| 质粒名称: | pBb(RSF1010)1k-GFPuv |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 106395 |
| 存储实验室: | Brian Pfleger |
|
载体骨架基本信息 |
|
| 载体名称: | pSEVA251 |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 卡那霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 低拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | GFPuv |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | trc |
| 标签/融合蛋白: | -- |
| 克隆方法: | Gibson克隆 |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 5’测序引物 | -- |
| 3’测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pBb(RSF1010)1k-GFPuv was a gift from Brian Pfleger (Addgene plasmid # 106395 ; http://n2t.net/addgene:106395 ; RRID:Addgene_106395) |
| 参考文献部分: | Genetic tools for reliable gene expression and recombineering in Pseudomonas putida. Cook TB, Rand JM, Nurani W, Courtney DK, Liu SA, Pfleger BF. J Ind Microbiol Biotechnol. 2018 Jan 3. pii: 10.1007/s10295-017-2001-5. doi: 10.1007/s10295-017-2001-5. 10.1007/s10295-017-2001-5 [pii] PubMed 29299733 |
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
1k-GFPuv载体图谱.png)
载体简介
载体序列
LOCUS pBb(RSF1010)1k-GFPuv 7107 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION IPTG-inducible trc promoter expressing gfpuv on RSF1010 origin.
ACCESSION .
VERSION .
KEYWORDS pBb(RSF1010)1k-GFPuv
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7107)
AUTHORS Cook TB, Rand JM, Nurani W, Courtney DK, Liu SA, Pfleger BF
TITLE Genetic tools for reliable gene expression and recombineering in
Pseudomonas putida.
JOURNAL J Ind Microbiol Biotechnol. 2018 Jan 3. pii:
10.1007/s10295-017-2001-5. doi: 10.1007/s10295-017-2001-5.
PUBMED 29299733
REFERENCE 2 (bases 1 to 7107)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7107
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 7..84
/gene="lacI (mutant)"
/label=lacIq promoter
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 85..1167
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind complement(104..123)
/label=LacI-R
/note="LacI, reverse primer"
promoter 1399..1428
/label=trc promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 1436..1452
/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)."
CDS 1493..2209
/codon_start=1
/product="GFP variant optimized for excitation by UV light"
/label=GFPuv
/translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
primer_bind complement(1523..1551)
/label=GFP-R
/note="GFP, reverse primer. Does NOT anneal to EGFP"
primer_bind 2146..2166
/label=GFP-F
/note="GFP, forward primer. Does NOT anneal to EGFP"
terminator 2242..2313
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 2329..2356
/label=T7Te terminator
/note="phage T7 early transcription terminator"
CDS complement(3207..4046)
/codon_start=1
/gene="repA"
/product="replication protein A of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/label=RSF1010 RepA
/translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
CDS complement(4557..5528)
/codon_start=1
/gene="repB"
/product="replication protein B of the broad-host-range
plasmid RSF1010 (Scholz et al., 1989)"
/label=RSF1010 RepB
/translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
rep_origin 5635..6029
/label=RSF1010 oriV
/note="replication origin of the broad-host-range plasmid
RSF1010; requires the RSF1010 RepA/B/C proteins for
replication (Scholz et al., 1989)"
terminator 6052..6146
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
CDS complement(6177..6971)
/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(6288..6307)
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind 6898..6917
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
ORIGIN
1 gacgtcgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
61 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
121 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
181 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
