| 质粒名称: | pTRE-Tight-MitoTimer |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 50547 |
| 存储实验室: | Roberta Gottlieb |
|
载体骨架基本信息 |
|
| 载体名称: | pTRE Tight |
| 载体出品公司: | Clontech |
| 空载体大小: | 2516 bp |
| 完整质粒大小: | 3325 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素100 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | XL1 Blue |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | MitoTimer |
| 插入大小: | 809 bp |
| 基因突变: | F12S mutation in Timer |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | EcoRI(未破坏) |
| 3’克隆位点: | XbaI(未破坏) |
| 5’测序引物 | AGG CGT ATC ACG AGG CCC TTT CGT |
| 3’测序引物 | TAT TAC CGC CTT TGA GTG AGC TGA |
|
文献引用方法 |
|
| 材料和方法部分: | pTRE-Tight-MitoTimer was a gift from Roberta Gottlieb (Addgene plasmid # 50547 ; http://n2t.net/addgene:50547 ; RRID:Addgene_50547) |
| 参考文献部分: | MitoTimer: A novel tool for monitoring mitochondrial turnover. Hernandez G, Thornton C, Stotland A, Lui D, Sin J, Ramil J, Magee N, Andres A, Quarato G, Carreira RS, Sayen MR, Wolkowicz R, Gottlieb RA. Autophagy. 2013 Nov 1;9(11):1852-61. doi: 10.4161/auto.26501. Epub 2013 Oct 11. 10.4161/auto.26501 PubMed 24128932 |
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 pTRE-Tight-MitoTimer 3325 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses the Timer protein targeted to the mitochondria under a
Tet-inducible promoter.
ACCESSION .
VERSION .
KEYWORDS pTRE-Tight-MitoTimer
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3325)
AUTHORS Hernandez G, Thornton C, Stotland A, Lui D, Sin J, Ramil J, Magee N,
Andres A, Quarato G, Carreira RS, Sayen MR, Wolkowicz R, Gottlieb RA
TITLE MitoTimer: A novel tool for monitoring mitochondrial turnover.
JOURNAL Autophagy. 2013 Nov 1;9(11):1852-61. doi: 10.4161/auto.26501. Epub
2013 Oct 11.
PUBMED 24128932
REFERENCE 2 (bases 1 to 3325)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..3325
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 4..318
/label=tight TRE promoter
/note="Tet-responsive promoter PTight, consisting of seven
tet operator sequences followed by the minimal CMV
promoter"
protein_bind 12..30
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 48..66
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 83..101
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 119..137
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 155..173
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 190..208
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
protein_bind 226..244
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
primer_bind 291..315
/label=LNCX
/note="Human CMV promoter, forward primer"
CDS 340..414
/codon_start=1
/product="mitochondrial presequence of human cytochrome c
oxidase subunit VIII (Rizutto et al., 1989)"
/label=COX8 presequence
/translation="MSVLTPLLLRGLTGSARRLPVPRAK"
CDS 448..1125
/codon_start=1
/product="green-to-red fluorescent timer derivative of
DsRed (Terskikh et al., 2000)"
/label=Timer
/note="mammalian codon-optimized"
/translation="VRSSKNVIKESMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVK
LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
ATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERTEGRHH
LFL"
primer_bind complement(628..648)
/label=DsRed1-N
/note="DsRed1, reverse primer"
primer_bind 1040..1063
/label=DsRed1-C
/note="DsRed1, forward primer"
polyA_signal 1239..1320
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1276..1295)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(1331..1348)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(1502..2090)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(1582..1601)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(2261..3121)
/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 2884..2903
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(3122..3226)
/gene="bla"
/label=AmpR promoter
primer_bind 3294..3312
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
ORIGIN
1 ctcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
61 tagagaacga tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
121 cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg
181 tcgagtttat ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
241 gagaacgtat gtcgaggtag gcgtgtacgg tgggaggcct atataagcag agctcgttta
301 gtgaaccgtc agatcgcctg gagaattcag atctccacca tgtccgtcct gacgccgctg
361 ctgctgcggg gcttgacagg ctcggcccgg cggctcccag tgccgcgcgc caagatccat
421 tcgttggggg atccccgggt accggtcgtg cgctcctcca agaacgtcat caaggagtcc
481 atgcgcttca aggtgcgcat ggagggcacc gtgaacggcc acgagttcga gatcgagggc
541 gagggcgagg gccgccccta cgagggccac aacaccgtga agctgaaggt gaccaagggc
601 ggccccctgc ccttcgcctg ggacatcctg tccccccagt tccagtacgg ctccaaggtg
661 tacgtgaagc accccgccga catccccgac tacaagaagc tgtccttccc cgagggcttc
721 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg cgaccgtgac ccaggactcc
781 tccctgcagg acggctgctt catctacaag gtgaagttca tcggcgtgaa cttcccctcc
841 gacggccccg tgatgcagaa gaagaccatg ggctgggagg cctccaccga gcgcctgtac
901 ccccgcgacg gcgtgctgaa gggcgagatc cacaaggccc tgaagctgaa ggacggcggc
961 cactacctgg tggagttcaa gtccatctac atggccaaga agcccgtgca gctgcccggc
1021 tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta caccatcgtg
1081 gagcagtacg agcgcaccga gggccgccac cacctgttcc tgtagtctag aggatcataa
1141 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
1201 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
1261 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
1321 ctcgagcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
1381 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
1441 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
1501 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
1561 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
1621 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
1681 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
1741 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
1801 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
1861 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
1921 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
1981 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
2041 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
2101 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
2161 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
2221 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
2281 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
2341 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
2401 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
2461 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
2521 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
2581 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
2641 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
2701 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
2761 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
2821 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac
2881 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
2941 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
3001 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
3061 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
3121 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
3181 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
3241 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
3301 gtatcacgag gccctttcgt cttca
//
