质粒名称: | pMRX-IP-GFP-LC3-RFP |
---|---|
出品公司: | Addgene |
目录编号: | 84573 |
存储实验室: | Noboru Mizushima |
载体骨架基本信息 |
|
载体名称: | pMRX-IP |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | 6100 bp |
完整质粒大小: | 7900 bp |
载体修饰: | -- |
载体类型: | 哺乳动物表达,逆转录病毒 |
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: | microtubule-associated protein 1 light chain 3 beta,Map1lc3b,LC3,LC3B |
物种来源: | R. norvegicus (rat) |
插入大小: | 1800 bp |
基因突变: | -- |
GenBank编号 | U05784 NM_022867.2 |
启动子 | -- |
标签/融合蛋白: |
EGFP(插入的N端)
mRFP1(插入件上的C端子)
|
克隆方法: | 限制性内切酶 |
5' 克隆位点: | Bgl II(销毁时未知) |
3' 克隆位点: | Bgl II(销毁时未知) |
5' 测序引物 | CGACCACTACCAGCAGAACA |
3' 测序引物 | AAAAGACGGCAATATGGTGG |
文献引用方法 |
|
材料和方法部分: | pMRX-IP-GFP-LC3-RFP was a gift from Noboru Mizushima (Addgene plasmid # 84573 ; http://n2t.net/addgene:84573 ; RRID:Addgene_84573) |
参考文献部分: | An Autophagic Flux Probe that Releases an Internal Control. Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T, Mizushima N. Mol Cell. 2016 Nov 17;64(4):835-849. doi: 10.1016/j.molcel.2016.09.037. Epub 2016 Nov 3. 10.1016/j.molcel.2016.09.037 PubMed 27818143 |
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 pMRX-IP-GFP-LC3-RFP 8546 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Expresses GFP-LC3-RFP in mammalian cells to measure autophagic flux. ACCESSION . VERSION . KEYWORDS pMRX-IP-GFP-LC3-RFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8546) AUTHORS Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T, Mizushima N TITLE An Autophagic Flux Probe that Releases an Internal Control. JOURNAL Mol Cell. 2016 Nov 17;64(4):835-849. doi: 10.1016/j.molcel.2016.09.037. Epub 2016 Nov 3. PUBMED 27818143 REFERENCE 2 (bases 1 to 8546) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8546 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" LTR 734..1203 /note="long terminal repeat from Moloney murine leukemia virus" misc_feature 1268..1625 /label=MMLV Psi /note="packaging signal of Moloney murine leukemia virus (MMLV)" CDS 1690..2106 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 2036..2058 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 2074..2090 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" misc_feature 2116..2490 /label=pol region /note="Moloney murine leukemia virus (MMLV) pol region containing the splice acceptor site" primer_bind 2428..2444 /label=pBMN 5' /note="MMLV sequence, for inserts in pBMN retroviral vector" regulatory 2505..2514 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2511..3227 /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2556..2577) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2817..2836) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3164..3185 /label=EGFP-C /note="EGFP, forward primer" CDS 3651..4328 /codon_start=1 /product="monomeric derivative of DsRed (Campbell et al., 2002)" /label=mRFP1 /note="mammalian codon-optimized" /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS TGA" primer_bind complement(3781..3799) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 4049..4068 /label=mCherry-F /note="mCherry, forward primer" primer_bind 4243..4266 /label=DsRed1-C /note="DsRed1, forward primer" misc_feature 4462..5048 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(4629..4646) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 4856..4875 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 5049..5648 /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(5049..5068) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 5545..5565 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 5872..6341 /note="long terminal repeat from Moloney murine leukemia virus" primer_bind complement(6471..6488) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6642..7230) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6722..6741) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7394..8254) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 8017..8036 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8255..8359) /gene="bla" /label=AmpR promoter primer_bind 8427..8445 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg ctagagaacc atcagatgtt tccagggtgc cccaaggacc 421 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 481 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc 541 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 601 ccgacttgtg gtctcgaaga ccccacctgt aggtttggca agctagctta agtaacgcca 661 ttttgcaagg catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga 721 acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc 781 ggctcagggc caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta 841 agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct 901 cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc 961 tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct 1021 ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgattgac 1081 tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg 1141 tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt 1201 tcatttgggg gctcgtccgg gatcgggaga cccctgccca gggaccaccg acccaccacc 1261 gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg tctatgactg 1321 attttatgcg cctgcgtcgg tactagttag ctaactagct ctgtatctgg cggacccgtg 1381 gtggaactga cgagttcgga acacccggcc gcaaccctgg gagacgtccc agggacttcg 1441 ggggccgttt ttgtggcccg acctgagtcc aaaaatcccg atcgttttgg actctttggt 1501 gcacccccct tagaggaggg atatgtggtt ctggtaggag acgagaacct aaaacagttc 1561 ccgcctccgt ctgaattttt gctttcggtt tgggaccgaa gccgcgccgc gcgtcttgtc 1621 tgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt gtctgaaaat 1681 atgggcccgg gccagactgt taccactccc ttaagtttga ccttaggtca ctggaaagat 1741 gtcgagcgga tcgctcacaa ccagtcggta gatgtcaaga agagacgttg ggttaccttc 1801 tgctctgcag aatggccaac ctttaacgtc ggatggccgc gagacggcac ctttaaccga 1861 gacctcatca cccaggttaa gatcaaggtc ttttcacctg gcccgcatgg acacccagac 1921 caggtcccct acatcgtgac ctgggaagcc ttggcttttg acccccctcc ctgggtcaag 1981 ccctttgtac accctaagcc tccgcctcct cttcctccat ccgccccgtc tctccccctt 2041 gaacctcctc gttcgacccc gcctcgatcc tccctttatc cagccctcac tccttctcta 2101 ggcgccccca tatggccata tgagatctta tatggggcac ccccgcccct tgtaaacttc 2161 cctgaccctg acatgacaag agttactaac agcccctctc tccaagctca cttacaggct 2221 ctctacttag tccagcacga agtctggaga cctctggcgg cagcctacca agaacaactg 2281 gaccgaccgg tggtacctca cccttaccga gtcggcgaca cagtgtgggt ccgccgacac 2341 cagactaaga acctagaacc tcgctggaaa ggaccttaca cagtcctgct gaccaccccc 