241 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
301 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
361 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
421 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
481 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
541 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
601 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
661 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
721 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
781 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
841 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
901 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
961 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
1021 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
1081 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
1141 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt
1201 gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca
1261 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
1321 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa
1381 atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg
1441 agcggataac aatttcagaa ttcaaaagat cttttaagaa ggagatatac atatgagtaa
1501 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa
1561 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac
1621 ccttaaattt atttgcacta ctggaaaact acctgttccg tggccaacac ttgtcactac
1681 tttctcttat ggtgttcaat gcttttcccg ttatccggat cacatgaaac ggcatgactt
1741 tttcaagagt gccatgcccg aaggttatgt acaggaacgc actatatctt tcaaagatga
1801 cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat
1861 cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt ctcggacaca aactggagta
1921 caactataac tcacacaatg tatacatcac ggcagacaaa caaaagaatg gaatcaaagc
1981 taacttcaaa attcgccaca acattgaaga tggctccgtt caactagcag accattatca
2041 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac
2101 acaatctgcc ctttcgaaag atcccaacga aaagcgtgac cacatggtcc ttcttgagtt
2161 tgtaactgct gctgggatta cacatggcat ggatgagctc tacaaataag gatccaaact
2221 cgagtaagga tctccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
2281 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
2341 ggtgggcctt tctgcgttta tacctaggtc agcctgccgc cttgggccgg gtgatgtcgt
2401 acttgcccgc cgcgaactcg gttaccgtcc agcccagcgc gaccagctcc ggcaacgcct
2461 cgcgcacccg ctggcggcgc ttgcgcatgg tcgaaccact ggcctctgac ggccagacat
2521 agccgcacaa ggtatctatg gaagccttgc cggttttgcc ggggtcgatc cagccacaca
2581 gccgctggtg cagcaggcgg gcggtttcgc tgtccagcgc ccgcacctcg tccatgctga
2641 tgcgcacatg ctggccgcca cccatgacgg cctgcgcgat caaggggttc agggccacgt
2701 acaggcgccc gtccgcctcg tcgctggcgt actccgacag cagccgaaac ccctgccgct
2761 tgcggccatt ctgggcgatg atggatacct tccaaaggcg ctcgatgcag tcctgtatgt
2821 gcttgagcgc cccaccacta tcgacctctg ccccgatttc ctttgccagc gcccgatagc
2881 tacctttgac caccatggca tcagcggtga cggcctccca cttgggttcc aggaacagcc
2941 ggagctgccg tccgccttcg gtcttgggtt ccgggccaag cactaggcca ttaggcccag
3001 ccatggccac cagcccttgc aggatgcgca gatcatcagc gcccagcggc tccgggccgc
3061 tgaactcgat ccgcttgccg tcgccgtagt catacgtcac gtccagcttg ctgcgcttgc
3121 gctcgccccg cttgagggca cggaacaggc cgggggccag acagtgcgcc gggtcgtgcc
3181 ggacgtggct gaggctgtgc ttgttcttag gcttcaccac ggggcacccc cttgctcttg
3241 cgctgcctct ccagcacggc gggcttgagc accccgccgt catgccgcct gaaccaccga
3301 tcagcgaacg gtgcgccata gttggccttg ctcacaccga agcggacgaa gaaccggcgc
3361 tggtcgtcgt ccacacccca ttcctcggcc tcggcgctgg tcatgctcga caggtaggac
3421 tgccagcgga tgttatcgac cagtaccgag ctgccccggc tggcctgctg ctggtcgcct
3481 gcgcccatca tggccgcgcc cttgctggca tggtgcagga acacgataga gcacccggta
3541 tcggcggcga tggcctccat gcgaccgatg acctgggcca tggggccgct ggcgttttct
3601 tcctcgatgt ggaaccggcg cagcgtgtcc agcaccatca ggcggcggcc ctcggcggcg
3661 cgcttgaggc cgtcgaacca ctccggggcc atgatgttgg gcaggctgcc gatcagcggc
3721 tggatcagca ggccgtcagc cacggcttgc cgttcctcgg cgctgaggtg cgccccaagg
