| 质粒名称: | pcDNA3.1_AktPH-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 67301 |
| 存储实验室: | Moritoshi Sato |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1(+) |
| 载体出品公司: | Invitrogen |
| 空载体大小: | 5348 bp |
| 完整质粒大小: | 7750 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达 |
| 筛选标记: | 新霉素（G418筛选） |
| 载体抗性: | 氨苄青霉素，100μg/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: | AktPH-mCherry |
| 物种来源: | H. sapiens (human), Synthetic |
| 插入大小: | 2042 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | CMV |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | HindIII(未破坏) |
| 3’克隆位点: | ApaI (未破坏) |
| 5’测序引物 | CMV-F |
| 3’测序引物 | BGH-rev |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3.1_AktPH-mCherry was a gift from Moritoshi Sato (Addgene plasmid # 67301 ; http://n2t.net/addgene:67301 ; RRID:Addgene_67301) |
| 参考文献部分: | Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Kawano F, Suzuki H, Furuya A, Sato M. Nat Commun. 2015 Feb 24;6:6256. doi: 10.1038/ncomms7256. 10.1038/ncomms7256 PubMed 25708714 |
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pcDNA3.1_AktPH-mCherry载体质粒基本信息
pcDNA3.1_AktPH-mCherry质粒图谱载体图谱和pcDNA3.1_AktPH-mCherry载体序列质粒序列多克隆位点信息
pcDNA3.1_AktPH-mCherry质粒载体简介
pcDNA3.1_AktPH-mCherry质粒序列载体序列
LOCUS pcDNA3.1_AktPH-mCherry 7750 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Fluorescent indicator for PI(3,4,5)P3.
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1_AktPH-mCherry
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7750)
AUTHORS Kawano F, Suzuki H, Furuya A, Sato M
TITLE Engineered pairs of distinct photoswitches for optogenetic control
of cellular proteins.
JOURNAL Nat Commun. 2015 Feb 24;6:6256. doi: 10.1038/ncomms7256.
PUBMED 25708714
REFERENCE 2 (bases 1 to 7750)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7750
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 769..789
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 863..882
/label=T7
/note="T7 promoter, forward primer"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 1475..2185
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(1620..1638)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 1888..1907
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind complement(2246..2266)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(2664..2675)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(2872..2891)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 2937..2992
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(2991..3010)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
primer_bind complement(3344..3361)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 3350..3574
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3620..4048
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3707..3726)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3917..3938
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4057..4077)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 4062..4391
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 4242..4377
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 4304..4323
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 4458..5252
/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
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(4512..4531)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 5122..5141
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 5426..5547
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5463..5482)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5517..5536
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5596..5612)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5596..5612)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5609..5631)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5620..5636
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(5644..5674)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5689..5710
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5827..5844)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5998..6583)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(6078..6097)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6754..7614)
/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 7377..7396
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7615..7719)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
901 gtttaaactt aagcttgccg ccaccatgag cgacgtggct attgtgaagg agggttggct
961 gcacaaacga ggggagtaca tcaagacctg gcggccacgc tacttcctcc tcaagaatga
1021 tggcaccttc attggctaca aggagcggcc gcaggatgtg gaccaacgtg aggctcccct
1081 caacaacttc tctgtggcgc agtgccagct gatgaagacg gagcggcccc ggcccaacac
1141 cttcatcatc cgctgcctgc agtggaccac tgtcatcgaa cgcaccttcc atgtggagac
1201 tcctgaggag cgggaggagt ggacaaccgc catccagact gtggctgacg gcctcaagaa
1261 gcaggaggag gaggagatgg acttccggtc gggctcaccc agtgacaact caggggctga
1321 agagatggag gtgtccctgg ccaagcccaa gcaccgcgtg accatgaacg agtttgagta
1381 cctgaagctg ctgggcaagg gcactttcgg caaggtggaa ttcggagggt ctggaggggg
1441 tggctcaggt ggcggcggaa gtggcggaag atctatggtg agcaagggcg aggaggataa
1501 catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg
1561 ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc
1621 caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca
1681 gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa
1741 gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt
1801 ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct
1861 gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga
1921 ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag
1981 gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa
2041 gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca
2101 caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg
2161 cggcatggac gagctgtaca agtaatctag atgatcaacc tctggattac aaaatttgtg
2221 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
2281 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
2341 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
2401 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
2461 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
2521 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
2581 cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg
2641 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
2701 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
2761 ccctttgggc cgcctccccg catcggtaca attcactcct caggtgcagg ctgcctatca
2821 gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag atctttttcc
2881 ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata
2941 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
3001 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
3061 aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta
3121 tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta
3181 gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt
3241 acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc
3301 tcttctctta tggagatcgg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
3361 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
3421 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
3481 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
3541 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
3601 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
3661 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
3721 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
3781 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
3841 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
3901 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
3961 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
4021 ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg
4081 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
4141 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
4201 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
4261 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
4321 aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
4381 gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag
4441 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
4501 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
4561 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
4621 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
4681 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
4741 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
4801 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
4861 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
4921 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
4981 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
5041 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
5101 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
5161 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
5221 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
5281 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
5341 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
5401 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
5461 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
5521 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
5581 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
5641 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
5701 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
5761 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
5821 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
5881 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
5941 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
6001 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
6061 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
6121 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
6181 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
6241 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
6301 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
6361 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
6421 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
6481 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtttttt
6541 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
6601 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
6661 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
6721 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
6781 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
6841 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
6901 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
6961 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
7021 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
7081 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
7141 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
7201 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
7261 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
7321 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
7381 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
7441 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
7501 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
7561 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
7621 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
7681 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
7741 acctgacgtc
//
