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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：
p426gal
- 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：p426gal酿酒酵母质粒
别称: p426gal
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 6412 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6412)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 12, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..6412
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 201..416
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
CDS 417..1220
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/note="URA3"
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
rep_origin complement(1351..1806)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 1951..1967
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1977..1995
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
terminator 2014..2261
/gene="S. cerevisiae CYC1"
/note="CYC1 terminator"
/note="transcription terminator for CYC1"
primer_bind 2264..2280
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2314..2330)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2345..2786)
/gene="S. cerevisiae GAL1"
/note="GAL1 promoter"
/note="inducible promoter, regulated by Gal4"
protein_bind 2669..2786
/bound_moiety="Gal4"
/note="UAS"
/note="upstream activating sequence mediating
Gal4-dependent induction"
promoter complement(2810..2828)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2849..2865)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2873..2889
/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(2897..2927)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(3251..3839)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4010..4870)
/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(4871..4975)
/gene="bla"
/note="AmpR promoter"
rep_origin 5002..6344
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(5965..6012)
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
1981 acgactcact atagggcgaa ttgggtaccg gccgcaaatt aaagccttcg agcgtcccaa
2041 aaccttctca agcaaggttt tcagtataat gttacatgcg tacacgcgtc tgtacagaaa
2101 aaaaagaaaa atttgaaata taaataacgt tcttaatact aacataacta taaaaaaata
2161 aatagggacc tagacttcag gttgtctaac tccttccttt tcggttagag cggatgtggg
2221 gggagggcgt gaatgtaagc gtgacataac taattacatg actcgaggtc gacggtatcg
2281 ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga atccggggtt
2341 ttttctcctt gacgttaaag tatagaggta tattaacaat tttttgttga tacttttatt
2401 acatttgaat aagaagtaat acaaaccgaa aatgttgaaa gtattagtta aagtggttat
2461 gcagtttttg catttatata tctgttaata gatcaaaaat catcgcttcg ctgattaatt
2521 accccagaaa taaggctaaa aaactaatcg cattatcatc ctatggttgt taatttgatt
2581 cgttcatttg aaggtttgtg gggccaggtt actgccaatt tttcctcttc ataaccataa
2641 aagctagtat tgtagaatct ttattgttcg gagcagtgcg gcgcgaggca catctgcgtt
2701 tcaggaacgc gaccggtgaa gacgaggacg cacggaggag agtcttcctt cggagggctg
2761 tcacccgctc ggcggcttct aatccgtact agagctccag cttttgttcc ctttagtgag
2821 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
2881 cgctcacaat tccacacaac ataggagccg gaagcataaa gtgtaaagcc tggggtgcct
2941 aatgagtgag gtaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
3001 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
3061 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
3121 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
3181 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
3241 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
3301 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
3361 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
3421 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
3481 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
3541 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
3601 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
3661 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
3721 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
3781 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
3841 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
3901 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
3961 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
4021 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
4081 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
4141 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
4201 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
4261 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
4321 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
4381 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
4441 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
4501 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
4561 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
4621 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
4681 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
4741 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
4801 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
4861 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
4921 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
4981 ttccccgaaa agtgccacct gaacgaagca tctgtgcttc attttgtaga acaaaaatgc
5041 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
5101 tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa
5161 aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac
5221 agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct
5281 acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc
5341 tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag
5401 gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact
5461 tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc
5521 ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt
5581 tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac
5641 tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt
5701 actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg
5761 agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca
5821 cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata
5881 ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc
5941 gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata ggaacttcgg
6001 aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg
6061 cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta tatatatata
6121 catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat gcgtctattt
6181 atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca tgcggggtat
6241 cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc ctcaattgga
6301 ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatacta agaaaccatt
6361 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
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：p426gal酿酒酵母质粒
别称: p426gal
| 启动子: | GAL1 |
|---|---|
| 复制子: | 2U |
| 终止子: | CYC1 |
| 质粒分类: | 酵母系列，酿酒酵母表达载体 |
| 质粒大小: | 6412bp |
| 原核抗性: | Amp |
| 筛选标记: | URA3 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 诱导方式: | 半乳糖诱导 |
| 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： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | URA3 |
