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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：
Plasmid#22733
- 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：pLCA.66/2272同源重组质粒
别称: Plasmid#22733
质粒属性
质粒简介
This vector was designed for assembling gene targeting constructs using the BAC recombineering approach. The vector contains a puromycin-(delta)thymidine kinase fusion gene driven by the mouse phosphoglycerol kinase promoter (pUDTK) and a neomycin resistant gene driven by the bacterial EM7 promoter (EM7neo). These selectable markers are flanked by lox66 and lox2272 sites (Cre-recombinase recognition sequences) in a pBluescript KS+ backbone with a modified multi-cloning region.
质粒图谱
质粒序列
LOCUS Exported 6678 bp ds-DNA circular SYN 15-AUG-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..6678
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 11..510
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 530..1129
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 1136..2131
/codon_start=1
/product="truncated herpes simplex virus thymidine kinase
(Salomon et al., 1995)"
/label=-Delta-TK
/note="confers sensitivity to nucleoside analogs such as
acyclovir or ganciclovir"
/translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
polyA_signal 2159..2383
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 2429..2476
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 2495..3298
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Kan
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGSAIEQDGLHAGSPAACVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATC-PFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3336..3560
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind complement(3626..3659)
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites."
promoter complement(3736..3754)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin 3919..4374
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4400..4504
/gene="bla"
/label=AmpR promoter
CDS 4505..5365
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5536..6124
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 6547..6565
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
protein_bind complement(6623..6656)
/label=lox66
/bound_moiety="Cre recombinase"
/note="loxP mutant. Cre-mediated recombination occurs in
the 8-bp core sequence (GCATACAT)."
ORIGIN
1 taattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
61 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
121 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
181 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
241 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
301 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
361 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
421 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
481 ttctcctctt cctcatctcc gggcctttcg acctgcagcc aacgccacca tggggaccga
541 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct
601 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat
661 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
721 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
781 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
841 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
901 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
961 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
1021 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
1081 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatccatgcc
1141 cacgctactg cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca
1201 actgctggtg gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta
1261 ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct
1321 cgaccagggt gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac
1381 aatgggcatg ccttatgccg tgaccgacgc cgttctggct cctcatatcg ggggggaggc
1441 tgggagctca catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc
1501 cgccctcctg tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt
1561 gctggcgttc gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc
1621 ccttccggag gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct
1681 tgacctggct atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg
1741 gtatctgcag ggcggcgggt cgtggcggga ggattgggga cagctttcgg ggacggccgt
1801 gccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac
1861 gttatttacc ctgtttcggg cccccgagtt gctggccccc aacggcgacc tgtataacgt
1921 gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct
1981 ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat
2041 ggtccagacc cacgtcacca cccccggctc cataccgacg atctgcgacc tggcgcgcac
2101 gtttgcccgg gagatggggg aggctaactg agctctagag ctcgctgatc agcctcgact
2161 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
2221 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
2281 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
2341 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
2401 accatctggg gctgaattcc tgcagcctgt tgacaattaa tcatcggcat agtatatcgg
2461 catagtataa tacgacaagg tgaggaacta aaccatggga tcggccattg aacaagatgg
2521 attgcacgca ggttctccgg ccgcttgcgt ggagaggcta ttcggctatg actgggcaca
2581 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
2641 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
2701 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
2761 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
2821 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
2881 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
2941 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
3001 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt
3061 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
3121 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
3181 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
3241 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg
3301 ggatcaattc tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
3361 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
3421 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
3481 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
3541 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct cgactagagc
3601 ttgcggaacc cttgaattcc tgcagataac ttcgtataaa gtatcctata ggaagttatg
3661 gatccaggta ccttcgaaac gcgtcctagg catatgatct agagcggccg ccaccgcggt
3721 ggagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
3781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
3841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
3901 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
3961 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
4021 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
4081 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
4141 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
