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc atccaccttt
7201 cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat ctctagtatt
7261 actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga aaacaatacg
7321 aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa ccgttcataa
7381 ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag tatgcgcaat
7441 ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc acccgcagct
7501 tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga agagaaaata
7561 gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag ttatcaagag
7621 actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt gttagaaaaa
7681 tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct ttgttggtaa
7741 aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga
7801 aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag attcttcgtt
7861 ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg
7921 tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc acagatgctt
7981 cgttgct
//
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
| P2593/pENTR223-NMRK1人源基因质粒 |
| P2594/pENTR223-IL11人源基因质粒 |
| P2595/pENTR223-DLK2（538-1152bp）人源基因质粒 |
| P2596/pENTR223-ATP6V0B人源基因质粒 |
| P2597/pENTR223-RAB1A人源基因质粒 |
| P2598/pENTR223-MTHFS人源基因质粒 |
| P2599/pENTR223-ASF1B人源基因质粒 |
| P2600/pENTR223-DNAJC12-T19C人源基因质粒 |
| P2601/pENTR223-CAV1人源基因质粒 |
| P2602/pENTR223-PPM1B人源基因质粒 |
| P2603/pENTR223-SRSF10人源基因质粒 |
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文献和实验相关实验
构建 ① 设定温度梯度(26-35°C)和时间梯度(12-72 h)，对龙须菜进行高温胁迫。 ② 提取龙须菜总RNA(RNA提取按照Qiagen试剂盒说明书进行，RNeasy Plant Mini Kit，Qiagen)。 ③ 应用SMART技术构建龙须菜高温胁迫表达cDNA文库，3’和5’引物分别加接酶切位点。 4.文库筛选载体构建 将龙须菜高温胁迫表达文库cDNA双酶切，连入同样双酶切的AD 克隆质粒pGADT7中，用PEG/LiAc 转化
表达的调控，单单靠GLA4不知道能不能起到调控蛋白表达的目的？有这方面的文献吗？先谢谢各位大侠的帮助了！！！！ shilei5794749 类似GAL4-UAS系统，楼主可以借鉴这个系统。GLA4可以用pGBKT7或者pBIND上的，做成删除NLS序列的GAL4(BD+AD)或者GAL4BD-VP16。此质粒还可融合你要研究的蛋白，上游可以加调控元件。 另一个质粒用UAS序列打头，后置报告基因就行啦！ 版主dnazyme留言:
的功能蛋白编码基因，验证反式转录调控因子的DNA结合结构域，准确定位参与特定蛋白质结合的核苷酸序列。 酵母单杂交用的cDNA文库用什么试剂盒构建? 单杂交文库可以用酵母双杂交试剂盒中的SMART试剂盒来构建 用BD公司的酵母单杂交文库构建和筛选试剂盒进行酵母单杂交文库的构建。具体操作步骤为：在15 mL试管中依次加入如下成分：20 μL SMART cDNA、6 μL pGADT7-Rec2(0.5 μg/μL，SmaI-线性化)、5 μg pHIS2-PBE
pGADT7-AD酵母双杂交质粒
￥100 - 1000
