| 质粒名称: | cytoplasmic EKAR (EGFP-mRFP) |
|---|---|
| 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 |
|
| 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 |
|
| 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）
|
| 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 |
|
| 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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cytoplasmic EKAR (EGFP-mRFP)载体质粒基本信息
cytoplasmic EKAR (EGFP-mRFP)质粒图谱载体图谱和cytoplasmic EKAR (EGFP-mRFP)载体序列质粒序列多克隆位点信息
质粒图谱.png)
cytoplasmic EKAR (EGFP-mRFP)质粒载体简介
cytoplasmic EKAR (EGFP-mRFP)质粒序列载体序列
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
//