2401 accgccctca aagtagacgg catcgcagct tggatacacg ccgcccacgt gaaggctgcc 2461 gaccccgggg gtggaccatc ctctagactg ccggatctcc cgccgccacc atggtgagca 2521 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 2581 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 2641 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 2701 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 2761 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 2821 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 2881 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 2941 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 3001 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 3061 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 3121 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 3181 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggt ggcggtggct 3241 cgggcggtgg tgggtcgggt ggcggcggat ctatcgatga attgaattcc atgccgtccg 3301 agaagacctt caaacagcgc cggagcttcg aacaaagagt ggaagatgtc cggctcatcc 3361 gggagcagca ccccaccaag atcccagtga ttatagagcg atacaagggt gagaagcagc 3421 tgcccgtcct ggacaagacc aagttccttg tacctgatca cgtgaatatg agcgaactca 3481 tcaagataat tagaaggcgc ctgcagctca atgctaacca agccttcttc ctcctggtga 3541 atgggcacag catggtgagt gtgtccacac ccatctctga agtgtacgag agcgagagag 3601 atgaagacgg cttcctgtac atggtctatg cctcccagga gacgttcggg atggcctcct 3661 ccgaggacgt catcaaggag ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg 3721 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 3781 ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccctc 3841 agttccagta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 3901 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 3961 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc 4021 tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 4081 aggcctccac cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagatga 4141 ggctgaagct gaaggacggc ggccactacg acgccgaggt caagaccacc tacatggcca 4201 agaagcccgt gcagctgccc ggcgcctaca agaccgacat caagctggac atcacctccc 4261 acaacgagga ctacaccatc gtggaacagt acgagcgcgc cgagggccgc cactccaccg 4321 gcgcctaaag atcccagtgt ggtggtacgg gaattcctgc aggcctcgag ggccggcgcg 4381 ccgcggccgc tacgtaaatt ccgccccccc cccccccycc cccccccccc cccccccccc 4441 ccccmcmccc cccccccccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag 4501 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc 4561 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg 4621 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc 4681 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc 4741 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa 4801 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct 4861 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg 4921 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg 4981 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg 5041 ccacaaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc 5101 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg 5161 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg 5221 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca 5281 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt 5341 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc 5401 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg 5461 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg 5521 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg 5581 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg 5641 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg 5701 gctcgacgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc 5761 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 5821 cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 5881 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 5941 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 6001 ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 6061 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 6121 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 6181 cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 6241 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 6301 ctcctctgag tgattgacta cccgtcagcg ggggtctttc acatgcagca tgtatcaaaa 6361 ttaatttggt tttttttctt aagtatttac attaaatggc catagttgca ttaatgaatc 6421 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 6481 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 6541 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 6601 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 6661 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 6721 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 6781 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 6841 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 6901 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 6961 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 7021 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 7081 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 7141 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 7201 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 7261 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 7321 atcttcacct agatcctttt gcggccggcc gcaaatcaat ctaaagtata tatgagtaaa 7381 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 7441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 7501 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 7561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 7621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 7681 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 7741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 7801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 7861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 7921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 7981 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa 8041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 8101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 8161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 8221 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 8281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 8341 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca 8401 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcact 8461 cgaggagctt ggcccattgc atacgttgta tccatatcat aatatgtaca tttatattgg 8521 ctcatgtcca acattaccgc catgtt //