| 质粒名称: | pcDNA GNSTM-3-Flag-10-Lamp2b-HA |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 71293 |
| 存储实验室: | Joshua Leonard |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA 3.1+ Hygro |
| 载体出品公司: | Clontech |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5578 bp |
| 完整质粒大小: | 6925 bp |
| 载体修饰: | 从MCS中删除了一些限制性位点 |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 潮霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Lamp2b |
| 物种来源: | H. sapiens (human) |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: |
Lamp2信号肽（N端插入）
GNSTM糖基化基序（N端插入）
FLAG （插入N端子）
HA（插入件上的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | NheI（克隆过程中销毁） |
| 3' 克隆位点: | BamHI（克隆过程中销毁） |
| 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: | pcDNA GNSTM-3-Flag-10-Lamp2b-HA was a gift from Joshua Leonard (Addgene plasmid # 71293 ; http://n2t.net/addgene:71293 ; RRID:Addgene_71293) |
| 参考文献部分: | Stabilization of exosome-targeting peptides via engineered glycosylation. Hung ME, Leonard JN. J Biol Chem. 2015 Mar 27;290(13):8166-72. doi: 10.1074/jbc.M114.621383. Epub 2015 Feb 5. 10.1074/jbc.M114.621383 PubMed 25657008 |
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 pcDNA GNSTM-3-Flag-10-Lamp2b-HA 6925 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Encodes (N to C): GNSTM glycosylation motif, 3 residue spacer, Flag
tag,10 residue spacer, Lamp2b (exosomal transmembrane protein), 3
residue spacer, HA tag..
ACCESSION .
VERSION .
KEYWORDS pcDNA GNSTM-3-Flag-10-Lamp2b-HA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6925)
AUTHORS Hung ME, Leonard JN
TITLE Stabilization of exosome-targeting peptides via engineered
glycosylation.
JOURNAL J Biol Chem. 2015 Mar 27;290(13):8166-72. doi:
10.1074/jbc.M114.621383. Epub 2015 Feb 5.
PUBMED 25657008
REFERENCE 2 (bases 1 to 6925)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6925
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 110..489
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 490..693
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 644..664
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 738..757
/label=T7
/note="T7 promoter, forward primer"
promoter 738..756
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
regulatory 779..788
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 893..916
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 2102..2128
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
primer_bind complement(2225..2242)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2231..2455
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 2501..2929
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2588..2607)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2798..2819
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(2938..2958)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 2943..3272
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3123..3258
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3185..3204
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3321..4346
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
polyA_signal 4476..4597
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(4513..4532)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 4567..4586
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(4646..4662)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4646..4662)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4659..4681)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4670..4686
/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(4694..4724)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4739..4760
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4877..4894)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5048..5633)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5128..5147)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5804..6664)
/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 6427..6446
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(6665..6769)
/gene="bla"
/label=AmpR promoter
primer_bind complement(6844..6863)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
ORIGIN
1 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
61 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
481 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
541 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
601 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
661 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
721 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagtgccgc
781 cgccatggtg tgcttccgcc tcttcccggt tccgggctca gggctcgttc tggtctgcct
841 agtcctggga gctgtgcggt cttatgcagg taactcgact atgggcagtg gagactataa
901 agatgacgat gacaagggca gtggatctgg atccggtggc tcgagtttgg aacttaattt
961 gacagattca gaaaatgcca cttgccttta tgcaaaatgg cagatgaatt tcacagttcg
1021 ctatgaaact acaaataaaa cttataaaac tgtaaccatt tcagaccatg gcactgtgac
1081 atataatgga agcatttgtg gggatgatca gaatggtccc aaaatagcag tgcagttcgg
1141 acctggcttt tcctggattg cgaattttac caaggcagca tctacttatt caattgacag
1201 cgtctcattt tcctacaaca ctggtgataa cacaacattt cctgatgctg aagataaagg
1261 aattcttact gttgatgaac ttttggccat cagaattcca ttgaatgacc tttttagatg
1321 caatagttta tcaactttgg aaaagaatga tgttgtccaa cactactggg atgttcttgt
1381 acaagctttt gtccaaaatg gcacagtgag cacaaatgag ttcctgtgtg ataaagacaa
1441 aacttcaaca gtggcaccca ccatacacac cactgtgcca tctcctacta caacacctac
1501 tccaaaggaa aaaccagaag ctggaaccta ttcagttaat aatggcaatg atacttgcct
1561 gctggctacc atggggctgc agctgaacat cactcaggat aaggttgctt cagttattaa
1621 catcaacccc aatacaactc actccacagg cagctgccgt tctcacactg ctctacttag
1681 actcaatagc agcactatta agtatctaga ctttgtcttt gctgtgaaaa atgaaaaccg
1741 attttatctg aaggaagtga acatcagcat gtatttggtt aatggctccg ttttcagcat
1801 tgcaaataac aatctcagct actgggatgc ccccctggga agttcttata tgtgcaacaa
1861 agagcagact gtttcagtgt ctggagcatt tcagataaat acctttgatc taagggttca
1921 gcctttcaat gtgacacaag gaaagtattc tacagcccaa gagtgttcgc tggatgatga
1981 caccattcta atcccaatta tagttggtgc tggtctttca ggcttgatta tcgttatagt
2041 gattgcttac gtaattggca gaagaaaaag ttatgctgga tatcagactc tgggaagcgg
2101 atacccatac gatgtgccag attacgctta aggatccact agtccagtgt ggtggaattc
2161 tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
2221 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
2281 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
2341 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
2401 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
2461 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
2521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
2581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
2641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
2701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
2761 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
2821 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
2881 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
2941 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
3001 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
3061 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
3121 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
3181 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
3241 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
3301 tcggatctga tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
3361 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
3421 ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
3481 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
3541 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
3601 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
3661 cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg
3721 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
3781 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
3841 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
3901 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
3961 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
4021 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
4081 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
4141 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
4201 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
4261 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
4321 gcactcgtcc gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct
4381 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
4441 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
4501 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
4561 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
4621 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
4681 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
4741 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
4801 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
4861 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
4921 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
4981 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
5041 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
5101 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
5161 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
5221 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
5281 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
5341 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
5401 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
5461 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
5521 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
5581 gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
5641 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
5701 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
5761 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
5821 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
5881 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
5941 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
6001 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
6061 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
6121 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
6181 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
6241 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
6301 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
6361 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
6421 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
6481 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
6541 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
6601 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
6661 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
6721 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
6781 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
6841 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
6901 ggaggtcgct gagtagtgcg cgagc
//
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