| 质粒名称: | pmGFP-P2A-K(AAG)20-P2A-RFP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 105689 |
| 存储实验室: | Ramanujan Hegde |
|
载体骨架基本信息 |
|
| 载体名称: | pEGFP-N1 |
| 载体出品公司: | Clontech |
| 空载体大小: | 4700bp |
| 完整质粒大小: | 6030bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | Kanamycin, 50 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称1: | P2A-3xFLAG-VHPbeta |
| 物种来源: | Synthetic |
| 插入大小: | 479bp |
| 启动子: | CMV (from vector) |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BspEI (not destroyed) |
| 3’克隆位点: | SalI (not destroyed) |
| 5’测序引物 | GGCATCAAGGTGAACTTCAAGA |
| 3’测序引物 | ACAGGATGTCCCAGGCGAA |
| 插入基因名称2: | K(AAG)20 |
| 插入大小: | (bp)103 |
| 启动子: | CMV (from vector) |
| 克隆方法: | Restriction Enzyme |
| 5’克隆位点: | SalI (not destroyed) |
| 3’克隆位点: | KpnI (not destroyed) |
| 5’测序引物 | GGCATCAAGGTGAACTTCAAGA |
| 3’测序引物 | ACAGGATGTCCCAGGCGAA |
| 插入基因名称3: | P2A-mCherry |
| 插入大小: | (bp)795 |
| 启动子: | CMV (from vector) |
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | GGCATCAAGGTGAACTTCAAGA |
| 3’测序引物 | ACCACAACTAGAATGCAG |
|
文献引用方法 |
|
| 材料和方法部分: | pmGFP-P2A-K(AAG)20-P2A-RFP was a gift from Ramanujan Hegde (Addgene plasmid # 105689 ; http://n2t.net/addgene:105689 ; RRID:Addgene_105689) |
| 参考文献部分: | Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination. Juszkiewicz S, Hegde RS. Mol Cell. 2017 Feb 16;65(4):743-750.e4. doi: 10.1016/j.molcel.2016.11.039. Epub 2017 Jan 5. 10.1016/j.molcel.2016.11.039 PubMed 28065601 |
买家导航
pmGFP-P2A-K(AAG)20-P2A-RFP载体质粒基本信息
pmGFP-P2A-K(AAG)20-P2A-RFP质粒图谱载体图谱和pmGFP-P2A-K(AAG)20-P2A-RFP载体序列质粒序列多克隆位点信息
20-P2A-RFP质粒图谱.png)
pmGFP-P2A-K(AAG)20-P2A-RFP质粒载体简介
pmGFP-P2A-K(AAG)20-P2A-RFP质粒序列载体序列
LOCUS pmGFP-P2A-K(AAG)20-P2A-RFP 6024 bp DNA circular SYN 25-AUG-2024
DEFINITION Double fluorescent stalling reporter K(AAG)20.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6024)
AUTHORS Juszkiewicz S, Hegde RS
TITLE Initiation of Quality Control during Poly(A) Translation Requires
Site-Specific Ribosome Ubiquitination.
JOURNAL Mol Cell. 2017 Feb 16;65(4):743-750.e4. doi:
10.1016/j.molcel.2016.11.039. Epub 2017 Jan 5.
PUBMED 28065601
REFERENCE 2 (bases 1 to 6024)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.molcel.2016.11.039"; journalName: "Mol Cell"; date:
"2017-02-16- 16"; volume: "65"; issue: "4"; pages: "743-750.e4"
FEATURES Location/Qualifiers
source 1..6024
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..304
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 305..508
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 459..479
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 547..556
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 553..1269
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
EFVTAAGITLGMDELYK"
primer_bind complement(598..619)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(859..878)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
CDS 1285..1341
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 1348..1413
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1849..1905
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 1912..2619
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
GRHSTGGMDELYK"
primer_bind complement(2054..2072)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 2322..2341
/label=mCherry-F
/note="mCherry, forward primer"
polyA_signal 2752..2873
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2789..2808)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2843..2862
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2880..3335)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3017..3038)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 3229..3248
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 3362..3466
/gene="bla"
/label=AmpR promoter
promoter 3468..3825
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
enhancer 3468..3659
/label=SV40 enhancer
/note="enhancer for the SV40 early promoter (Herr, 1993)"
primer_bind complement(3491..3511)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 3676..3811
/label=SV40 ori
/note="SV40 origin of replication"
CDS 3860..4654
/codon_start=1
/product="aminoglycoside phosphotransferase"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
CDS 3860..4654
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3914..3933)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4524..4543
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4842..4861)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4886..4933
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 5262..5850
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5751..5770
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
541 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
601 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
1201 gatcacatgg tccttaagga gttcgtgacc gccgccggga tcactctcgg catggacgag
1261 ctgtacaagt ccggaggcag cggcgccacc aacttttccc tgctcaagca ggccggcgac
1321 gtggaagaga atcccggccc cggtactgac tacaaagacc atgacggtga ttataaagat
1381 catgacatcg attacaagga tgacgatgac aaggccggat ctcctggtcc gacccccagt
1441 ggcactaacg tgggatcctc actctctgac gaggacttca aggctgtttt tggcatgacc
1501 cgctctgcct ttgccaactt gcccttgtgg aaacagcaga acctcaagaa ggagaaagga
1561 ctcttcggat cagggcgctc tcccagcaaa gcagtggccg cccgggcggc gggatccact
1621 gtccggcaga ggaaaaatgc cagctgtggg acaaggagtg caggccgcac aacctcggca
1681 ggcaccgggg ggatgtggcg attctacaca gaagattcac ctgggctcaa agttggccct
1741 aggtcgaccg ccatggcgac caagaagaag aagaagaaga agaagaagaa gaagaagaag
1801 aagaagaaga agaagaagaa gcccggggga tccggtaccg gaagcggagc tactaacttc
1861 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctctcga ggtgagcaag
1921 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
1981 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
2041 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
2101 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
2161 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
2221 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
2281 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
2341 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
2401 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
2461 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
2521 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
2581 cgccactcca ccggcggcat ggacgagctg tacaagtaaa ctggatccac cggatctaga
2641 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
2701 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
2761 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
2821 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta
2881 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
2941 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
3001 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
3061 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
3121 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
3181 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
3241 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
3301 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
3361 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
3421 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
3481 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
3541 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
3601 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
3661 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
3721 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
3781 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
3841 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
3901 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
3961 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
4021 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
4081 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
4141 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
4201 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
4261 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
4321 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
4381 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
4441 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
4501 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
4561 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
4621 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
4681 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
4741 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
4801 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
4861 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
4921 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
4981 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
5041 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
5101 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
5161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
5221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
5281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
5341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
5401 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
5461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
5521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
5581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
5641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
5701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
5761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
5821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
5881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
5941 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
6001 tcatagccca tatatggagt tccg
//
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