| 荧光蛋白： |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 6412 bp ds-DNA circular SYN 12-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 9
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6412)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Monday, September 12, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..6412
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 201..416
/gene="S. cerevisiae URA3"
/note="URA3 promoter"
CDS 417..1220
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/note="URA3"
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
rep_origin complement(1351..1806)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 1951..1967
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
promoter 1977..1995
/note="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
terminator 2014..2261
/gene="S. cerevisiae CYC1"
/note="CYC1 terminator"
/note="transcription terminator for CYC1"
primer_bind 2264..2280
/note="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2314..2330)
/note="SK primer"
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2345..2786)
/gene="S. cerevisiae GAL1"
/note="GAL1 promoter"
/note="inducible promoter, regulated by Gal4"
protein_bind 2669..2786
/bound_moiety="Gal4"
/note="UAS"
/note="upstream activating sequence mediating
Gal4-dependent induction"
promoter complement(2810..2828)
/note="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(2849..2865)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 2873..2889
/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(2897..2927)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
rep_origin complement(3251..3839)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4010..4870)
/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(4871..4975)
/gene="bla"
/note="AmpR promoter"
rep_origin 5002..6344
/note="2u ori"
/note="yeast 2u plasmid origin of replication"
protein_bind complement(5965..6012)
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FRT"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
1981 acgactcact atagggcgaa ttgggtaccg gccgcaaatt aaagccttcg agcgtcccaa
2041 aaccttctca agcaaggttt tcagtataat gttacatgcg tacacgcgtc tgtacagaaa
2101 aaaaagaaaa atttgaaata taaataacgt tcttaatact aacataacta taaaaaaata
2161 aatagggacc tagacttcag gttgtctaac tccttccttt tcggttagag cggatgtggg
2221 gggagggcgt gaatgtaagc gtgacataac taattacatg actcgaggtc gacggtatcg
2281 ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga atccggggtt
2341 ttttctcctt gacgttaaag tatagaggta tattaacaat tttttgttga tacttttatt
2401 acatttgaat aagaagtaat acaaaccgaa aatgttgaaa gtattagtta aagtggttat
2461 gcagtttttg catttatata tctgttaata gatcaaaaat catcgcttcg ctgattaatt
2521 accccagaaa taaggctaaa aaactaatcg cattatcatc ctatggttgt taatttgatt
2581 cgttcatttg aaggtttgtg gggccaggtt actgccaatt tttcctcttc ataaccataa
2641 aagctagtat tgtagaatct ttattgttcg gagcagtgcg gcgcgaggca catctgcgtt
2701 tcaggaacgc gaccggtgaa gacgaggacg cacggaggag agtcttcctt cggagggctg
2761 tcacccgctc ggcggcttct aatccgtact agagctccag cttttgttcc ctttagtgag
2821 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
2881 cgctcacaat tccacacaac ataggagccg gaagcataaa gtgtaaagcc tggggtgcct
2941 aatgagtgag gtaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
3001 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
3061 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
3121 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
3181 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
3241 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
3301 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
3361 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
3421 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
3481 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
3541 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
3601 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
3661 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
3721 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
3781 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
3841 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
3901 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
3961 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
4021 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
4081 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
4141 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
4201 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
4261 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
4321 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
4381 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
4441 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
4501 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
4561 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
4621 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
4681 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
4741 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
4801 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
4861 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
4921 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
4981 ttccccgaaa agtgccacct gaacgaagca tctgtgcttc attttgtaga acaaaaatgc
5041 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
5101 tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa
5161 aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac
5221 agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct
5281 acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc
5341 tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag
5401 gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact
5461 tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc
5521 ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt
5581 tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac
5641 tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt
5701 actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg
5761 agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca
5821 cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata
5881 ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc
5941 gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagagaata ggaacttcgg
6001 aataggaact tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg
6061 cacatacagc tcactgttca cgtcgcacct atatctgcgt gttgcctgta tatatatata
6121 catgagaaga acggcatagt gcgtgtttat gcttaaatgc gtacttatat gcgtctattt
6181 atgtaggatg aaaggtagtc tagtacctcc tgtgatatta tcccattcca tgcggggtat
6241 cgtatgcttc cttcagcact accctttagc tgttctatat gctgccactc ctcaattgga
6301 ttagtctcat ccttcaatgc tatcatttcc tttgatattg gatcatacta agaaaccatt
6361 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
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
| P2131/pENTR223-LYPLA1人源基因质粒 |
| P2132/pENTR223-FAM3A人源基因质粒 |
| P2133/pENTR223-STX8人源基因质粒 |
| P2145/pENTR223-KLC3人源基因质粒 |
| P2146/pENTR223-UGT3A2-T1467人源基因质粒 |
| P2147/pENTR223-SLC16A9人源基因质粒 |
| P2171/pENTR223-ECI1-T722C人源基因质粒 |
| P2172/pENTR223-DAZ2人源基因质粒 |
| P2182/pENTR223-THAP10人源基因质粒 |
| P2207/pENTR223-VNN2人源基因质粒 |
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