4201 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
4261 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
4321 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
4381 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
4441 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
4501 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
4561 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
4621 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
4681 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
4741 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
4801 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
4861 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
4921 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
4981 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
5041 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
5101 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
5161 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
5221 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
5281 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
5341 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
5401 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
5461 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
5521 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
5581 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
5641 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
5701 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
5761 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
5821 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
5881 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
5941 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
6001 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
6061 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
6121 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
6181 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
6241 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
6301 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
6361 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
6421 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
6481 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
6541 gctcgaaatt aaccctcact aaagggaaca aaagctgcgg gccccccctc gaagatctct
6601 cgaggctagc atgcatatcg attaccgttc gtataatgta tgctatacga agttatacta
6661 gtaagcttga tatcgaat
//
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：pLCA.66/2272同源重组质粒
别称: Plasmid#22733
| 启动子: | PGK |
|---|---|
| 复制子: | ori，F1 ori |
| 终止子: | bGH poly(A) signal |
| 质粒分类: | 基因文库质粒；其它基因质粒；其它种源质粒 |
| 质粒大小: | 6678bp |
| 原核抗性: | Amp |
| 筛选标记: | Puro |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 哺乳细胞 |
|---|---|
| 载体用途： | 基因敲除 |
| 基因种属： | |
| 基因类型： | |
| 原核抗性： | Amp |
| 真核抗性： | Puro |
| 荧光蛋白： |
质粒简介
This vector was designed for assembling gene targeting constructs using the BAC recombineering approach. The vector contains a puromycin-(delta)thymidine kinase fusion gene driven by the mouse phosphoglycerol kinase promoter (pUDTK) and a neomycin resistant gene driven by the bacterial EM7 promoter (EM7neo). These selectable markers are flanked by lox66 and lox2272 sites (Cre-recombinase recognition sequences) in a pBluescript KS+ backbone with a modified multi-cloning region.
质粒图谱
质粒序列
LOCUS Exported 6678 bp ds-DNA circular SYN 15-AUG-2017
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..6678
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 11..510
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 530..1129
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
CDS 1136..2131
/codon_start=1
/product="truncated herpes simplex virus thymidine kinase
(Salomon et al., 1995)"
/label=-Delta-TK
/note="confers sensitivity to nucleoside analogs such as
acyclovir or ganciclovir"
/translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQV
LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAV
PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFI
LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
polyA_signal 2159..2383
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
promoter 2429..2476
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 2495..3298
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=Kan
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MGSAIEQDGLHAGSPAACVERLFGYDWAQQTIGCSDAAVFRLSAQ
GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
LLSSHLAPAEKVSIMADAMRRLHTLDPATC-PFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3336..3560
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind complement(3626..3659)
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites."
promoter complement(3736..3754)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin 3919..4374
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4400..4504
/gene="bla"
/label=AmpR promoter
CDS 4505..5365
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 5536..6124
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 6547..6565
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
protein_bind complement(6623..6656)
/label=lox66
/bound_moiety="Cre recombinase"
/note="loxP mutant. Cre-mediated recombination occurs in
the 8-bp core sequence (GCATACAT)."
ORIGIN
1 taattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
61 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
121 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
181 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
241 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
301 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
361 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
421 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
481 ttctcctctt cctcatctcc gggcctttcg acctgcagcc aacgccacca tggggaccga
541 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc ccccgggccg tacgcaccct
601 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgacccgg accgccacat
661 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
721 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
781 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
841 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
901 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
961 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
1021 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
1081 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gatccatgcc
1141 cacgctactg cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca
1201 actgctggtg gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta
1261 ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct
1321 cgaccagggt gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac
