| 质粒名称: | pcDNA3-PKA-SPARK |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 106920 |
| 存储实验室: | Xiaokun Shu |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3 |
| 完整质粒大小: | 7045 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | PKA-SPARK |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3-PKA-SPARK was a gift from Xiaokun Shu (Addgene plasmid # 106920 ; http://n2t.net/addgene:106920 ; RRID:Addgene_106920) |
| 参考文献部分: | Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter. Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis A, Coughlin SR, Kornberg TB, Shu X. Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi: 10.1016/j.molcel.2017.12.008. 10.1016/j.molcel.2017.12.008 PubMed 29307513 |
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 pcDNA3-PKA-SPARK 106920 7045 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Express PKA-SPARK, an activity reporter of protein kinase A in
mammalian cells..
ACCESSION .
VERSION .
KEYWORDS pcDNA3-PKA-SPARK
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7045)
AUTHORS Zhang Q, Huang H, Lu Q, Wu R, Chung CI, Zhang SQ, Torra J, Schepis
A, Coughlin SR, Kornberg TB, Shu X
TITLE Visualizing Dynamics of Cell Signaling In Vivo with a Phase
Separation-Based Kinase Reporter.
JOURNAL Mol Cell. 2018 Jan 3. pii: S1097-2765(17)30936-X. doi:
10.1016/j.molcel.2017.12.008.
PUBMED 29307513
REFERENCE 2 (bases 1 to 7045)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7045
/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"
regulatory 901..910
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 997..1713
/codon_start=1
/product="the original enhanced GFP (Yang et al., 1996)"
/label=EGFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
primer_bind complement(1042..1063)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1303..1322)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 1650..1671
/label=EGFP-C
/note="EGFP, forward primer"
CDS 1909..1962
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
primer_bind complement(2636..2653)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2642..2866
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2912..3340
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2999..3018)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3209..3230
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3349..3369)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3354..3683
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3534..3669
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3596..3615
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3750..4544
/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(3804..3823)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4414..4433
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
polyA_signal 4718..4839
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4755..4774)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4809..4828
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4888..4904)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4888..4904)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4901..4923)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4912..4928
/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(4936..4966)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4981..5002
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5119..5136)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5290..5878)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5370..5389)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6049..6909)
/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 6672..6691
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6910..7014)
/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 gccaccatgg gcggtggagg gagcctgcgt cgcgccaccc tggtggacgg cggactcgag
961 tccggactca gatctggcag cggtggaggc gatatcatgg tgagcaaggg cgaggagctg
1021 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
1081 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
1141 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
1201 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
1261 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
1321 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
1381 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
1441 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
1501 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
1561 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
1621 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
1681 gggatcactc tcggcatgga cgagctgtac aagggatccg gcagtgctgg tggtagtgct
1741 ggtggtagtg ctggtggtag tgctggtggc agtgctggtg gtagtgctgg tggtagtgct
1801 ggtggctcta gaggagaaat cgcaaagagt ctgaaagaaa ttgcaaaatc cttgaaggaa
1861 atagcgtggt cactgaaaga aattgccaag tccctcaaag ggggtaccga gggcagagga
1921 agtctgctaa catgcggtga cgtcgaggag aatcctggcc caaagcttgc caccatgcat
1981 aagttttctc aagaacagat cggcgaaaac attgtgtgca gggtcatttg taccacgggt
2041 caaattccca tccgagattt gtcagctgat atttcacaag tgcttaagga aaaacgatcc
2101 ataaagaaag tttggacatt tggtagaaac ccagcctgtg actatcattt aggaaacatt
2161 tcaagactgt caaataagca tttccaaata ctactaggag aagacggtaa ccttttattg
2221 aatgacattt ccactaatgg gacctggtta aatgggcaaa aagtcgagaa gaacagcaat
2281 cagttactgt ctcaaggtga tgaaataacc gttggtgtag gcgtggaatc agatatttta
2341 tctctggtca ttttcataaa cgacaaattt aagcagtgcc tcgaacagaa caaagttgat
2401 cgcggatccg gcagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggc
2461 agtgctggtg gtagtgctgg tggtagtgct ggtggctcta gaaccttgag agaaatcgag
2521 gaattgttga ggaaaatcat tgaagacagc gtgcgcagtg tggccgagct ggaggacatt
2581 gagaagtggc tgaagaaaat ctaagaattc gggcccgttt aaacccgctg atcagcctcg
2641 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
2701 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
2761 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
2821 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
2881 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
2941 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
3001 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
3061 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
3121 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
3181 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
3241 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
3301 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
3361 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
3421 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
3481 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
3541 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
3601 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
3661 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
3721 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
3781 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
3841 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
3901 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
3961 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
4021 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
4081 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
4141 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
4201 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
4261 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
4321 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
4381 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
4441 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
4501 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
4561 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
4621 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
4681 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
4741 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
4801 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
4861 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
4921 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
4981 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
5041 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
5101 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
5161 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
5221 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
5281 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
5341 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
5401 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
5461 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
5521 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
5581 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
5641 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
5701 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
5761 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
5821 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
5881 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
5941 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
6001 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
6061 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
6121 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
6181 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
6241 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
6301 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
6361 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
6421 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
6481 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
6541 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
6601 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
6661 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
6721 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
6781 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
6841 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
6901 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
6961 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
7021 tccccgaaaa gtgccacctg acgtc
//
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