3781 gcgtgcaggc ggtgatgaat ggcggtgggc gggtcttcgg cgggcaggta gatcaccggg
3841 ccggtgggca gttcgcccac ctccagcaga tccggcccgc ctgcaatctg tgcggccagt
3901 tgcagggcca gcatggattt accggcacca ccgggcgaca ccagcgcccc gaccgtaccg
3961 gccaccatgt tgggcaaaac gtagtccagc ggtggcggcg ctgctgcgaa cgcctccaga
4021 atattgatag gcttatgggt agccattgat tgcctccttt gcaggcagtt ggtggttagg
4081 cgctggcggg gtcactaccc ccgccctgcg ccgctctgag ttcttccagg cactcgcgca
4141 gcgcctcgta ttcgtcgtcg gtcagccaga acttgcgctg acgcatccct ttggccttca
4201 tgcgctcggc atatcgcgct tggcgtacag cgtcagggct ggccagcagg tcgccggtct
4261 gcttgtcctt ttggtctttc atatcagtca ccgagaaact tgccggggcc gaaaggcttg
4321 tcttcgcgga acaaggacaa ggtgcagccg tcaaggttaa ggctggccat atcagcgact
4381 gaaaagcggc cagcctcggc cttgtttgac gtataaccaa agccaccggg caaccaatag
4441 cccttgtcac ttttgatcag gtagaccgac cctgaagcgc ttttttcgta ttccataaaa
4501 cccccttctg tgcgtgagta ctcatagtat aacaggcgtg agtaccaacg caagcactac
4561 atgctgaaat ctggcccgcc cctgtccatg cctcgctggc ggggtgccgg tgcccgtgcc
4621 agctcggccc gcgcaagctg gacgctgggc agacccatga ccttgctgac ggtgcgctcg
4681 atgtaatccg cttcgtggcc gggcttgcgc tctgccagcg ctgggctggc ctcggccatg
4741 gccttgccga tttcctcggc actgcggccc cggctggcca gcttctgcgc ggcgataaag
4801 tcgcacttgc tgaggtcatc accgaagcgc ttgaccagcc cggccatctc gctgcggtac
4861 tcgtccagcg ccgtgcgccg gtggcggcta agctgccgct cgggcagttc gaggctggcc
4921 agcctgcggg ccttctcctg ctgccgctgg gcctgctcga tctgctggcc agcctgctgc
4981 accagcgccg ggccagcggt ggcggtcttg cccttggatt cacgcagcag cacccacggc
5041 tgataaccgg cgcgggtggt gtgcttgtcc ttgcggttgg tgaagcccgc caagcggcca
5101 tagtggcggc tgtcggcgct ggccgggtcg gcgtcgtact cgctggccag cgtccgggca
5161 atctgccccc gaagttcacc gcctgcggcg tcggccacct tgacccatgc ctgatagttc
5221 ttcgggctgg tttccactac cagggcaggc tcccggccct cggctttcat gtcatccagg
5281 tcaaactcgc tgaggtcgtc caccagcacc agaccatgcc gctcctgctc ggcgggcctg
5341 atatacacgt cattgccctg ggcattcatc cgcttgagcc atggcgtgtt ctggagcact
5401 tcggcggctg accattcccg gttcatcatc tggccggtgg tggcgtccct gacgccgata
5461 tcgaagcgct cacagcccat ggccttgagc tgtcggccta tggcctgcaa agtcctgtcg
5521 ttcttcatcg ggccaccaag cgattcccac acattatacg agccggaagc ataaagtgta
5581 gctagatccg aaggatgagc cgggctgaat gatcgaccga gacaggccct gcggggctgc
5641 acacgcgccc ccacccttcg ggtaggggga aaggccgcta aagcggctaa aagcgctcca
5701 gcgtatttct gcggggtttg gtgtggggtt tagcgggctt tgcccgcctt tccccctgcc
5761 gcgcagcggt ggggcggtgt gtagcctagc gcagcgaata gaccagctat ccggcctctg
5821 gccgggcata ttgggcaagg gcagcagcgc cccacaaggg cgctgataac cgcgcctagt
5881 ggattattct tagataatca tggatggatt tttccaacac cccgccagcc cccgcccctg
5941 ctgggtttgc aggtttgggg gcgtgacagt tattgcaggg gttcgtgaca gttattgcag
6001 gggggcgtga cagttattgc aggggttcgt gacagttaga ctagtgcttg gattctcacc
6061 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt
6121 cattactgga tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag
6181 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg
6241 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta
6301 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca
6361 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg
6421 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc
6481 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt
6541 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta
6601 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat
6661 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg
6721 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct
6781 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg
6841 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc
6901 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct
6961 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcatga tcccctgcgc
7021 catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta
7081 ccagagggcg ccccagctgg caattcc
//
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