1381 aatgggcatg ccttatgccg tgaccgacgc cgttctggct cctcatatcg ggggggaggc
1441 tgggagctca catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc
1501 cgccctcctg tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt
1561 gctggcgttc gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc
1621 ccttccggag gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct
1681 tgacctggct atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg
1741 gtatctgcag ggcggcgggt cgtggcggga ggattgggga cagctttcgg ggacggccgt
1801 gccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac
1861 gttatttacc ctgtttcggg cccccgagtt gctggccccc aacggcgacc tgtataacgt
1921 gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct
1981 ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat
2041 ggtccagacc cacgtcacca cccccggctc cataccgacg atctgcgacc tggcgcgcac
2101 gtttgcccgg gagatggggg aggctaactg agctctagag ctcgctgatc agcctcgact
2161 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
2221 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
2281 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
2341 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
2401 accatctggg gctgaattcc tgcagcctgt tgacaattaa tcatcggcat agtatatcgg
2461 catagtataa tacgacaagg tgaggaacta aaccatggga tcggccattg aacaagatgg
2521 attgcacgca ggttctccgg ccgcttgcgt ggagaggcta ttcggctatg actgggcaca
2581 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
2641 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
2701 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
2761 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
2821 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
2881 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
2941 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
3001 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg atctcgtcgt
3061 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
3121 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
3181 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
3241 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg
3301 ggatcaattc tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
3361 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
3421 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
3481 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
3541 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct cgactagagc
3601 ttgcggaacc cttgaattcc tgcagataac ttcgtataaa gtatcctata ggaagttatg
3661 gatccaggta ccttcgaaac gcgtcctagg catatgatct agagcggccg ccaccgcggt
3721 ggagctccaa ttcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac
3781 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
3841 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
3901 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
3961 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
4021 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
4081 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
4141 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
4201 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
4261 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
4321 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
4381 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
4441 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
4501 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
4561 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
4621 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
4681 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
4741 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
4801 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
4861 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
4921 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
4981 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
5041 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
5101 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
5161 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
5221 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
5281 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
5341 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
5401 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
5461 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
5521 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
5581 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
5641 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
5701 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
5761 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
5821 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
5881 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
5941 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
6001 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
6061 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
6121 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
6181 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
6241 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
6301 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
6361 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
6421 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
6481 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
6541 gctcgaaatt aaccctcact aaagggaaca aaagctgcgg gccccccctc gaagatctct
6601 cgaggctagc atgcatatcg attaccgttc gtataatgta tgctatacga agttatacta
6661 gtaagcttga tatcgaat
//
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
| P3387/pCMV-SPORT6-PPP1R7人源基因质粒 |
| P3388/pCMV-SPORT6-ACTB人源基因质粒 |
| P3389/pCMV-SPORT6-GATA3人源基因质粒 |
| P3390/pCMV-SPORT6-TIMP1（1同义突变）人源基因质粒 |
| P3391/pCMV-SPORT6-BRAF（2同义突变）人源基因质粒 |
| P3392/pCMV-SPORT6-MAGEA12人源基因质粒 |
| P3393/pCMV-SPORT6-ODR4人源基因质粒 |
| P3394/pCMV-SPORT6-FNDC11人源基因质粒 |
| P3395/pCMV-SPORT6-AURKA（1点突变）人源基因质粒 |
| P3396/pCMV-SPORT6-SGK1人源基因质粒 |
| P3397/pCMV-SPORT6-MRPL28（1点突变2同义突变）人源基因质粒 |
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文献和实验相关实验
5XmJWvdiDThyvxpaXCh0iUY72ZkiLAvXv3lJhjxhpDgCFQcRH4etIrfPr0qUSRTStXrlzIZeSQ8OwG3sIQdRpYEl1vVuGS8XzfZjwgNY16uUJXUs++8yDANB95IFGeinrE2jqVuNhKCg0TfOXKZckp+2YIMAQYAqWHgEx6hSEYbpOl8izGDNnpFdYnZqVXKMbgUux6/vx5cZRlRUKr0y0X1zp1CuEmBcHXXoAjFh1x/juw6X9kmzk1Bm/vncKh66HQb7cEC
酸的siRNA瞬时转染哺乳动物细胞，可以获得基因特异性抑制。这些siRNA的长度足以诱导产生基因特异性抑制，又不至于引起宿主的干扰反应。另外，siRNA也可以来自于含有茎环发夹结构的 dsRNA。这些短发夹RNA(shRNAS) 可以用含有RNA聚合酶Ⅲ(PolⅢ)启动子的质粒DNA在体外表达。9-14 接着shRNA被Dicer切割成siRNA，然后在胞浆参与构成RISC，降解mRNA。这些研究表明在哺乳动物细胞产生 si/shRNA是可行的。然而，沉默效果是短暂的，转染原代细胞效率相对较低。为了提高
就可以把目的片断定向、高效地插入LIC载体上。 一般的pET载体是先在不含DE3片段的菌株中进行克隆筛选，之后再 把阳性克隆转化进入表达菌株，如BL21(DE3)中，通过IPTG诱导进 行表达。而pETcoco载体它引入了低拷贝控制元件，F附加体上的oriS和 repE元件与parABC共同作用下使质粒在细菌中保持单拷贝，这样即可 以使质粒在细菌内稳定存在又可以减少外源蛋白对细胞的毒害作用。 当我们需要大量表达时加入树胶醛醣诱导trfA基因表达，质粒在细胞内 的拷贝数就可以增加到25-50
pLCA.66/2272同源重组质粒
￥100 - 1000
