| 质粒名称: | pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 32703 |
| 存储实验室: | Hans Clevers |
|
载体骨架基本信息 |
|
| 载体名称: | MSCV |
| 载体出品公司: | Clontech |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 6295 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，逆转录病毒，Cre/Lox |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | -- |
| 标签/融合蛋白: | 3x HA标签（主干上的C终端） |
| 克隆方法: | -- |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | -- |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE was a gift from Hans Clevers (Addgene plasmid # 32703 ; http://n2t.net/addgene:32703 ; RRID:Addgene_32703) |
| 参考文献部分: | Controlled gene expression in primary Lgr5 organoid cultures. Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, Clevers H. Nat Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802. 10.1038/nmeth.1802 PubMed 22138822 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE 7889 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pMSCV-loxp-dsRed-loxp-3xHA-Puro-WPRE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7889)
AUTHORS Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH,
Clevers H
TITLE Controlled gene expression in primary Lgr5 organoid cultures.
JOURNAL Nat Methods. 2011 Dec 4. doi: 10.1038/nmeth.1802.
PUBMED 22138822
REFERENCE 2 (bases 1 to 7889)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7889
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 579..920
/label=MESV Psi
/note="packaging signal of murine embryonic stem cell
virus"
CDS 987..1403
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
primer_bind 1333..1355
/label=pLXSN 5'
/note="Murine stem cell virus, forward primer. Also called
MSCV"
primer_bind 1371..1387
/label=pBABE 5'
/note="Psi packaging signal, forward primer for pBABE
vectors"
protein_bind 1437..1470
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
regulatory 1499..1508
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1505..2182
/codon_start=1
/product="noncytotoxic tetrameric variant of DsRed
fluorescent protein (Strack et al., 2008)"
/label=DsRed-Express2
/note="mammalian codon-optimized"
/translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
LFQ"
primer_bind complement(1685..1705)
/label=DsRed1-N
/note="DsRed1, reverse primer"
primer_bind 1903..1922
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2097..2120
/label=DsRed1-C
/note="DsRed1, forward primer"
protein_bind 2207..2240
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS 2291..2317
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 2327..2353
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 2363..2389
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
promoter 2417..2916
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
primer_bind complement(2452..2475)
/label=MSCV-rev
/note="Murine stem cell virus, reverse primer"
primer_bind 2849..2868
/label=mPGK-F
/note="Mouse PGK promoter, forward primer"
CDS 2937..3536
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind complement(2937..2956)
/label=Puro-R
/note="Puromycin resistance gene, reverse primer. Also
called puro-variant-R"
primer_bind 3433..3453
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 3606..4194
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3659..3679)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4077..4088)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 4251..4765
/label=3' LTR
/note="3' long terminal repeat from murine embryonic stem
cell virus"
primer_bind complement(4934..4950)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4934..4950)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4947..4969)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4958..4974
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(4982..5012)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5027..5048
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5165..5182)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5336..5924)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5416..5435)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6095..6955)
/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"
primer_bind 6718..6737
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6956..7060)
/gene="bla"
/label=AmpR promoter
primer_bind 7128..7146
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(7184..7206)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 7306..7325
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7519..7541
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 7669..7688
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
LTR 7888..515
/label=5' LTR
/note="5' long terminal repeat from murine embryonic stem
cell virus"
ORIGIN
1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
1381 ccctcactcc ttctctaggc gccggaatta gatcactagt ccagtgtggt ggaattataa
1441 cttcgtatag catacattat acgaagttat aattctgcag atatccagca cagtggcggc
1501 caccatggat agcactgaga acgtcatcaa gcccttcatg cgcttcaagg tgcacatgga
1561 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggca agccctacga
1621 gggcacccag accgccaagc tgcaggtgac caagggcggc cccctgccct tcgcctggga
1681 catcctgtcc ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat
1741 ccccgactac aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
1801 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcaccttcat
1861 ctaccacgtg aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa
1921 gactctgggc tgggagccct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg
1981 cgagatccac aaggcgctga agctgaaggg cggcggccac tacctggtgg agttcaagtc
2041 aatctacatg gccaagaagc ccgtgaagct gcccggctac tactacgtgg actccaagct
2101 ggacatcacc tcccacaacg aggactacac cgtggtggag cagtacgagc gcgccgaggc
2161 ccgccaccac ctgttccagt agagctagct gaataaggcc gctcgaataa cttcgtatag
2221 catacattat acgaagttat tcgagtctag agggcccgtt agatctctcg aggttaacga
2281 attcgactac tacccctacg acgtgcccga ctacgccgct ggagcatacc cctacgacgt
2341 gcccgactac gccgctggag cataccccta cgacgtgccc gactacgcct gagtcgacgc
2401 ggccgccaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
2461 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
2521 acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct
2581 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
2641 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
2701 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
2761 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
2821 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
2881 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcagcccaag cttaccatga
2941 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
3001 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
3061 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
3121 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
3181 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
3241 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
3301 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
3361 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
3421 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
3481 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc
3541 cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcgataattc
3601 ccgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
3661 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
3721 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
3781 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
3841 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
3901 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
3961 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
4021 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
4081 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
4141 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcggg
4201 aattatcgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc
4261 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaatac
4321 ataactgaga atagagaagt tcagatcaag gttaggaaca gagagacagc agaatatggg
4381 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt
4441 ccccagatgc ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc
4501 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
4561 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
4621 cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt
4681 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
4741 ctacccgtca gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc
4801 tgagggtagg agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac
4861 atactccaat actcctgaaa tagttcatta tggacagcgc agaagagctg gggagaatta
4921 attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
4981 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
5041 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
5101 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
5161 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
5221 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
5281 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
5341 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
5401 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
5461 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
5521 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
5581 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
5641 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
5701 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
5761 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
5821 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
5881 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
5941 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
6001 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
6061 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
6121 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
6181 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
6241 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
6301 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
6361 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
6421 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
6481 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
6541 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
6601 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
6661 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
6721 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
6781 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
6841 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
6901 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
6961 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
7021 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
7081 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
7141 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
7201 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
7261 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
7321 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
7381 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
7441 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
7501 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc
7561 atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc
7621 gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc
7681 gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc
7741 gtagaggcga ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt
7801 gactcaacaa tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat
7861 tttatttagt ctccagaaaa aggggggaa
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载体质粒应用举例
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