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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：
pLVX-IRES-tdTomato
- 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：pLVX-IRES-tdTomato慢病毒表达质粒
别称: pLVX-IRES-tdTomato
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 8892 bp ds-DNA circular SYN 09-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8892)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Friday, September 9, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8892
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/note="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2028..2143
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)"
enhancer 2201..2504
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2505..2708
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 2843..3415
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 2866..3417
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 3417..4847
/codon_start=1
/product="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
/note="tdTomato"
/note="mammalian codon-optimized"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLYGMDELYK"
misc_feature 4861..5449
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5332..5343)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
LTR 5656..6289
/note="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6417..6433)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 6441..6457
/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(6465..6495)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 6510..6531
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6819..7407)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7578..8438)
/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(8439..8543)
/gene="bla"
/note="AmpR promoter"
polyA_signal 8591..8725
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
ORIGIN
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
2161 acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa
2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
2761 tccatagaag acaccgactc tactagagga tctatttccg gtgaattcct cgagactagt
2821 tctagagcgg ccgcggatcc cgcccctctc cctccccccc ccctaacgtt actggccgaa
2881 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
2941 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
3001 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
3061 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
3121 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
3181 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
3241 tcacctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
3301 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg
3361 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
3421 tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
3481 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
3541 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
3601 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
3661 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
3721 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
3781 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
3841 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
3901 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct
3961 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
4021 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
4081 accacctgtt cctggggcat ggcaccggca gcaccggcag cggcagctcc ggcaccgcct
4141 cctccgagga caacaacatg gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg
4201 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
4261 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
4321 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
4381 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
4441 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
4501 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
4561 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
4621 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga
4681 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
4741 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
4801 gccgccacca cctgttcctg tacggcatgg acgagctgta caagtgaacg cgtctggaac
4861 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
4921 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
4981 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
5041 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
5101 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
5161 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
5221 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
5281 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
5341 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
5401 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat
5461 tctgcagtcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
5521 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
5581 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
5641 aagaaaagag gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
5701 tgtggatcta ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag
5761 gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata
5821 aggtagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg
5881 ggatggatga cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc
5941 atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct
6001 acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt
6061 ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg
6121 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
6181 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
6241 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
6301 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
6361 ccttgacatt gctagcgttt accgtcgacc tctagctaga gcttggcgta atcatggtca
6421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
6481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
6541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
6601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
6661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
6721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
6781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
6841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
6901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
6961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
7021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
7081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
7141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
7201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
7261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
7321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
7381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
7441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
7501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
7561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
7621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
7681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
7741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
7801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
7861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
7921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
7981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
8041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
8101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
8161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
8221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
8281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
8341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
8401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
8461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
8521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
8581 tcgggagatc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
8641 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
8701 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
8761 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
8821 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
8881 aaacttagta gt
//
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：pLVX-IRES-tdTomato慢病毒表达质粒
别称: pLVX-IRES-tdTomato
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 终止子: | SV40 poly(A) signal |
| 质粒分类: | 病毒系列，慢病毒克隆载体 |
| 质粒大小: | 8892bp |
| 原核抗性: | Amp |
| 克隆菌株: | Stbl3 |
| 培养条件: | 37度 |
| 表达宿主: | 哺乳细胞 |
| 诱导方式: | 无须诱导，瞬时表达 |
| 5'测序引物: | CMV-F：CGCAAATGGGCGGTAGGCGTG |
| 3'测序引物: | WPRE-R：CATAGCGTAAAAGGAGCAACA |
质粒属性
| 载体宿主： | 哺乳细胞,慢病毒 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 真核抗性： | |
| 荧光蛋白： | 红色 |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 8892 bp ds-DNA circular SYN 09-SEP-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 4
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8892)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Friday, September 9, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..8892
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 1..634
/note="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 2028..2143
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)"
