质粒名称: | cytoplasmic EKAR (EGFP-mRFP) |
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出品公司: | 爱迪基因 |
目录编号: | 18680 |
存储实验室: | Karel Svoboda |
载体骨架基本信息 |
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载体名称: | pCI |
载体出品公司: | Promega |
载体抗性: | 氨苄青霉素 |
空载体大小: | 4000 bp |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 哺乳细胞表达 |
筛选标记: | -- |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | ERK Activity Reporter,EKAR |
物种来源: | -- |
插入大小: | 550 bp |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: |
EGFP(插入件上的N端)
mRFP(插入件上的C端子)
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克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | CMV Forward |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | cytoplasmic EKAR (EGFP-mRFP) was a gift from Karel Svoboda (Addgene plasmid # 18680 ; http://n2t.net/addgene:18680 ; RRID:Addgene_18680) |
参考文献部分: | A genetically encoded fluorescent sensor of ERK activity. Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, Svoboda K.. PNAS 2008 Dec 9;105(49):19264-9 10.1073/pnas.0804598105 PubMed 19033456 |
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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 cytoplasmic EKAR (EGFP-mRFP) 5981 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS cytoplasmic EKAR (EGFP-mRFP) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5981) AUTHORS Harvey CD, Ehrhardt AG, Cellurale C, Zhong H, Yasuda R, Davis RJ, Svoboda K. TITLE A genetically encoded fluorescent sensor of ERK activity JOURNAL PNAS 2008 Dec 9;105(49):19264-9 PUBMED 19033456 REFERENCE 2 (bases 1 to 5981) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5981 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 138..517 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 518..721 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 672..692 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 718..742 /label=LNCX /note="Human CMV promoter, forward primer" intron 857..989 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 972..991 /label=pMT2-F /note="Synthetic intron, forward primer" promoter 1034..1052 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1058..1732 /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(1188..1206) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 1456..1475 /label=mCherry-F /note="mCherry, forward primer" primer_bind 1650..1673 /label=DsRed1-C /note="DsRed1, forward primer" CDS 2300..3016 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 2300..3016 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(2345..2366) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2606..2625) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2953..2974 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 3095..3216 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3106..3125) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3160..3179 /label=SV40pA-R /note="SV40 polyA, reverse primer" rep_origin 3397..3852 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3484..3503) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3694..3715 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(3919..3938) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4038..4060 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4098..4116) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 4184..4288 /gene="bla" /label=AmpR promoter CDS 4289..5149 /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 complement(4507..4526) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5320..5908 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5809..5828 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc 661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 721 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat 781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc 841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa 901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac 961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta 1021 aggctagagt acttaatacg actcactata ggctagcatg gcctcctccg aggacgtcat 1081 caaggagttc atgcgcttca aggtgcgcat ggagggctcc gtgaacggcc acgagttcga 1141 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt 1201 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tccagtacgg 1261 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc 1321 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac 1381 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa 1441 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga 1501 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagatgaggc tgaagctgaa 1561 ggacggcggc cactacgacg ccgaggtcaa gaccacctac atggccaaga agcccgtgca 1621 gctgcccggc gcctacaaga ccgacatcaa gctggacatc acctcccaca acgaggacta 1681 caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg ccgagctcaa 1741 gcttatggcg gacgaggaga agctgccgcc cggctgggag aagcgcatga gccgcagctc 1801 aggccgagtg tactacttca accacatcac taacgccagc cagtgggagc ggcccagcgg 1861 caacagcagc agtggtggca aaaacgggca gggggagcct gccaggaagc ttcgaattct 1921 gcagtcgact agggagcaag ggcttgtgtc tagaggcgga ggtggtggag gaggtggagg 1981 tggtggaggt ggaggtggag gtggtggcgg tggtggagga ggaggagcta gaggcggagg 2041 tggtggagga ggtggaggtg gtggaggtgg aggtggaggt ggtggcggtg gtggaggagg 2101 aggagctagc ggcggaggtg gtggaggagg tggaggtggt ggaggtggag gtggaggtgg 2161 tggcggtggt ggaggaggag gaatggctga ccaactgact gaagagtggt cgacggtacc 2221 gcggaccggt cctgacgtac cacgtacacc agtaggaaag gcaaagctgt cattccaatt 2281 cccgcgggcc cgggatccca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 2341 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 2401 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct 2461 gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg 2521 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt 2581 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 2641 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 2701 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 2761 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 2821 cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 2881 gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga 2941 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 3001 ggacgagctg tacaaggatg ctcttcaaaa aaaacttgaa gaacttgaac ttgacgaatg 3061 aactagtatc tgcggccgct tcgagcagac atgataagat acattgatga gtttggacaa 3121 accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 3181 ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 3241 atgtttcagg ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa 3301 tgtggtaaaa tcgataagga tccgggctgg cgtaatagcg aagaggcccg caccgatcgc 3361 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa 3421 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 3481 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 3541 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca 3601 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 3661 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 3721 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 3781 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 3841 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 3901 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 3961 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 4021 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 4081 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 4141 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 4201 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 4261 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 4321 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 4381 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4441 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 4501 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 4561 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 4621 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 4681 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 4741 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 4801 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 4861 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 4921 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 4981 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 5041 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 5101 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 5161 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 5221 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 5281 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5341 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 5401 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 5461 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 5521 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 5581 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 5641 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 5701 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 5761 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 5821 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 5881 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 5941 ggccttttgc tggccttttg ctcacatggc tcgacagatc t //