| 质粒名称: | GalT-RpHLuorin2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 171719 |
| 存储实验室: | Geert van den Bogaart |
|
载体骨架基本信息 |
|
| 载体名称: | pRpHluorin-N1 |
| 载体出品公司: | Clontech |
| 空载体大小: | 4730 bp |
| 完整质粒大小: | 4959 bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | Kanamycin |
| 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: | Ratiometric pHLuorin2 fused to B4GALT1 / B4GAL-T1, CDG2D, GGTB2, GT1, GTB, beta4Gal-T1, GalT |
| 物种来源: | H. sapiens (human) |
| GenBank编号 | NM_001497.3 |
| 启动子: | CMV |
| 标签/融合蛋白: | ratiometric pHluorin2 (C terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | SmaI (not destroyed) |
| 3’克隆位点: | BamHI (not destroyed) |
| 5’测序引物 | TAACAACTCCGCCCCATT |
| 3’测序引物 | GTCCAGCTCGACCAGGATGGG |
|
文献引用方法 |
|
| 材料和方法部分: |
GalT-RpHLuorin2 was a gift from Geert van den Bogaart (Addgene plasmid # 171719 ; http://n2t.net/addgene:171719 ; RRID:Addgene_171719)
|
| 参考文献部分: | Fluorescence Lifetime Imaging of pH along the Secretory Pathway. Linders PTA, Ioannidis M, Ter Beest M, van den Bogaart G. ACS Chem Biol. 2022 Jan 21;17(1):240-251. doi: 10.1021/acschembio.1c00907. Epub 2022 Jan 10. 10.1021/acschembio.1c00907 PubMed 35000377 |
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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 GalT-RpHLuorin2 4959 bp ds-DNA circular SYN 03-MAR-2022
DEFINITION Trans-Golgi expression of ratiometric pHLuorin2.
ACCESSION .
VERSION .
KEYWORDS GalT-RpHLuorin2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4959)
TITLE Fluorescence lifetime imaging of pH along the secretory pathway
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 4959)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Mar 3, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..4959
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 108..411
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 412..615
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 566..586
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind complement(1258..1277)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
polyA_signal 1794..1915
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(1831..1850)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 1885..1904
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(1922..2377)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2059..2080)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2271..2290
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2404..2508
/gene="bla"
/label=AmpR promoter
promoter 2510..2867
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2533..2553)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 2718..2853
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 2780..2799
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 2902..3696
/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
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(2956..2975)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3566..3585
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(3884..3903)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 3928..3975
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4304..4892
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4793..4812
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag
61 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
121 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
181 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
241 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
301 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
361 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
421 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
481 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
541 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
601 tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg gactcagatc
661 tcgagctcaa gcttcgaatt catgaggctt cgggagccgc tcctgagcgg cagcgccgcg
721 atgccaggcg cgtccctaca gcgggcctgc cgcctgctcg tggccgtctg cgctctgcac
781 cttggcgtca ccctcgttta ctacctggct ggccgcgacc tgagccgcct gccccaactg
841 gtcggagtct ccacaccgct gcagggcggc tcgaacagtg ccgccgccat cgggcagtcc
901 tccggggagc tccggaccgg aggggccgtg gatccaggcg gcagcggcgg cagcgctagc
961 aagggcgagg agctgttcac cggcgtggtg cccatcctgg tggagctgga cggcgacgtg
1021 aacggccaca agttcagcgt gagcggcgag ggcgagggcg acgccaccta cggcaagctg
1081 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctggtgacc
1141 accctgagct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
1201 ttcttcaaga gcgccatgcc cgagggctac gtgcaggagc gcaccatctt cttcaaggac
1261 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
1321 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctgggcca caagctggag
1381 tacaactaca acgagcacct ggtctacatc atggccgaca agcagaagaa cggcaccaag
1441 gccatcttcc aggtgcacca caacatcgag gacggcggcg tgcagctggc cgaccactac
1501 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgcac
1561 acccagagcg ccctgagcaa ggaccccaac gagaagcgcg accacatggt gctgctggag
1621 ttcgtgaccg ccgccggcat cacccacggc atggacgagc tgtacaagta aagcggccgc
1681 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
1741 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
1801 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
1861 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
1921 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
1981 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
2041 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
2101 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
2161 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
2221 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
2281 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
2341 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
2401 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
2461 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
2521 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
2581 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
2641 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
2701 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
2761 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
2821 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
2881 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
2941 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
3001 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
3061 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
3121 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
3181 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
3241 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
3301 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
3361 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
3421 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
3481 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
3541 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
3601 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
3661 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
3721 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
3781 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
3841 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
3901 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
3961 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
4021 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
4081 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
4141 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
4201 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
4261 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
4321 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
4381 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
4441 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
4501 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
4561 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
4621 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
4681 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
4741 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
4801 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
4861 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
4921 tcctggcctt ttgctggcct tttgctcaca tgttctttc
//
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