| 质粒名称: | pRK5-FLAG-p14 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 42330 |
| 存储实验室: | David Sabatini |
|
载体骨架基本信息 |
|
| 载体名称: | pRK5 |
| 载体出品公司: | BD PharMingen |
| 空载体大小: | 4661 bp |
| 完整质粒大小: | 5096 bp |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄青霉素，100μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | p14 |
| 物种来源: | H. sapiens (human) |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | NM_014017.3 NP_054736.1 |
| 启动子: | CMV, SP6 |
| 标签/融合蛋白: | Flag (N terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | EcoRI(未破坏) |
| 3’克隆位点: | HindIII (未破坏) |
| 5’测序引物 | Sp6 |
| 3’测序引物 | SV40pA-R |
|
文献引用方法 |
|
| 材料和方法部分: | pRK5-FLAG-p14 was a gift from David Sabatini (Addgene plasmid # 42330 ; http://n2t.net/addgene:42330 ; RRID:Addgene_42330) |
| 参考文献部分: | Ragulator Is a GEF for the Rag GTPases that Signal Amino Acid Levels to mTORC1. Bar-Peled L, Schweitzer LD, Zoncu R, Sabatini DM. Cell. 2012 Sep 14;150(6):1196-208. doi: 10.1016/j.cell.2012.07.032. 10.1016/j.cell.2012.07.032 PubMed 22980980 |
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 pRK5-FLAG-p14 5096 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pRK5-FLAG-p14
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5096)
AUTHORS Bar-Peled L, Schweitzer LD, Zoncu R, Sabatini DM
TITLE Ragulator Is a GEF for the Rag GTPases that Signal Amino Acid Levels
to mTORC1.
JOURNAL Cell. 2012 Sep 14;150(6):1196-208. doi: 10.1016/j.cell.2012.07.032.
PUBMED 22980980
REFERENCE 2 (bases 1 to 5096)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5096
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..380
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 381..584
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 535..555
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 581..605
/label=LNCX
/note="Human CMV promoter, forward primer"
promoter 815..833
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 815..832
/label=SP6
/note="SP6 promoter, forward primer"
primer_bind 846..865
/label=pMT2-F
/note="Synthetic intron, forward primer"
CDS 924..947
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
regulatory 1383..1392
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
polyA_signal 1396..1530
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1433..1452)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1487..1506
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
promoter 1599..1956
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(1622..1642)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 1807..1942
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 1869..1888
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
primer_bind complement(1976..1993)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1976..1992)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1985..2007)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2205..2660
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2292..2311)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2502..2523
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2677..2696)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2796..2818
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2856..2874)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2942..3046
/gene="bla"
/label=AmpR promoter
CDS 3047..3907
/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(3265..3284)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 4078..4666
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4567..4586
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4820..4837
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 4954..4975
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 4990..5020
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5028..5044
/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)."
primer_bind 5033..5055
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 5052..5068
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 5052..5068
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
ORIGIN
1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
901 cgattgaatt caccatggaa atggactaca aggatgacga tgacaagggt ggtggtggtg
961 gtgcgtcgac gatgctgcgc cccaaggctt tgacccaggt gctaagccaa gccaacactg
1021 gaggcgtcca gagcaccctg ctgctgaata acgagggatc actgctggcc tactctggtt
1081 acggggacac tgacgcccgg gtcaccgctg ccatagccag taacatctgg gccgcctacg
1141 accggaacgg gaaccaagcg tttaatgaag acaatctcaa attcatcctc atggactgca
1201 tggagggccg tgtagccatc acccgagtgg ccaaccttct gctgtgtatg tatgccaagg
1261 agaccgtggg ctttggaatg ctcaaggcca aggcccaggc tttggtgcag tacctggagg
1321 agcccctcac ccaagtggcg gcatcttaag cggccgctaa gtaagtaagg atccccagct
1381 tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
1441 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
1501 ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc accatggcct
1561 gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg
1621 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
1681 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
1741 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
1801 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
1861 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
1921 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc ttggcactgg
1981 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
2041 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
2101 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
2161 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
2221 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
2281 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
2341 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
2401 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
2461 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
2521 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
2581 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
2641 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
2701 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
2761 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
2821 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
2881 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
2941 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
3001 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
3061 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
3121 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
3181 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
3241 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
3301 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
3361 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
3421 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
3481 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
3541 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
3601 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
3661 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
3721 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
3781 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
3841 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
3901 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
3961 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
4021 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
4081 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
4141 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
4201 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
4261 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
4321 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
4381 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
4441 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
4501 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
4561 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
4621 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
4681 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
4741 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
4801 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
4861 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
4921 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
4981 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
5041 acaatttcac acaggaaaca gctatgacat gattacgaat taattcgagc tcgccc
//
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