enhancer 2201..2504
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2505..2708
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 2843..3415
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
misc_feature 2866..3417
/note="IRES"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 3417..4847
/codon_start=1
/product="tandem dimeric (pseudo-monomeric) derivative of
DsRed (Shaner et al., 2004)"
/note="tdTomato"
/note="mammalian codon-optimized"
/translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLYGMDELYK"
misc_feature 4861..5449
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(5332..5343)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
LTR 5656..6289
/note="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6417..6433)
/note="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 6441..6457
/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(6465..6495)
/note="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 6510..6531
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6819..7407)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7578..8438)
/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(8439..8543)
/gene="bla"
/note="AmpR promoter"
polyA_signal 8591..8725
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
ORIGIN
1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag
2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac
2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg
2161 acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa
2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
2761 tccatagaag acaccgactc tactagagga tctatttccg gtgaattcct cgagactagt
2821 tctagagcgg ccgcggatcc cgcccctctc cctccccccc ccctaacgtt actggccgaa
2881 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
2941 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
3001 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
3061 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
3121 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
3181 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
3241 tcacctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
3301 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg
3361 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
3421 tgagcaaggg cgaggaggtc atcaaagagt tcatgcgctt caaggtgcgc atggagggct
3481 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
3541 cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc tgggacatcc
3601 tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc gacatccccg
3661 attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
3721 acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg ctgatctaca
3781 aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag aagaagacca
3841 tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg aagggcgaga
3901 tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc aagaccatct
3961 acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc aagctggaca
4021 tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc gagggccgcc
4081 accacctgtt cctggggcat ggcaccggca gcaccggcag cggcagctcc ggcaccgcct
4141 cctccgagga caacaacatg gccgtcatca aagagttcat gcgcttcaag gtgcgcatgg
4201 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
4261 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
4321 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
4381 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
4441 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
4501 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
4561 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
4621 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga
4681 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
4741 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
4801 gccgccacca cctgttcctg tacggcatgg acgagctgta caagtgaacg cgtctggaac
4861 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
4921 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
4981 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
5041 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
5101 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
5161 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
5221 ttgggcactg acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
5281 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
5341 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
5401 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat
5461 tctgcagtcg agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca
5521 atgctgattg tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac
5581 ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
5641 aagaaaagag gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc
5701 tgtggatcta ccacacacaa ggctacttcc ctgattagca gaactacaca ccagggccag
5761 gggtcagata tccactgacc tttggatggt gctacaagct agtaccagtt gagccagata
5821 aggtagaaga ggccaataaa ggagagaaca ccagcttgtt acaccctgtg agcctgcatg
5881 ggatggatga cccggagaga gaagtgttag agtggaggtt tgacagccgc ctagcatttc
5941 atcacgtggc ccgagagctg catccggagt acttcaagaa ctgctgatat cgagcttgct
6001 acaagggact ttccgctggg gactttccag ggaggcgtgg cctgggcggg actggggagt
6061 ggcgagccct cagatcctgc atataagcag ctgctttttg cctgtactgg gtctctctgg
6121 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
6181 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
6241 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
6301 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
6361 ccttgacatt gctagcgttt accgtcgacc tctagctaga gcttggcgta atcatggtca
6421 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
6481 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
6541 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
6601 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
6661 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
6721 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
6781 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
6841 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
6901 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
6961 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
7021 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
7081 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
7141 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
7201 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
7261 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
7321 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
7381 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
7441 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
7501 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
7561 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
7621 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
7681 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
7741 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
7801 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
7861 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
7921 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
7981 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
8041 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
8101 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
8161 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
8221 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
8281 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
8341 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
8401 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
8461 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
8521 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga
8581 tcgggagatc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
8641 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
8701 caatgtatct tatcatgtct ggatcaactg gataactcaa gctaaccaaa atcatcccaa
8761 acttcccacc ccatacccta ttaccactgc caattacctg tggtttcatt tactctaaac
8821 ctgtgattcc tctgaattat tttcatttta aagaaattgt atttgttaaa tatgtactac
8881 aaacttagta gt
//
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
| P3956/pCMV-SPORT6-MAP1LC3A人源基因质粒 |
| P3957/pCMV-SPORT6-UBE2C人源基因质粒 |
| P3958/pCMV-SPORT6-EEF1G（1同义突变）人源基因质粒 |
| P3959/pCMV-SPORT6-MRPL10（点突变）人源基因质粒 |
| P3960/pCMV-SPORT6-MATR3人源基因质粒 |
| P3961/pCMV-SPORT6-PIGT（2同义突变）人源基因质粒 |
| P3962/pCMV-SPORT6-CENPQ人源基因质粒 |
| P3963/pCMV-SPORT6-PAIP1（1点突变）人源基因质粒 |
| P3964/pCMV-SPORT6-TRIM63（1-975bpA709G）人源基因质粒 |
| P3965/pCMV-SPORT6-GALT（1点突变）人源基因质粒 |
| P3966/pCMV-SPORT6-SPSB1人源基因质粒 |
| P3967/pCMV-SPORT6-ZNF408人源基因质粒 |
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文献和实验相关实验
作为细胞生物学专业的博士生，在基因功能研究上已经有五年的经验了，今天我给大家讲讲其中经常用到并且非常重要的一种技术--慢病毒包装的过表达体系。下面我就从引物设计，慢病毒表达质粒构建等方面详细讲述。下面以 plex-MCS 载体为例。1. 选择酶切位点，为了避免移码突变，上游的酶切位点选择起始密码子 ATG 前面的 spel，下游选择 xho1。2. 构建方法的选择。连接法对于连接法，首先设计引物将目的基因 PCR 出来，即酶切位点加基因引物，此时应注意，为了防止酶切将基因破坏，通常习惯在两端
但感觉自己包装效率还是不太高，做起来不好弄 kinguangjun 你可以考虑clontech的pLVX-ShRNA2和pLVX-IRES-ZsGreen1慢病毒系统，比invitrogen的慢病毒系统要高两个数量级以上，我们实验室一直都用这个系统。 jinanfendou 看你做什么实验了，慢病毒载体系统很多公司有卖的，但是你自己包装的话，滴度可能达不到这么高，好转染的细胞还是可以的，但是要是难转染的细胞和动物
sunyuzhu10 包装质粒是pMD2.G pMDLg/pRRE pRSV-Rev 过表达载体是pLVX-IRES-ZsGreen1 这样的四个质粒能包装出慢病毒么？ 请有经验的战友帮忙看下 暴君 可以的 644414915 pMD2.G是VSVG吗 我现在正在包病毒，别人给了我这四个质粒，别的什么都没说，我不知道该怎么包装装染 谁有具体步骤 转染
pLVX-IRES-tdTomato慢病毒表达质粒
￥100 - 1000
